Abstracts from the 53rd European Society of Human Genetics (ESHG) Conference: Interactive e-Posters

Interactive e-Posters

P01 Reproductive Genetics/Prenatal Genetics
P01.001.A Prenatal diagnostics of 15q11.2 microdeletion syndrome - the possibilities and challenges

K. Belemezova 1,2 , P. Chaveeva 1 , V. Stratieva 3,4 , M. Yankova 3,5 , V. Peycheva 6 , R. Bozhilova 6 , M. Rizov 1 , S. Yovinska 1 , M. Hristova-Savova 1 , R. Kaneva 6 , I. Dimova 1,6

1 SAGBAL „Dr Shterev“, Sofia, Bulgaria, 2 University Hospital “St. Ivan Rilski”, Department of Immunology, Medical University of Sofia, Sofia, Bulgaria, 3 O.S.С.A.R. Clinic, Sofia, Bulgaria, 4 First Specialized Hospital of Obstetrics and Gynecology „St. Sofia”, Sofia, Bulgaria, 5 University Hospital "Lozenetz", Sofia, Bulgaria, 6 Center of Molecular medicine, Medical University of Sofia, Sofia, Bulgaria
Microdeletion in 15q11.2 has been described as a distinct syndrome encompassing an area between two fragile sites in 15q (BP1 and BP2), with approximately 500 bp size, containing NIPA1, NIPA2, TUBGCP5 , and CYFIP1 genes. It is a multisystemic disease affecting mostly the nervous system (intellectual deficits, delayed psychomotor development, ataxia, epilepsy, behavioral problems, etc.), followed by congenital heart defects and various dysmorphisms. This microdeletion was found in 0.57-1.27% of pediatric patients targeted for microarray analysis, mainly with developmental delay and intellectual deficits. However, not all of the deletion carriers have a clinical manifestation - it is found in 0.25% of the population of healthy controls. The penetrance of 15q11.2 microdelection syndrome is estimated at 10.4%, which is significantly higher in de novo occurrence. We report 4 cases of prenatally established 15q11.2 microdeletion, in 3 of the cases the microdeletion was inherited from clinically healthy mothers. The indications for aCGH prenatal diagnosis were: 1) ultrasound data for unilateral cleft lip and palate in combination with an increased nuchal translucency of 3.3 mm- mother was the carrier; 2) second pregnancy after the first case; 3) increased risk from the biochemical screening (1:6) and tricuspid regurgitation, brother with neuropsychiatric disability - mother was a carrier; 4) Increased NT of 7.3 mm. Our results highlight the importance of microarray analysis in the diagnostic refinement of pregnancies which are abnormal in early fetal screening. We emphasize the difficulties of genetic counseling in the inheritance and the incomplete penetrance of the aberrations. K. Belemezova: None. P. Chaveeva: None. V. Stratieva: None. M. Yankova: None. V. Peycheva: None. R. Bozhilova: None. M. Rizov: None. S. Yovinska: None. M. Hristova-Savova: None. R. Kaneva: None. I. Dimova: None. P01.002.B Improvement of prenatal care in case of fetal anomaly of the corpus callosum using exome sequencing during the pregnancy

S. Heide 1 , M. Spentchian 1 , S. Valence 2 , J. Buratti 3 , C. Mach 3 , E. Lejeune 3 , V. Olin 3 , M. Massimello 1 , C. Garel 4 , E. Blondiaux 4 , G. Quenum-Miraillet 5 , S. Chantot-Bastaraud 5 , M. Milh 6 , V. des Portes 7 , M. Spodenkiewic 8 , V. Layet 9 , R. Dard 10 , S. Moutton 11 , M. Gorce 12 , M. Nizon 13 , S. Mercier 13 , M. Vincent 13 , C. Beneteau 13 , J. Jouannic 14 , M. Moutard 2 , B. Keren 3 , D. Héron 3

1 UF de Génétique Médicale et CRMR « Déficience intellectuelle », Département de Génétique, Groupe Hospitalier Pitié-Salpêtrière, APHP Sorbonne Université, Paris, France, 2 Service de Neurologie Pédiatrique, Hôpital Armand Trousseau, APHP Sorbonne Université, Paris, France, 3 UF de Génomique du Développement, Département de Génétique, Groupe Hospitalier Pitié-Salpêtrière, APHP Sorbonne Université, Paris, France, 4 Service de Radiologie Pédiatrique, Hôpital Armand Trousseau, APHP Sorbonne Université, Paris, France, 5 Service de Cytogénétique, Hôpital Armand Trousseau, HUEP, APHP Sorbonne Université, Paris, France, 6 Service de Neurologie Pédiatrique, Hôpital La Timone, APHM, Marseille, France, 7 Service de Neurologie Pédiatrique, Hospices Civils de Lyon, Bron, France, 8 Service de Génétique Clinique, CHU de Reims, Reims, France, 9 Service de Génétique Clinique, Hôpital du Havre, Le Havre, France, 10 Service de Génétique Clinique, Hôpital de Poissy, Poissy, France, 11 Centre de Référence des anomalies du développement, Service de Génétique Médicale, CHU de Dijon, Dijon, France, 12 Service de génétique clinique, CHU d’Angers, Angers, France, 13 Service de Génétique Clinique, CHU de Nantes, Nantes, France, 14 Service de Médecine Fœtale, Hôpital Armand Trousseau, HUEP, APHP Sorbonne Université, Paris, France
Purpose: Anomalies of the corpus callosum (ACC) are usually diagnosed by prenatal ultrasound. This malformation is characterized by its uncertain prognosis especially when isolated which makes the prenatal counseling difficult. Among genetic etiologies, only chromosomal anomalies are investigated during the prenatal period while ACC is due to single gene mutation in most cases and neurodevelopmental prognosis depends on the underlying cause. Methods: In order to evaluate the feasibility and contribution of prenatal exome sequencing (pES) during the pregnancy, we included 66 fetuses with ACC for trio analysis. Only pathogenic variants in known ACC and/or ID genes were considered. Results: pES results were available within a median delay of 22 days. A pathogenic SNV was identified in 12 cases (18%). Moreover, a pathogenic CNV was identified in 4 cases. Thus, the genetic cause was determined in 22% of cases. In all cases with diagnoses, pES results enlightened the neurodevelopmental prognosis of the fetus and enabled the parents to choose to continue with or terminate the pregnancy knowingly. Conclusions: This is the first study of pES focusing on a single malformation characterized by an uncertain prognosis. Our results demonstrate the feasibility of pES in fetuses with prenatal diagnosis of ACC. pES has an evident clinical impact. The impact of pES when negative remains to be evaluated S. Heide: None. M. Spentchian: None. S. Valence: None. J. Buratti: None. C. Mach: None. E. Lejeune: None. V. Olin: None. M. Massimello: None. C. Garel: None. E. Blondiaux: None. G. Quenum-Miraillet: None. S. Chantot-Bastaraud: None. M. Milh: None. V. des Portes: None. M. Spodenkiewic: None. V. Layet: None. R. Dard: None. S. Moutton: None. M. Gorce: None. M. Nizon: None. S. Mercier: None. M. Vincent: None. C. Beneteau: None. J. Jouannic: None. M. Moutard: None. B. Keren: None. D. Héron: None. P01.003.C Genetic management of a small deletion in the PWS/AS imprinting center in a prenatal case

C. Pérez 1 , E. Pestaña 2 , C. Rivera 2 , M. Augé 1 , L. Barranco 1 , A. Canellas 1 , M. Costa 1 , C. de la Iglesia 1 , D. Fernández 1 , N. Palau 1 , B. Méndez 1 , M. Piqué 1 , M. Herrero 1 , D. Yeste 1 , L. Puig 1 , R. Ferreti 1 , M. Punzón 1 , S. Martín 1 , J. Mendoza 1 , N. Pinedo 1 , M. Rodríguez 1 , L. Linares 1 , E. Lloveras 1

1 Synlab, Esplugues de Llobregat, Spain, 2 Synlab, Madrid, Spain
Introduction: Imprinted genes involved in PWS and AS are under control of an imprinting center comprising two regulatory regions, PWS-SRO (around the SNRPN promoter) and the AS-SRO (35 kb upstream). A very few AS patients have small and/or atypical deletions in AS-SRO. Patient and Results: Pregnant patient (13 weeks) was referred because a daughter of 4 years old with development delay and peculiar phenotype and a 62Kb copy number loss at 15q11.2, arr[GRCh37] 15q11.2(25119854_25177138)x1. In extended familial studies, mother (pregnant) and grandfather have both the same deletion. MS-MLPA was performed in the daughter and the pregnant. The 15q11.2 deletion was confirmed in both and an altered methylation pattern in the daughter was detected correlating with an AS diagnosis. ArrayCGH analysis (CytoSure™ Constitutional v3 8x60k, OGT, UK) was performed in chorionic villus sample and the same deletion of 62Kb involving U5 exon of AS-SRO (exon 4 according NM sequence of SNRPN gene) was detected. MS-MLPA (PW/AS MS-MLPA Kit, MRC Holland) was performed in amniotic fluid with alteration of the methylation pattern, compatible with hypomethylation of the probes located in the CpG islands region of SNRPN gene was detected. Considering all the results, a prenatal diagnosis of AS was performed. Comments: IC deletion have been described in a small fraction of AS patients with ID, with no disease-causing variants detected to date.Deletions involving AS-SRO are a molecular defect that must be determined as the recurrence risk in case of a familial IC deletion. Caution should be made in prenatal diagnosis and is appropriate recommending MS-MLPA analysis. C. Pérez: None. E. Pestaña: None. C. Rivera: None. M. Augé: None. L. Barranco: None. A. Canellas: None. M. Costa: None. C. de la Iglesia: None. D. Fernández: None. N. Palau: None. B. Méndez: None. M. Piqué: None. M. Herrero: None. D. Yeste: None. L. Puig: None. R. Ferreti: None. M. Punzón: None. S. Martín: None. J. Mendoza: None. N. Pinedo: None. M. Rodríguez: None. L. Linares: None. E. Lloveras: None. P01.005.B START: Thestudy of assistedreproductive technologies

R. Lyle 1 , C. Page 2 , Y. Lee 2 , K. L. Haftorn 2 , A. Jugessur 2 , J. Bohlin 2 , H. K. Gjessing 2 , P. Magnus 2 , S. Håberg 2

1 Oslo University Hospital, Oslo, Norway, 2 Norwegian Institute of Public Health, Oslo, Norway
In the last few decades there have been marked changes in patterns of fertility and family structure in rich countries. These include increasing age at childbirth, a lower number of children born to each woman or man, greater use of assisted reproductive technology (ART), higher frequency of family disruptions and increasingly complex family structures. The Centre for Fertility and Health (CeFH; www.cefh.no ) consists of a multidisciplinary research team of epidemiologists, geneticists, demographers, sociologists and economists with research themes including fertility problems and intergenerational transmission of health . The START project aims to understand the causes and health consequences of subfertility and how genetic influences and epigenetic differences are associated with subfertility and Assisted Reproductive Technologies (ART). ART may introduce epigenetic changes in the embryo since the procedures are during a period of extensive epigenetic repatterning. It is unknown which genes may be affected and the potential health consequences for the children. START uses data from the Norwegian Mother, Father and Child Cohort Study (MoBa; https://www.fhi.no/en/studies/moba/ ), which follows more than 110 000 children and their parents. We have collected genetic and epigenetic data from 1000 trios with children born after ART, and 1000 trios from naturally conceived children analyzed with the Illumina EPIC array and the Illumina HumanCoreExome chip. Investigations are focussed on the effect of ART treatment on imprinted genes and fertility genes, and how epigenetic age acceleration interacts with fertility and ART treatment. Our results show DNA methylation changes due to both parental subfertility and ART procedures. R. Lyle: None. C. Page: None. Y. Lee: None. K.L. Haftorn: None. A. Jugessur: None. J. Bohlin: None. H.K. Gjessing: None. P. Magnus: None. S. Håberg: None. P01.006.C Beckwith Wiedemann Syndrome: a prenatal case report with a novel CDKN1C mutation

P. Maia Almeida 1,2 , S. Moreira Ribeiro 1 , L. Abreu 3 , A. Isabel Rei 3 , M. Branco 3 , E. Galhano 3 , J. Saraiva 1,4,5 , F. Ramos 1,3

1 Medical Genetics Unit, Centro Hospitalar e Universitário de Coimbra, Coimbra, Portugal, 2 Faculty of Health Sciences, University of Beira Interior, Covilhã, Portugal, 3 Prenatal Diagnostic Center, Maternidade Bissaya Barreto, Centro Hospitalar e Universitário de Coimbra, Coimbra, Portugal, 4 University Clinic of Pediatrics, Faculty of Medicine, University of Coimbra, Coimbra, Portugal, 5 Clinical Academic Center of Coimbra, Coimbra, Portugal
Introduction: Beckwith Wiedemann Syndrome (BWS) is a genomic imprinting disorder typically diagnosed after birth. Some ultrasound features that include polyhydramnios, omphalocele, and visceromegaly may raise the clinical suspicion in prenatal. Most reported BWS cases are sporadic, but around 15% may be familial. Delineation of the molecular defects can predict familial recurrence risks and make invasive prenatal diagnosis possible. Case Report: We report a case of a 41-year-old pregnant woman in whom ultrasound revealed at 24-weeks corpus callosum hypoplasia, macroglossia, omphalocele, short long bones and enlarged kidneys. The pregnancy was terminated and autopsy confirmed the ultrasound findings and also enlarged ovaries and pancreas, cytomegaly of the adrenal cortex and a placental mesenchymal dysplasia. Family history was irrelevant. Materials and Methods: Clinical and molecular characterization of a prenatal case with diagnosis of BWS. We performed array-CGH, methylation analysis at 11p15.5 BWS locus and Sanger sequencing of CDKN1C gene. The presence of the variant identified in the foetus was tested in the parents. Results: Array-CGH and 11p15.5 methylation analysis ( MS-MLPA ) were normal. CDKN1C Sanger sequencing found a heterozygous novel probable pathogenic frameshift variant: NM_000076.2:c.479del p.(Pro160Argfs*112) of maternal origin. Conclusions: This case demonstrates that search for a clear specific molecular diagnosis is key not only to confirm the diagnosis but also to provide proper genetic counselling. Although present in only 5% of the patients with no family history the identification of a CDKN1C pathogenic variant of maternal origin increases the recurrence risk to 50%. P. Maia Almeida: None. S. Moreira Ribeiro: None. L. Abreu: None. A. Isabel Rei: None. M. Branco: None. E. Galhano: None. J. Saraiva: None. F. Ramos: None. P01.007.A Prenatal phenotype in Beckwith-Wiedemann spectrum

D. Carli, C. Bertola, S. Cardaropoli, V. P. Ciuffreda, M. Pieretto, G. B. Ferrero , A. Mussa

Department of Public Health and Pediatrics, University of Torino, Torino, Italy
Introduction: the prenatal phenotype of Beckwith-Wiedemann spectrum (BWSp) is not well characterized. Here we investigate the prenatal features of large series of BWSp patients. Methods: postnatal phenotype, genotype, prenatal ultrasound (US) features, pregnancy course and biochemical screening tests of 88 patients with BWSp (51 females) were reviewed. Results: molecular tests had been performed in 86 patients, 80 resulted positives for BWSp-related anomalies, 6 negative with a clinical diagnosis. Seventeen (19.3%) were conceived through artificial reproductive techniques. Twinning occurred in 9 pregnancies and resulted in 13 BWSp newborns. Pregnancy biochemical screening tests showed increased serum markers: PAPP-A at 10-13 weeks of gestational age ( n = 44, 1.13 ± 0.61 MoM, p < 0.001), and αFP (1.52 ± 0.80 MoM, p = 0.003), uEstriol (1.41 ± 0.37 MoM, p < 0.001) and total hCG (2.10 ± 2.17 MoM, p < 0.001) at 15-18 weeks ( n = 27). Morphology US scan (available in 64) revealed abdominal and head circumferences higher than normal (1.41 ± 1.10 SD, p < 0.001 and 0.44 ± 1.04, p = 0.003, respectively) with normal femur lengths. Fifty-eight patients (66%) had a various combination of pathologic US findings, including enlarged abdominal circumference at 20 weeks’ scan ( n = 17), macrosomia at a subsequent scan ( n = 35, of these 14 normal at 20 weeks’ scan), omphalocele ( n = 12), enlargement of abdominal organs ( n = 6), macroglossia ( n = 15), adrenal cysts/masses ( n = 2), nephroureteral anomalies ( n = 11), polyhydramnios ( n = 24), placental enlargement ( n = 2) or mesenchymal dysplasia ( n = 4). Thirty cases had no detected anomaly at any US. Conclusions: on the basis of these findings, we propose a new embryo-fetal BWSp scoring system, mirroring the post-natal one, that will implement the prenatal clinical approach of this developmental syndrome. D. Carli: None. C. Bertola: None. S. Cardaropoli: None. V.P. Ciuffreda: None. M. Pieretto: None. G.B. Ferrero: None. A. Mussa: None. P01.008.B Prenatal diagnosis of a severe form of BICD2-opathy : further evidence of an extreme phenotypic spectrum

E. Marchionni 1 , E. Agolini 2 , G. Mastromoro 1 , D. Guadagnolo 1 , F. Di Palma 1 , C. Pajno 3 , V. D’Ambrosio 3 , M. Roggini 4 , A. Giancotti 3 , A. Novelli 2 , A. Pizzuti 1

1 Department of Experimental Medicine, Sapienza University of Rome, Rome, Italy, 2 Laboratory of Medical Genetics, Ospedale Pediatrico Bambino Gesù, Rome, Italy, 3 Department of Maternal and Child Health and Urologic Science, Sapienza University of Rome, Rome, Italy, 4 Pediatrics and Child Neuropsychiatry Department, Sapienza University of Rome, Rome, Italy
Introduction: Arthrogryposis multiplex congenita (AMC) is characterized by the development of multiple joint contractures. Neurogenic and myogenic-types have been described, resulting in complete or partial movement restriction. In prenatal diagnosis decreased fetal movement is observed, the most severe forms being lethal in utero or after birth. We report a case of a male fetus at 22 gestational weeks with evidence of AMC on ultrasound, presenting fixed extended legs and flexed arms, bilateral clubfoot and absent fetal movements. Frontal oedema was also observed. After amniocentesis, karyotype and SNParray resulted normal. Materials and Methods: Trio-based Whole Exome Sequencing (WES) was performed on fetus genomic DNA and his unaffected parents. Results: WES analysis disclosed a de novo heterozygous pathogenic deletion (NM_015250.3: c.1636_1638delAAT; p. Asn546del) in BICD2 gene, associated with autosomal dominant spinal muscular atrophy, lower extremity-predominant 2 (SMALED2). The couple decided to terminate the pregnancy. Babygram showed bilateral genu recurvatum, bilateral clubfoot and clubhand and multiple joints contractures. Conclusions: BICD2 mutations are associated with severe prenatal-onset forms of spinal muscular atrophy, lower extremity-predominant 2B (SMALED2B) or milder forms with childhood-onset (SMALED2A). In literature few prenatal cases are reported, mostly with early lethal outcome. The same variant was previously described in two children with a severe postnatal phenotype but belated or milder prenatal signs. This observation expands BICD2 phenotypic spectrum, with further evidence of early-onset extreme prenatal forms. To date, the localization of the disease-causing mutations does not seem to correlate with disease severity, other molecular mechanisms should be investigated to elucidate genotype-phenotype correlations. E. Marchionni: None. E. Agolini: None. G. Mastromoro: None. D. Guadagnolo: None. F. Di Palma: None. C. Pajno: None. V. D’Ambrosio: None. M. Roggini: None. A. Giancotti: None. A. Novelli: None. A. Pizzuti: None. P01.010.A Development and validation of an expanded pan-ethnic carrier screening test

S. Kyriakou , A. Achilleos, M. Georgiadou, C. Loizides, M. Nicoalou, V. Soteriou, C. Kkoufou, L. Constantinou, G. Billioud, K. Tsangaras, E. Kypri, M. Ioannides, G. Koumbaris, P. C. Patsalis

NIPD Genetics, Nicosia, Cyprus
Introduction: Carrier screening increasingly becomes more prevalent as prospective parents become more aware that the cumulative risk of having an affected child with an autosomal or X-linked condition can be higher than that for common aneuploidies. The changing admixture of populations, and the increase in gamete donation in assisted reproductive technologies have led professional organizations and professional bodies to recommend expanded carrier screening to the general population. Materials and Methods: The test was developed using targeted in-solution hybridization enrichment followed by next generation sequencing covering all gene exons, and intron-exon boundaries, associated with 229 autosomal recessive and X-linked conditions. The sensitivity and specificity for single nucleotide variant (SNV), indel and copy number variant (CNV) detection was ascertained in a blind validation study. Results: SNVs and indels were detected at high sensitivity (>99.0%) and specificity (>99.9%). The algorithm was also designed to detect CNVs to a single or a few exons with high sensitivity and specificity. Each positive call was confirmed with an orthogonal method. Conclusions: We have developed and validated an expanded pan-ethnic carrier screening test which includes 229 autosomal recessive and X-linked conditions. The test provides individuals and prospective parents with clinical information, helping them understand and evaluate their reproductive options. S. Kyriakou: A. Employment (full or part-time); Significant; NIPD Genetics. A. Achilleos: A. Employment (full or part-time); Significant; NIPD Genetics. M. Georgiadou: A. Employment (full or part-time); Significant; NIPD Genetics. C. Loizides: A. Employment (full or part-time); Significant; NIPD Genetics. M. Nicoalou: A. Employment (full or part-time); Significant; NIPD Genetics. V. Soteriou: A. Employment (full or part-time); Significant; NIPD Genetics. C. Kkoufou: A. Employment (full or part-time); Significant; NIPD Genetics. L. Constantinou: A. Employment (full or part-time); Significant; NIPD Genetics. G. Billioud: A. Employment (full or part-time); Significant; NIPD Genetics. K. Tsangaras: A. Employment (full or part-time); Significant; NIPD Genetics. E. Kypri: A. Employment (full or part-time); Significant; NIPD Genetics. M. Ioannides: A. Employment (full or part-time); Significant; NIPD Genetics. G. Koumbaris: A. Employment (full or part-time); Significant; NIPD Genetics. P.C. Patsalis: A. Employment (full or part-time); Significant; NIPD Genetics. P01.014.B CGH-array as first-tier test in all undergoing invasive prenatal samples: an experience for a reference Centre .

M. A. Mori 1 , J. Nevado 1 , E. Mansilla 1 , F. A. Garcia-Santiago 1 , E. Antolín 2 , C. Peña 1 , C. Adam 1 , P. D. Lapunzina 1

1 INGEMM-IdiPaz, Madrid, Spain, 2 Sº Fisiopatología Fetal, HULP, Madrid, Spain
Introduction: In the last years microarrays (CMA) has demonstrated to be a significant tool useful in the prenatal area, mainly in fetuses with sonography anomalies. However, using array-CGH in an invasive prenatal context is still under a significant and controversial debate especially regarding the use of CMA in advance maternal age or maternal anxiety samples. Previous studies supported the evidence on the feasibility of introducing a-CGH as a first-line diagnostic testing routine prenatal diagnosis practice (1). Indeed, others and our laboratory have reported significant diagnostic yields also in low-risk pregnancies, between 1.6-4% (2,3). Methods : We used customized Karyoarray®v3.0 platform (4) to study consecutive invasive prenatal samples along two-year period. QF-PCR positive cases were excluded. Results : We analyzed 800 samples and we found globally 6% of pathogenic CNVs and 2% of VUS. We segregated low-risk pregnancy samples (positive biochemical screening, soft markers, maternal anxiety, and antenatal death without sonographic anomalies). Diagnostic yield in this group was 4.5% (17/379). Discussion : In conclusion, the use of the array-CGH in the prenatal context demonstrated a greater diagnostic capacity not only in fetuses with ultrasound abnormalities but also in low-risk pregnancies. Therefore, we definitively recommend the possibility of using the array-CGH in all those gestations that undergo invasive prenatal diagnosis, after excluding by QF-PCR the most common aneuploidies. (1)Kan AS. et al., 2014. PLoS One. 9(2):e87988. (2)Wapner RJ. et al., 2012. N Engl J Med .367:2175-2184. (3)Armengol L. et al. 2012. Hum Genet . 131:513-523. (4)Vallespin E. et al,. 2013. Am J Med Genet A.161A:1950-60. M.A. Mori: None. J. Nevado: None. E. Mansilla: None. F.A. Garcia-Santiago: None. E. Antolín: None. C. Peña: None. C. Adam: None. P.D. Lapunzina: None. P01.016.A DERIVATIVE(9)T(2;9)(P23;P22) IN HIGH RISK TWIN PREGNANCY

M. Kadkhoda zadeh , s. seyedhassani, f. harrazi zadeh yazdi, a. danafar, m. mirjalili, f. saeb

yazdgenetic, yazd, Iran, Islamic Republic of
Introduction: A 39- year- old mother was referred with GA=16 w + 5 d and high risk pregnancy screening test for T 21. The first child of this patient was a 14- years old suffering from developmental delay and mental retardation. On the other hand, this family had two spontaneous abortion. These evidences revealed enough reasons for probability of chromosomal abnormalities such as Down syndrome, monosomy X, and other chromosomal disorder in fetus. Amniocentesis was suggested for complementary investigation. Methods and materials: Twenty metaphases cells were studied on the basis of G-Banding technique from culture of amniocytes in two flasks. Then, karyotypig was done on the basis of G-Banding for the parents. Result: In this study, we described a paternal chromosomal aberration of twins. Through a combination of cytogenetic assessment of fetus and parents 46, XY, der (9) t(2;9) (p23;p22)pat was shown. Mother’s karyotype was 46XX but father’s karyotype revealed an abnormal chromosome complement in all cells with balanced 46,XY,t(2;9)(p23;p22). Conclusions: Paternal chromosomal balanced translocation can explain repeated abortions in this family. On the other hand, unbalanced chromosomal translocation resulting from paternal inheritance lead to developmental delay in his son. This study was highly emphasized the priority of genetic counseling and attention to the family history and genetic basis of parents in evolution of repeated spontaneous abortion. Legal abortion was recommended to unbalance chromosomal abnormalities. M. Kadkhoda zadeh: None. S. seyedhassani: None. F. harrazi zadeh yazdi: None. A. danafar: None. M. mirjalili: None. F. saeb: None. P01.017.B Karyotype analysis of 8531 pregnancies in prenataly identified cases with amniocentesis from south of Turkey

A. Pazarbasi 1 , D. Alptekin 1 , N. S. Ilgaz 1 , I. N. Uslu 1 , G. Ay 1 , S. Kocaturk-Sel 1 , O. Demirhan 1 , U. Luleyap 1 , M. B. Yilmaz 1 , N. Çetinel 1 , G. Evyapan 1 , G. Comertpay-Tufan 1 , S. Buyukkurt 2

1 Çukurova University, Faculty of Medicine, Dept. of Medical Biology, Adana, Turkey, 2 Çukurova University, Faculty of Medicine, Dept. of Obstetrics and Gynecology, Adana, Turkey
Amniocentesis is a very crucial diagnostic procedure for preventing the birth of genetically defective fetuses in order to decrease the prevalence of genetic diseases in populations. A retrospective review of our amniocentesis database for the period from January 2000 to February 2020 was carried out. The karyotyping of 8531 fetuses was carried out in Department of Medical Biology from the samples of amniotic fluids which were sent from Department of Gynecology and Obstetrics of Balcali Hospital. A standart nomenclature has been developed to describe each of types of abnormality found in human chromosomes. A total of 8531 amniocentesis specimens were processed during the study period. 620 fetuses (7.26%) had various chromosomal abnormalities. 54.51% of abnormal karyotypes (338 cases) were numerical and 43.38% (269 cases) were structural. Both numerical and structural chromosomal aberrations were observed in 13 cases (2.09%). The ratios were as: trisomy 21 (49.70%), trisomy 18 (17.75%), monosomy X (9.46%), trisomy 13 (6.80%), Triploidy (4.73%), Klinefelter Syndrome (3.25%), Trisomy X (1. 18%), XYY Syndrome (0.88%), and the others in all numerical abnormalities. The frequent structural abnormalities were as: 46,XX/XY, inv(9) (p11;q12)/(p11;q13)(28.25 %), 46,XX/XY, 1qh(+)(11.89%), 46,XY, Yqh(-)(7.43%), 46,XX/XY, 16qh(+)(7.06%), 46,XX/XY, 9qh(+)(4.46%) and 46,XY, Yqh(+)(4.08%). Balanced and unbalanced translocations, deletions and duplications were also found in less ratio. According to the literature and our results, advanced maternal age is the main cause of fetal chromosomal abnormalities. Fetal chromosomal abnormality ratio that we found was 7.26%. This ratio emphasize the importance of prenatal diagnosis. A. Pazarbasi: None. D. Alptekin: None. N.S. Ilgaz: None. I.N. Uslu: None. G. Ay: None. S. Kocaturk-Sel: None. O. Demirhan: None. U. Luleyap: None. M.B. Yilmaz: None. N. Çetinel: None. G. Evyapan: None. G. Comertpay-Tufan: None. S. Buyukkurt: None. P01.018.C Microarray testing in pregnancies with nuchal translucency 3-3.4 mm - time to change the cut-off .

L. Sagi-Dain 1 , I. Maya 2 , A. Singer 3

1 Carmel Medical Center, Haifa, Israel, 2 Rabin Medical Center, Petach Tikva, Israel, 3 Sheba Medical Center, Ramat Gan, Israel
Objective: To examine the risk for chromosomal microarray analysis (CMA) aberrations in fetuses with nuchal translucency (NT) of 3 to 3.4 mm. Methods: The results of all CMA tests performed due to an NT of 3 to 3.4 mm were retrospectively retrieved from the Ministry of Health computerized database. The rates of clinically significant (pathogenic and likely pathogenic) microarray findings were compared to a previously published local control population, encompassing 5541 fetuses with normal ultrasound. Results: Overall, 619 CMA analyses were performed due to a sonographic indication of NT of 3 to 3.4 mm. Of these, 29 (4.7%) clinically significant copy number variants were detected, a considerably increased risk compared to control pregnancies. Fetal karyotyping would have missed 31% of the abnormal cases, while non-invasive prenatal screening (NIPS) would have overlooked 31-49% of the anomalies, depending on the method. Conclusions: The outcomes of our study, representing the largest number of CMA results in fetuses with NT of 3 to 3.4 mm, suggest that the rate of abnormal CMA findings in these pregnancies is significantly higher compared to pregnancies with normal ultrasound. Our results support the consideration to perform invasive prenatal testing for CMA in pregnancies with NT above 3 mm. In addition, as several of the CMA findings in these pregnancies are not associated with major sonographic anomalies, it seems that NIPS with later sonographic survey cannot adequately replace NT measurement. L. Sagi-Dain: None. I. Maya: None. A. Singer: None. P01.020.B Three fetuses with achondrogenesis type 2 explained by maternal mosaicism

S. Z. Jan 1 , R. F. Suijkerbuijk 1 , Y. J. Vos 1 , P. M. Grootscholten 1 , L. Zijlstra 1 , K. Bouman 1 , M. T. M. Franssen 2 , M. H. Schoots 3 , N. Corsten-Janssen 1 , B. Sikkema-Raddatz 1

1 Univeristy Medical Center Groningen, Department of Genetics, Groningen, Netherlands, 2 Univeristy Medical Center Groningen, Department of Obstetrics, Groningen, Netherlands, 3 Univeristy Medical Center Groningen, Department of Pathology and Medical Biology, Groningen, Netherlands
Background: Adding whole exome sequencing (WES) to the standard diagnostic procedure in fetuses with ultrasound anomalies improves the diagnostic yield. Identifying a diagnosis is important for parental choice and informed reproductive decision making, amongst other factors. Here we describe three fetuses in whom a diagnosis of achondrogenesis type 2 ( COL2A1 variant) due to maternal mosaicism was made using WES. Materials & Methods: A couple expecting dichorionic diamniotic twins, both with severe limb shortening and fetal hydrops, was referred to our department. A previous pregnancy had been terminated for the same reasons. QF-PCR and SNP array were performed on DNA of chorionic villi from both fetuses of the current pregnancy. WES was performed on parental DNA (extracted from blood) and fetal DNA. Results: QF-PCR and SNP-array showed no abnormalities for both fetuses. WES revealed a pathogenic, apparently de novo, COL2A1 variant c.3062_3079del p.(Pro1021_Gly1026del) in both samples, which explained the ultrasound abnormalities. Retrospective analysis confirmed the presence of the same COL2A1 variant in the previous pregnancy. Re-analysis of parental WES data revealed the variant in less than 1% of maternal reads, implying an extremely low grade of mosaicism, which was confirmed by Sanger sequencing. Conclusions: This case study highlights the added value of WES in prenatal diagnostics, including the capacity to identify extremely low-grade parental mosaicism that profoundly impact the recurrence risk of a genetic disease. It also suggests that the possibility of very low-grade parental mosaicism should be closely investigated when an apparently de novo variant is identified. S.Z. Jan: None. R.F. Suijkerbuijk: None. Y.J. Vos: None. P.M. Grootscholten: None. L. Zijlstra: None. K. Bouman: None. M.T.M. Franssen: None. M.H. Schoots: None. N. Corsten-Janssen: None. B. Sikkema-Raddatz: None. P01.021.C Prenatal diagnostics in Estonia: a 2-year prospective study assessing the performance of combined first-trimester screening, chromosomal microarray analysis, and next-generation sequencing

K. Ridnõi 1,2 , K. Muru 1,3 , M. Keernik 1 , S. Pajusalu 1,3,4 , E. Ustav 5 , P. Tammur 1 , T. Mölter-Väär 1 , T. Kahre 1,3 , U. Šamarina 1 , K. Asser 5 , F. Szirko 2 , T. Reimand 1,3,6 , K. Õunap 1,3

1 Department of Clinical Genetics, United Laboratories, Tartu University Hospital, Tartu, Estonia, 2 Centre for Perinatal Care, Women’s Clinic, East-Tallinn Central Hospital, Tallinn, Estonia, 3 Department of Clinical Genetics, Institute of Clinical Medicine, University of Tartu, Tartu, Estonia, 4 Yale University School of Medicine, Department of Genetics, New Haven, CT, United States, 5 Women’s Clinic, Tartu University Hospital, Tartu, Estonia, 6 Department of Biomedicine, Institute of Biomedicine and Translational Medicine, University of Tartu, Tartu, Estonia
Introduction: The implementation of cell-free fetal DNA (cff-DNA) testing in maternal blood opened possibilities to improve performance of combined first trimester screening (cFTS). On the other hand, use of new molecular methods, such as chromosomal microarray analysis (CMA) and next-generation sequencing (NGS), have shown benefits in diagnosis of chromosomal and genetic diseases, which are not detectable with cff-DNA screening, but require invasive procedure. We prospectively evaluated performance of cFTS in Estonia during 2 years using national guidelines with application of CMA and NGS in high-risk pregnancies. Material and methods: We investigated 14,566 singleton pregnancies (60% of all pregnancies). We selected 334 high-risk pregnancies for CMA diagnostic performance evaluation and 28 cases of highly dysmorphic fetuses for NGS analysis. CMA study group was divided into two groups based on the indications for testing: group A patients with high-risk for trisomies after cFTS, but normal ultrasound and group B patients who met criteria for CMA as a first-tier diagnostic test. Results: We found the first trimester detection rate 94%, 100% and 100% for trisomies 21, 18 and 13, respectively, with false-positive rate of 3.1%. The diagnostic yield of CMA was overall 3.6% (1.7% in Group A and 6.0% in Group B). In NGS analysis group we report diagnostic yield of 17.9%. Combining these three methods, overall detection rate of all genetic anomalies was 101 (0.7%). Conclusions: the use of CMA and NGS in high-risk pregnancies after cFTS is justified and increase diagnostic yield. Funding: Estonian Research Council grants PUT355, PRG471, and PUTJD827. K. Ridnõi: None. K. Muru: None. M. Keernik: None. S. Pajusalu: None. E. Ustav: None. P. Tammur: None. T. Mölter-Väär: None. T. Kahre: None. U. Šamarina: None. K. Asser: None. F. Szirko: None. T. Reimand: None. K. Õunap: None. P01.023.B Higher incidence of congenital anomalies in children born after assisted reproduction in the Czech Republic: Population based study

A. Sipek Jr 1,2,3 , V. Gregor 2,4 , J. Klaschka 5,6 , M. Maly 5,7 , A. Sipek Sr 2,4,8

1 Institute of Biology and Medical Genetics, First Faculty of Medicine, Charles University, Prague, Czech Republic, 2 Department of Medical Genetics, Thomayer Hospital, Prague, Czech Republic, 3 Institute of Medical Genetics, Third Faculty of Medicicne, Charles University, Prague, Czech Republic, 4 Department of Medical Genetics, Pronatal Sanatorium, Prague, Czech Republic, 5 Institute of Computer Science of the Czech Academy of Sciences, Prague, Czech Republic, 6 Institute of Biophysics and Informatics, First Faculty of Medicine, Charles University, Prague, Czech Republic, 7 National Institute of Public Health, Prague, Czech Republic, 8 GENNET, Prague, Czech Republic
Introduction: The number of children conceived by assisted reproduction techniques (ART) is increasing in the Czech Republic. The main goal of this study was to evaluate the incidence of congenital anomalies in ART conceived children. Methods: The retrospective epidemiological analysis was performed using data from the National Registry of Congenital Anomalies and National Registry of Newborns, run by the Institute of Health Information and Statistics of the Czech Republic. All diagnoses of congenital anomalies (Q00-Q99) were included. We compared the incidences of congenital anomalies in naturally conceived children and ART conceived children born in the Czech Republic during 5 years period (2013-2017). Statistical analysis was performed by Fisher’s exact test. Results: During the selected period there were 547 675 children born in the Czech Republic (531 064 were naturally conceived children and 16 611 were ART conceived children). The incidence of congenital anomalies was 3.90% in naturally conceived children group and 4.35% in ART conceived children. This difference is statistically significant (p = 0.003). Analyzing different subgroups of congenital anomalies we have found a significantly increased incidence of anomalies in two subgroups: Congenital malformations of the circulatory system (Q20-Q28) and Congenital malformations of the respiratory system (Q30-Q34). Discussion: The increased risk of congenital anomalies in ART conceived children was highlighted in many studies however the results are often diverse. In our population-based study, we confirmed a significantly higher general incidence of congenital anomalies in ART conceived children. The study is supported by the RVO project: “Thomayerova nemocnice - TN, 00064190” . A. Sipek Jr: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Modest; Research grant provided by Ministry of Health of the Czech Republic, RVO project: “Thomayerova nemocnice TN, 00064190”. V. Gregor: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Modest; Research grant provided by Ministry of Health of the Czech Republic, RVO project: “Thomayerova nemocnice TN, 00064190”. J. Klaschka: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Modest; Research grant provided by Ministry of Health of the Czech Republic, RVO project: “Thomayerova nemocnice TN, 00064190”. M. Maly: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Modest; Research grant provided by Ministry of Health of the Czech Republic, RVO project: “Thomayerova nemocnice TN, 00064190”. A. Sipek Sr: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Modest; Research grant provided by Ministry of Health of the Czech Republic, RVO project: “Thomayerova nemocnice TN, 00064190”. P01.024.C A low-cost microsatellite marker method for quick andA low-cost microsatellite marker method for quick and reliable prenatal 22q11.2DS screening and detection in fetuses with suspected congenital heart defects

L. Torres-Juan 1 , M. Coll-Ferrer 1 , M. Prado-Farnos 1 , R. Martorell-Riera 1 , E. Amengual-Cloquell 1 , A. Perez-Granero 1 , A. Tubau 2 , M. Juan 2 , J. Rosell 1 , I. Martinez-Lopez 1 , D. Heine-Suñer 1

1 Institute of Health Research of the Balearic Islands (IDISBA) and Hospital Son Espases, Palma, Spain, 2 Institute of Health Research of the Balearic Islands (IDISBA) and Hospital Son Llatzer, Palma, Spain
Congenital heart defects (CHD) are the most common form of birth defects and affect 1% of newborns and an even higher percentage of unborn fetuses. CHDs are a frequent finding during routine ultrasound prenatal screening. Once detected, it is important to determine their etiology. The 22q11.2 deletion syndrome (22q11.2 DS) is the most frequent known genetic cause of CHDs (1.9% prevalence). We present a multiplex marker PCR kit to detect 22q11.2 deletions and duplications which we prospectively used in 1630 individuals and 450 fetal cases with a congenital heart defect referred by an obstetrician. Important features: 1) Low cost (there are 2 mixes of 11 pairs of primers and 2 conventional PCR reactions). 2) Fast, results can be given within hours of receiving the sample (DNA extraction -> PCR 1.5 hours -> automatic sequencer). 3) Safely rules out sample contamination by maternal DNA. 4) Uses small amounts of DNA (10 ng) and can be performed starting from amniotic fluid or non-cultured chorion biopsy or any other tissue. 5) In prenatal samples, it would be complementary to the QF-PCR (negative samples for trisomy 13, 18 or 21). 6) The parental origin of the deletion/duplication is determined when the fetus is analyzed with the parents. 7) Detects atypical and typical deletions, extending the analysis far beyond that given by the FISH probe that has been historically used (TUPLE1). 8) We have developed a panel of 31 additional markers, not included in the multiplex, to be used in case of lack of informativity. L. Torres-Juan: None. M. Coll-Ferrer: None. M. Prado-Farnos: None. R. Martorell-Riera: None. E. Amengual-Cloquell: None. A. Perez-Granero: None. A. Tubau: None. M. Juan: None. J. Rosell: None. I. Martinez-Lopez: None. D. Heine-Suñer: None. P01.026.B The effects of the CFTR genotype on the reproductive system in adult male patients with cystic fibrosis

S. Repina 1 , S. Krasovskiy 1,2 , M. Shtaut 1 , G. Shmarina 1 , T. Adyan 1 , V. Chernykh 1

1 Research Centre for Medical Genetics, Moscow, Russian Federation, 2 Pulmonology Research Institute, Moscow, Russian Federation
Background: Commonly male patients with Cystic Fibrosis (CF) is infertile due to obstructive azoospermia (OA), at that an influence of CFTR genotype on reproductive system is poor understood. Materials and methods: We examined 93 Russian male CF patients (17-45 y.o., mean - 24.9 ± 5.7 y.o.). Physical examination, scrotal ultrasonography, hormonal, biochemical, and semen analysis were done. Standard and biochemical semen examination were done according to the WHO’s (2010) recommendations. The CFTR gene variants were analyzed using by AFPL, MLPA and DNA sequencing (Sanger’s method and MPS). Results: CFTR mutations were detected in 100% CF alleles. Folowing genotypes were found: F508del/F508del (20.4%); F508del/3849+10kbC>T (15.1%); F508del/2789+5G>A (4.3%); F508del/CFTRdele2.3 (4.3%); F508del/E92K (3.2%); F508del/L138ins (3.2%); F508del/3272-16T>A (2.2%); F508del/W1282X (2.2%); others (45.3%). The spectrum of CFTR gene pathogenic variants and genotypes showed high frequency of “mild” pathogenic variants (25.8%) and genotypes (50.5%), as well as a relatively high frequency of 3849+10kbС>T variant (12.4%), which was identified in 8.2% patients with OA and 94.4% with no OA ( p < 0.00001). Statistically significant difference was found between patients with F508del/3849+10kbC>T and F508del/F508del genotypes for a prevalence of OA/seminal ducts obstruction (14.3% vs 100%, p < 0.05) and testicular hypoplasia (26.9% vs 66.7%, p = 0.03). Conclusions: CF dramatically affects reproductive organs, male fertility and semen parameters in CF patients, at that 3849+10kbС>T mutation in the CFTR genotype is major genetic factor of male fertility. S. Repina: None. S. Krasovskiy: None. M. Shtaut: None. G. Shmarina: None. T. Adyan: None. V. Chernykh: None. P01.029.B First and second trimester biochemical screening: additional value in the era of NIPS

R. Michaelson-Cohen 1,2,3 , A. Lahad 3,4

1 Medical Genetics Institute, Shaare Zedek Medical Center, Jerusalem, Israel, 2 Department of Obstetrics and Gynecology, Shaare Zedek Medcial Center, Jerusalem, Israel, 3 Hebrew University, Jerusalem, Israel, 4 Clalit Health Services, Jerusalem, Israel
Background: Since implementation of NIPS, first and second trimester biochemical tests may no longer be necessary for Down Syndrome screening. However, studies suggest their ability to predict pregnancy complications. We sought to evaluate whether screening tests have value for this purpose. Methods: Our cohort included all women who had first and/or second trimester screening from 2015-2018 and subsequently delivered in our center. Data was collected on demographics, medical history, biochemical screening and maternal-fetal pregnancy outcomes. Logistic regression was performed to examine correlations between biochemical markers and pregnancy complications using SPSS version 25.0 (SPSS, Inc). Results: Over 3.5 years, 5400 women had screening tests and later delivered in our center. Decreased PAPP-A was associated with preeclampsia, gestational diabetes, IUGR and preterm delivery (PTD), OR=1.792, 1.364, 1.403 and 1.277, respectively, p < 0.005. Elevated AFP was associated with PTD, maternal blood administration, IUGR and low 5-minute Apgar-score, OR=2.044, 1.95, 1.452 and 1.22, respectively, p < 0.033. Decreased HCG in second trimester was correlated with IUGR, OR=1.346, p < 0.05. Elevated HCG in second trimester was correlated with PTD and newborn NICU admission, OR=1.192, 1.285, respectively, p < 0.015. In both first and second trimesters, elevated HCG was associated with preeclampsia, OR= 1.169, 1.309, respectively, p < 0.05. Low estriol was correlated with IUGR, OR=3.597, p < 0.001. After controlling for confounders (age, ethnicity, IVF, parity, etc.) most associations remained significant. Discussion: Despite superiority of NIPS for Down Syndrome screening, biochemical screening is still valuable for prediction of pregnancy complications. Identifying women with high-risk pregnancies has clinical implications for prenatal care and risk-reducing management strategies. R. Michaelson-Cohen: None. A. Lahad: None. P01.034.A Use of prenatal exome sequencing in fetuses with ultrasound anomalies

M. Segura-Puimedon , B. Campos, J. Luna, C. Sintas, M. de Castro-Miró, H. Díez, S. B. Estruch, R. Garcia, L. Quintana, J. Rodríguez, L. Armengol

Quantitative Genomic Medicine Laboratories, qGenomics, Barcelona, Spain, Esplugues de Llobregat, Spain
Introduction : Whole exome sequencing (WES) is an established diagnostic tool in postnatal settings for individuals with a suspected genetic condition. Recently, it is increasingly used as a diagnostic tool in prenatal settings as well. We present here our experience using this technology in fetuses with ultrasound anomalies. Material and Methods : WES was performed in 166 fetal samples with ultrasound anomalies. 104 samples were from evolutive pregnancies and 62 were from legal interruptions or stillbirths. In 61 samples, previous prenatal CGH-array was performed with negative result. Segregation studies were performed in cases with a candidate variant when possible. Results: Common reasons for referral were skeletal anomalies, polymalformated fetuses, cerebral anomalies or specific syndrome suspicion. Pathogenic or likely pathogenic variants were identified in n = 42 (25%) of samples. In n = 40 (24%) cases, variants of unknown significance were identified, and in 13 of them inheritance was consistent with expected pattern, including a probable mosaicism case. In half of the cases (50,6%) no candidate variant was identified. Diagnostic yield was higher in fetuses with skeletal anomalies, where pathogenic or likely pathogenic variants were identified in ( n = 10) 31% of cases, and in fetuses with increased nuchal translucency or hidrops (Noonan syndrome suspicion), where a pathogenic variant was found in 24% ( n = 6) of the samples. No pathogenic variants were found in fetuses with cardiovascular anomalies ( n = 9). Conclusions: Exome sequencing is a valuable diagnostic tool in fetuses with ultrasound anomalies, especially when skeletal anomalies are present or when Noonan syndrome is suspected. M. Segura-Puimedon: A. Employment (full or part-time); Significant; Quantitative Genomic Medicine Laboratories, qGenomics, Barcelona, Spain. B. Campos: A. Employment (full or part-time); Significant; Quantitative Genomic Medicine Laboratories, qGenomics, Barcelona, Spain. J. Luna: A. Employment (full or part-time); Significant; Quantitative Genomic Medicine Laboratories, qGenomics, Barcelona, Spain. C. Sintas: A. Employment (full or part-time); Significant; Quantitative Genomic Medicine Laboratories, qGenomics, Barcelona, Spain. M. de Castro-Miró: A. Employment (full or part-time); Significant; Quantitative Genomic Medicine Laboratories, qGenomics, Barcelona, Spain. H. Díez: A. Employment (full or part-time); Significant; Quantitative Genomic Medicine Laboratories, qGenomics, Barcelona, Spain. S.B. Estruch: A. Employment (full or part-time); Significant; Quantitative Genomic Medicine Laboratories, qGenomics, Barcelona, Spain. R. Garcia: A. Employment (full or part-time); Significant; Quantitative Genomic Medicine Laboratories, qGenomics, Barcelona, Spain. L. Quintana: A. Employment (full or part-time); Significant; Quantitative Genomic Medicine Laboratories, qGenomics, Barcelona, Spain. J. Rodríguez: A. Employment (full or part-time); Significant; Quantitative Genomic Medicine Laboratories, qGenomics, Barcelona, Spain. L. Armengol: A. Employment (full or part-time); Significant; Quantitative Genomic Medicine Laboratories, qGenomics, Barcelona, Spain. P01.035.B AnDDI-Prenatome: The French exome sequencing study in prenatal diagnosis: causal diagnosis identified in nearly half of fetuses

F. Tran Mau Them 1,2 , A. Bruel 1,2 , A. Vitobello 1,2 , S. Nambot 3 , S. Moutton 3 , A. Sorlin 1,2,3 , J. Delanne 3 , A. Ziegler 4 , E. Colin 4 , A. Guichet 4 , M. Legendre 5 , C. Colson 6,7 , C. Abel 8 , A. Putoux 9 , J. Amiel 10,11 , A. Lavillaureix 12 , C. Quelin 12 , A. Guerrot 13 , A. Brehin 13 , L. Faivre 3,2 , C. Philippe 1,2 , V. Couturier 1,2 , Y. Duffourd 2 , C. Thauvin-Robinet 1,2,14

1 Unité Fonctionnelle d’Innovation diagnostique des maladies rares, FHU-TRANSLAD, Dijon, France, 2 Inserm – UB UMR 1231 GAD « Génétique des Anomalies du Développement », FHU-TRANSLAD, Dijon, France, 3 Centre de Référence Maladies rares « Anomalies du Développement et syndromes malformatifs », Centre de Génétique, FHU-TRANSLAD, Dijon, France, 4 Biochemistry and Genetics Department, University Hospital of Angers, Angers, France, 5 Service de Génétique Médicale, CHU de Bordeaux – Hopital Pellegrin, Bordeaux, France, 6 CHU Lille, Clinique de Génétique Guy Fontaine, Lille, France, 7 EA7364, RADEME, Université de Lille, Lille, France, 8 Service de génétique et centre de diagnostic anténatal, Lyon, France, 9 Service de Génétique, Centre de Référence Anomalies du Développement et Syndromes Malformatifs, Lyon, France, 10 Service de Génétique Médicale et Clinique, Hôpital Necker-Enfants Malades, Paris, France, 11 Equipe "Embryologie et Génétiques des malformations congénitales", Institut Imagine - INSERM U1163, Paris, France, 12 Centre de Référence Maladies Rares CLAD-Ouest, CHU Hôpital Sud, Rennes, France, 13 Normandie Univ, UNIROUEN, Inserm U1245 and Rouen University Hospital, Department of Genetics and Reference Center for Developmental Disorders, F 76000, Normandy Center for Genomic and Personalized Medicine, Rouen, France, 14 Centre de Référence « Déficiences Intellectuelles de causes rares », Centre de Génétique, FHU-TRANSLAD, Dijon, France
Introduction : Exome sequencing (ES) is the most powerful and current test for patients with developmental abnormalities (DA). It has considerably been deployed in postnatal indications with a diagnostic yield ranging from 30 to 40%. Several prenatal studies report a diagnostic yield ranging from 15% to 35%, depending on the exome strategy and the inclusion criteria. The French national network for rare diseases with AD deployed a pilot project of feasibility for rapid prenatal exome sequencing. Materials and methods: We aimed to include 61 fetuses and both parents. The main inclusion criteria were: 2 major malformations or, 1 major and 1 minor malformation or, 1 malformation and a strong genetic suspicion. The feasibility aim was to emit a diagnostic report in 5-6 weeks. Array-CGH and ES-trio were concomitantly performed. ES-trio was captured with the TWIST-v2 kit and sequenced on a NovaSeq6000 device with a 70X average depth. Results : We included 23 patients. The turnaround time from receipt of samples to the diagnostic report was on average of 27 days for the negative patients and of 40 days for the positive patients. The diagnostic yield was of 44% with 7 SNV and 3 CNV, including a tetrasomy 12p. Array-CGH allowed a diagnosis in 13% and exome in 44%. Results were concordant for CNV detection between array-CGH and ES-trio. Discussion : Prenatal trio-ES appears feasible in a useful time and strongly helpful for decision in 65 % and genetic counseling was impactful in at least 44%. F. Tran Mau Them: None. A. Bruel: None. A. Vitobello: None. S. Nambot: None. S. Moutton: None. A. Sorlin: None. J. Delanne: None. A. Ziegler: None. E. Colin: None. A. Guichet: None. M. Legendre: None. C. Colson: None. C. Abel: None. A. Putoux: None. J. Amiel: None. A. Lavillaureix: None. C. Quelin: None. A. Guerrot: None. A. Brehin: None. L. Faivre: None. C. Philippe: None. V. Couturier: None. Y. Duffourd: None. C. Thauvin-Robinet: None. P01.036.C Preconception carrier screening in consanguineous couples by whole exome sequencing

A. Abulí , I. Valenzuela, M. Codina, E. Rovira, D. Palau, S. Ourani, A. Cueto-González, I. Paramonov, E. García-Arumí, E. Tizzano, I. Cusco

Hospital Vall d’Hebron, Barcelona, Spain
Consanguineous couples have an increased risk of severe abnormalities and mortality in offspring due to autosomal recessive disorders. Exome-sequencing provides the possibility of wide preconception carrier screening (PCS) in consanguineous couples who may be at risk of rare genetic disorders. The efficacy of this strategy has been poorly explored. We retrospectively analyzed exome sequencing data from 27 affected children and 3 fetuses (after termination of pregnancy) of consanguineous couples. We explored diagnostic yield and carrier status for recessive disorders. Overall diagnostic yield was 67% (20/30 patients). We detected homozygous causative variants in 19 cases and one de novo heterozygous variant. Out of 20 causal variants, 18 were classified as pathogenic or likely pathogenic and two as unknown significance, which were reclassified as likely pathogenic because of highly specific phenotype. Based on our results and considering diagnostic yield and variant classification, causative pathogenic recessive mutations would have been detected in at least 60% of consanguineous couples by preconception exome-sequencing. Most of these conditions (16/20) would not have been detected in PCS panels for most prevalent diseases considering a carrier frequency of 1/250. Most of the patients were carriers of one additional heterozygous recessive mutation (70%, 21/30) and a smaller number were carriers of two (17%, 5/30) or more (13%, 4/30). The average carrier burden of autosomal recessive conditions was of 1.7 pathogenic mutations per patient. PCS by exome-sequencing increases the detection rate of rare recessive conditions in consanguineous couples and could potentially increase their reproductive autonomy and facilitate informed decision making. A. Abulí: None. I. Valenzuela: None. M. Codina: None. E. Rovira: None. D. Palau: None. S. Ourani: None. A. Cueto-González: None. I. Paramonov: None. E. García-Arumí: None. E. Tizzano: None. I. Cusco: None. P01.037.A Does maternal blood exosomal DNA effective for the fetal RHD and SRY genotyping?

B. Yaşa 1 , O. Şahin 2 , S. Sözer 1

1 Aziz Sancar Institute of Experimental Medicine, Istanbul, Turkey, 2 Clinic of Obstetrics and Gynecology, Okmeydanı Training and Research Hospital, Istanbul, Turkey
Introduction: Recent advances have led to the development of important techniques in extracellular fetal DNA derived from maternal blood for prenatal diagnosis. Exosomes’ DNA content is not affected structurally by nuclease activity and have a potential clinical application. Determination of fetal Rhesus D(RhD) and sex is important for prenatal diagnosis in some diseases. Material And Method: Fetal RHD and SRY genotyping was performed using exosomal DNA(exoDNA) from peripheral blood of 81 RhD negative pregnant women with exosome isolation kit. Specific primers were applied for the SRY and RHD genes amplifications by Real-Time polymerase chain reaction. The average age of the cases in the study was 28.5 ± 5.5(year ± SD). The mean gestational week of the cases was 25.81 ± 9.02. The exoDNA concentrations obtained were between 1.8-21.9 ng/μL with an average of 11.65 ± 8.08 ng/μL. Results: The results were confirmed with the hospital’s New Born Follow Up System. After analysis of the 81 cases, no amplification was observed in 16 samples, including the GAPDH gene. 100% accuracy was achieved in the RHD and SRY detection from the remaining 65 samples. Conclusions: It was determined that fetal RHD and SRY genotyping could be performed with exoDNA from maternal plasma. The isolation technique of exoDNA could be enhanced and improved for high concentration with more sensitive detection covering early pregnancy age. This technique might provide a new perspective to the prenatal diagnosis as well as to the treatment options all which require more detailed studies. The present work was supported by the Research Fund of Istanbul University. Project No. 29083 B. Yaşa: None. O. Şahin: None. S. Sözer: None. P01.038.B Population-based preconception expanded carrier screening for severe recessive disorders: Does a GP-provided test offer with couple results meet criteria for responsible implementation?

J. Schuurmans 1,2 , M. Plantinga 1 , E. Birnie 1 , K. M. Abbott 1 , A. D. Diemers 1 , D. M. Van der Kolk 1 , L. M. Van den Heuvel 1 , M. Y. Berger 1 , M. Verkerk 1 , A. Fenwick 2 , A. V. Ranchor 1 , A. Lucassen 2 , I. M. Van Langen 1

1 University Medical Center Groningen, Groningen, Netherlands, 2 University of Southampton, Southampton, United Kingdom
Introduction: Expanded carrier screening (ECS) enables the identification of couples who are at increased risk of having children with recessive conditions. Based on preliminary research, we conducted a pilot study of GP-provided population-based preconception ECS (2016-2018) to evaluate whether this approach would meet criteria for responsible implementation as specified by Henneman et al., EJHG, 2016. The free test-offer consisted of 50 severe early-onset childhood conditions. Only couple-results were reported. We discuss key insights to guide large-scale implementation. Methods: We used a mixed-methods approach. Trained GPs from nine practices invited their female patients ( n = 4295, aged 18-40) and partners. We addressed uptake, feasibility (e.g. pre-test counselling possible within 20 minutes), informed-choice, psychological outcomes (e.g. anxiety, decisional conflict) and reproductive intentions using a longitudinal survey (couples) and semi-structured interviews (GPs). Results: 190 couples were included. 130 (approximately 15% of the eligible population) accepted the test-offer within one month and attended pre-test counselling. 117 (90%) proceeded with testing. More than 90% of test-participants made an informed-choice. GPs felt confident to conduct counselling, although having to make a GP-appointment with both partners was a barrier. Most participants reported low anxiety and decisional conflict; this was relatively higher in test-offer decliners than acceptors. Conclusions: Population-based preconception ECS for a specific set of severe conditions provided by motivated and trained GPs is feasible. We will discuss how responsible implementation can best be facilitated. The possibility of prenatal ECS, ECS to high-risk couples or which HCPs should offer this (e.g. midwives, fertility clinics) should now also be investigated. J. Schuurmans: None. M. Plantinga: None. E. Birnie: None. K.M. Abbott: None. A.D. Diemers: None. D.M. Van der Kolk: None. L.M. Van den Heuvel: None. M.Y. Berger: None. M. Verkerk: None. A. Fenwick: None. A.V. Ranchor: None. A. Lucassen: None. I.M. Van Langen: None. P01.041.B Donor oocyte pregnancies and fetal fraction: managing patient expectations and providing accurate information

M. Pastrick , M. K. Maisenbacher, G. Goldring, W. DiNonno, A. Ryan

Natera, Inc, San Carlos, CA, United States
Objective: Retrospective analysis to determine if differences in fetal fraction (FF) are observed in donor oocyte pregnancies compared to the general population. Methods: Noninvasive prenatal testing samples from singleton pregnancies were analyzed at a single reference lab. NIPT was performed using a SNP-based method with FF measured as previously described. 1 FF from 1611 donor oocytes was analyzed and compared to a large set of reference cases matched for maternal weight (MW) and gestational age (GA). A z-score was calculated for each donor oocyte compared to its reference data. If no impact to FF from the use of donor or in vitro fertilization (IVF), the average z-score is expected to be zero. Statistical analysis was performed using a z-test to establish if this was the case. Results: The average z-score for the donor oocyte cases was -0.4. A z-test determined this deviation from normal to be significant (p < 0.00001), indicating lower FF in donor oocyte cases vs. corresponding reference data. Conclusions: Previous studies have reported lower FF in patients undergoing IVF and in donor oocyte populations. 2,3 Lower FF has also been associated with increased MW, early GA, certain maternal health conditions, and abnormal fetal results (T18/T13/triploidy). 2,3,4 Our results reveal statistically significant lower FF in donor oocyte pregnancies compared to matched reference data. References 1. Zimmerman et al., Prenatal Diagnosis 2012; 32: 1-9 2. Revello R. et al., Ultrasound Obstet Gynecol . 2016 Jun;47(6):698-704. 3. Yanaihara A. et al., J Assist Reprod Genet . 2018 Mar;35(3):431-434. 4. Ebbing C. et al., PLoS One . 2013 Jul 30;8(7):e70380. M. Pastrick: A. Employment (full or part-time); Significant; Natera, Inc. M.K. Maisenbacher: A. Employment (full or part-time); Significant; Natera, Inc. G. Goldring: A. Employment (full or part-time); Significant; Natera, Inc. W. DiNonno: A. Employment (full or part-time); Significant; Natera, Inc. A. Ryan: A. Employment (full or part-time); Significant; Natera, Inc. P01.042.C Repeat Associated Non-AUG (RAN)-Translation in fragile X associated Premature Ovarian Insufficiency (FXPOI): FMRpolyG as predictive tool?

X. Nguyen 1 , B. Messmer 1 , K. Hinderhofer 2 , T. Strowitzki 3 , J. Rehnitz 1,3

1 Division of Reproduction Genetics, Department of Gynecological Endocrinology and Fertility Disorders, University Women’s Hospital, Heidelberg, Germany, 2 Institute of Human Genetics, University Heidelberg, Laboratory of Molecular Genetics, Heidelberg, Germany, 3 Department of Gynecological Endocrinology and Fertility Disorders, University Women’s Hospital, Heidelberg, Germany
Fragile-X-associated-primary-ovarian-insufficiency (FXPOI) is characterized by oligo/amenorrhea and hypergonadotropic hypogonadism by reason of the expansion of CGG-repeats in the 5′UTR of FMR1 , called permutation (PM) (n: 55-200). RAN-translation depending on variable CGG-repeat length is also hypothesized to cause FXPOI due to the production of a polyglycine-containing FMR1-protein, FMRpolyG. FMRpolyG expression in granulosa cells (GCs) of FXPOI-model-mice leads to perturbed ovarian function. However, the expression of FMRpolyG in leukocytes and GCs of women with different ovarian response and variable CGG repeat length has not been investigated yet. We performed Western-blot (WB) to analyze the expression of FMRP and FMRpolyG in patient´s leukocytes and GCs in parallel with FMR1 -gene-expressionanalysis. Firstly, we detected FMRP, both in leukocytes and GCs of women with variable ovarian reserve and different CGG-repeat-length. FMR1 /FMRP was detected in a larger amount in GCs than in leukocytes, since it is a major regulator of folliculogenesis. We therefore started a pilot project investigating if variable expression-levels of FMRP and FMRpolyG detected by WB can be related to women with different ovarian reserve parameters and variable CGG repeats including “normal” and premutated ranges. Preliminary data already showed, in a PM patient undergoing oocytes cryopreservation because of her risk of developing FXPOI, a decreased level of FMRP, even though the FMR1 transcription level was within the normal range, fitting to the hypothesis of kind of a perturbed protein production in this patient. We plan to further evaluate if results made in GCs can be translated to leukocytes. This would offer prospectively a predictive tool or marker of progression. X. Nguyen: None. B. Messmer: None. K. Hinderhofer: None. T. Strowitzki: None. J. Rehnitz: None. P01.045.C Investigation of single nucleotide variants (SNVs) in a fertile population in association with oocyte and embryo quality

G. Kakourou 1 , T. Mamas 1 , C. Vrettou 1 , E. Tsorva 2 , S. Zacharia 2 , C. Oraiopoulou 3 , E. Chatzi 4 , G. Kourlaba 5 , D. Christopikou 2 , M. Mastrominas 2 , M. Moisidou 3 , A. Chatziparasidou 3 , I. Georgiou 4 , J. Traeger-Synodinos 1

1 Department of Medical Genetics, National & Kapodistrian University of Athens, St. Sophia’s Children’s Hospital, Athens, Greece, 2 Embryogenesis, Assisted Reproduction Unit, Athens, Greece, 3 Embryolab SA, IVF Unit, Thessaloniki, Greece, 4 Genetics and IVF Unit, Department of Obstetrics and Gynecology, Faculty of Medicine, University of Ioannina, Ioannina, Greece, 5 Center for Clinical Epidemiology and Outcomes Research (CLEO), Athens, Greece
Introduction: Human and animal studies have provided information on a number of genes potentially impacting gamete quality and preimplantation development (with a role on meiosis, mitosis, DNA repair etc.). SNVs in these genes have been investigated in mostly infertile couples undergoing IVF, in search of potential biomarkers of IVF success. Recently, maternal SNVs in infertile women (rs1801133/rs1801131 in MTHFR , rs2305957 on chromosome 4 linked to INTU-SLC25A31-HSPA4L-PLK4-MFSD8-LARP1B and PGRMC2 ) were associated with embryo quality or chromosomal status. We investigated the potential association of SNVs in 26 selected genes with a possible role in preimplantation development, with oocyte/embryo quality in fertile women undergoing Preimplantation Genetic Testing for Monogenic Disorder (PGT-M). Materials/Methods: Maternal DNA was genotyped using exonic NGS (QiaseqTM-Targeted Custom Panel, Miseq) for 18 genes ( AIRE-AMH-AURKA-AURKB-AURKC-FSHR-HSPA4L-HUWE1-INTU-KHDC3L-LARP1B-MFSD8-MTHFR-PGRMC2-PLK4-SENP7-SLC25A31-WBP1 ) and 9 selected SNVs in a further 8 genes: rs175080( MLH3 ), rs1799963( F2 ), rs6025( F5 ), rs5918( ITGB3 ), rs5985( F13A1 ), rs1805087( MTR ), rs1801394( MTRR ), rs28756992( MLH3 ), rs2305957( HSPA4L ). The study involved 85 women and 107 PGT-M cycles performed between 2013-2019. Maternal genotyping was investigated in association with number of oocytes collected/fertilized and blastocyst formation. Results: A 20x coverage was achieved in all regions investigated, identifying 121 variants. Significant associations (p < 0.05) were revealed for 16 SNVs in: AIRE-AMH-AURKB-HSPA4L-KHDC3L-MTHFR-PLK4-SENP7-MLH3 . No significant associations were revealed for rs1801131 and rs2305957. Conclusions: Our understanding of genes that may impact the preimplantation stage and the search for genomic biomarkers predicting IVF-success may benefit by investigating a fertile population, minimizing many confounding variables. This study was supported by the State Scholarships Foundation (IKY), co-funded by national grants and the European union (ESPA 2014-2020). G. Kakourou: None. T. Mamas: None. C. Vrettou: None. E. Tsorva: None. S. Zacharia: None. C. Oraiopoulou: None. E. Chatzi: None. G. Kourlaba: None. D. Christopikou: None. M. Mastrominas: None. M. Moisidou: None. A. Chatziparasidou: None. I. Georgiou: None. J. Traeger-Synodinos: None. P01.047.B Further indication of a role for de novo mutations in cases of severe idiopathic male infertility

H. E. Smith 1 , M. S. Oud 2 , P. F. de Vries 2 , B. Alobaidi 1 , A. Virdi 1 , B. J. Houston 3 , R. M. Smits 4 , L. Batty 1 , M. K. O’Bryan 3 , J. Coxhead 5 , S. Cockell 6 , J. Greenwood 7 , G. van der Heijden 2,4 , K. McEleny 7 , L. Ramos 4 , L. E. L. M. Vissers 2 , M. J. Xavier 1 , J. A. Veltman 1,2

1 Biosciences Institute, Newcastle University, Newcastle Upon Tyne, United Kingdom, 2 Department of Human Genetics, Donders Institute for Brain, Cognition and Behaviour, Radboudumc, Nijmegen, Netherlands, 3 School of Biological Sciences, Monash University, Melbourne, Australia, 4 Department of Obstetrics and Gynaecology, Radboudumc, Nijmegen, Netherlands, 5 Genomics Core Facility, Newcastle University, Newcastle Upon Tyne, United Kingdom, 6 Bioinformatics Support Unit, Newcastle University, Newcastle Upon Tyne, United Kingdom, 7 Newcastle Fertility Centre, Newcastle University, Newcastle Upon Tyne, United Kingdom
Infertility affects 1 in 6 couples worldwide and in approximately 50% of these cases the infertility can be attributed to a male factor. Whilst causes such as Klinefelter’s and Y chromosome microdeletions have been well distinguished, the genetic causes behind severe spermatogenic failure are largely unknown, with around 40% of all male infertility cases remaining idiopathic. De novo mutations (DNMs) arise spontaneously in the germline or post-zygotically, and are known to be associated with early onset disorders with reduced reproductive fitness. These mutations, however, are vastly understudied in the field of male infertility due to the difficulty in obtaining parental samples. Here we report on the continuation and expansion of exome sequencing in our cohort of infertile patients in order to identify novel genes involved in male infertility. The role of DNMs was investigated in a total of 194 patients suffering from non-obstructive azoospermia or severe oligozoospermia and their fertile parents by adding 95 additional trios to the previously reported 99 cases. In these additional 95 trios, we have identified and validated 75 protein-altering DNMs, of which 19 are likely to affect normal gene function. Genes of particular interest are PPP1R7 , RBM5 and MSH5 , all of which are involved in spermatogenesis and linked to infertility in mice. At this moment, none of these genes have been recognised as human male infertility genes. Our data further indicates that DNMs may play an important role in severe male infertility and emphasises the need for further replication studies in larger cohorts. H.E. Smith: None. M.S. Oud: None. P.F. de Vries: None. B. Alobaidi: None. A. Virdi: None. B.J. Houston: None. R.M. Smits: None. L. Batty: None. M.K. O ’ Bryan: None. J. Coxhead: None. S. Cockell: None. J. Greenwood: None. G. van der Heijden: None. K. McEleny: None. L. Ramos: None. L.E.L.M. Vissers: None. M.J. Xavier: None. J.A. Veltman: None. P01.049.A A case report of a rare nonsense ZP1 variant in a patient with oocyte maturation defect

E. G. Berkay , B. Karaman, G. Toksoy, B. Ozsait Selcuk, Z. O. Uyguner, S. Basaran

Istanbul Medical Faculty, Istanbul University, Istanbul, Turkey
Introduction: Oocyte maturation defect (OOMD) is a rare condition causing female infertility that can be diagnosed during assisted reproduction techniques (ART). OOMD related genes are ZP1 , ZP2 , ZP3 , PANX1 , PATL2 , TUBB8 , WEE2 (OMIM, 2020). We report a case of a 31-year-old woman who had four ART failures diagnosed as empty follicle syndrome and OOMD. She has short stature (-3 SD), bilateral limited extension-flexion on elbows. Materials and Methods: Chromosome analysis and fluorescence in-situ hybridization (FISH) using X chromosome centromeric and SHOX-probe on interphase nuclei of lymphocytes and mucosal cells was investigated. Whole-exome sequencing (WES) performed via the Illumina platform. Confirmation and familial segregation analysis were performed by Sanger sequencing. Results: Karyotyping and FISH resulted in normal, possible mosaicism was excluded. WES analysis revealed a known, rare, pathogenic homozygous variant in exon 3 (c.628C>T; p.Q210*) of ZP1 gene, and her parents being first degree cousins were carriers for this variant. Conclusions: ZP1 with autosomal recessive inheritance is related to OOMD-1 (MIM_615774). Zona pellucida (ZP) is a glycoprotein structure surrounding oocytes and is essential for oocyte development. ZP contains four types of receptor proteins (ZP1-4). Our variant in ZP1 is nonsense, premature stop codon causes to truncate ZP1 receptor proteins. This is the first homozygous occurrence of this variant associated with OOMD. WES findings were also analyzed for known genes related to short stature and no pathogenic variant has been observed. WES is a valuable method to identify the genetic origin in complex, multigenic conditions like in infertility. Istanbul University Project-Number:TSA-2018-32135 E.G. Berkay: None. B. Karaman: None. G. Toksoy: None. B. Ozsait Selcuk: None. Z.O. Uyguner: None. S. Basaran: None. P01.051.C Prenatal diagnosis and molecular characterization of a jumping translocation leading to 18p monosomy

J. B. Gaillard 1 , T. Guignard 1 , J. Puechberty 2 , A. Schneider 1 , V. Gatinois 1 , M. Duport-Percier 3 , J. M. Faure 3 , F. Fuchs 3 , F. Pellestor 1 , P. Blanchet 4 , C. Wells 4

1 Génétique Chromosomique, CHU, Montpellier, France, 2 Maladies Génétiques de l’Enfant et de l’Adulte, CHU, Montpellier, France, 3 Médecine Maternelle et Foetale, CHU, Montpellier, France, 4 Génétique Prénatale et Foetopathologie, CHU, Montpellier, France
Jumping translocations are rare events in cytogenetics, characterised by a donor chromosome and multiple recipient chromosomes. To date, only ten prenatal cases have been described. Patient: This pregnancy was a second spontaneous pregnancy of a non-consanguinous couple. A first trimester ultrasound showed an increased nuchal translucency persisting at 18 gestational weeks (GW) along with retrognatia, cleft palate and femoral shortening. Choriocentesis was performed at 14 GW. Results: Direct examination on cytotrophoblast found a der(18)t(18;18) whereas the cultured karyotype on mesenchyme showed a der(18)t(2;18). Due to tissue discordance, an amiocentesis was performed at 18 GW. Surprisingly, we found, neither of the two previously identified cell lines, but a mosaic der(18)t(12;18) with a ring 18. In addition, array CGH provided breakpoints for chromosomes 2 and 12 in REEP1 gene (2p11.2) and TAOK3 gene (12q24.23) respectively. Whole genome sequencing on minION (Nanopore) was conducted to clarify breakpoint in chromosome 18. Using data obtained by array CGH, a long read with hard clipping showed a breakpoint in repeated sequence with about 90% homology with D18Z1 alpha satellite. Finally, all identified cell lines carried a 18p monosomy which could explain ultrasound abnormalities. The pregnancy was terminated. Following fetal autopsy, FISH analysis was performed on blood, thymic, lung and liver tissue and confirmed 18p monosomy in all tissues. Conclusion: We report here a new case of jumping translocation with precise molecular characterization of breakpoints : D18Z1 as donor region and at least 3 recipient chromosomes : 2p11.2, 12q24.3 and 18q21. J.B. Gaillard: None. T. Guignard: None. J. Puechberty: None. A. Schneider: None. V. Gatinois: None. M. Duport-Percier: None. J.M. Faure: None. F. Fuchs: None. F. Pellestor: None. P. Blanchet: None. C. Wells: None. P01.052.A Molecular biomarkers of ageing in men with Klinefelter syndrome

E. Pohl 1 , S. Muschal 2 , S. Kliesch 3 , M. Zitzmann 3 , J. Rohayem 3 , J. Gromoll 2 , S. Laurentino 2

1 Institute of Human Genetics, University of Münster, Münster, Germany, 2 Centre of Reproductive Medicine and Andrology (CeRA), Institute of Reproductive and Regenerative Biology, University Hospital Münster, 48149 Münster, Germany, Münster, Germany, 3 Centre of Reproductive Medicine and Andrology (CeRA), Department of Clinical and Surgical Andrology, University Hospital Münster, 48149 Münster, Germany, Münster, Germany
Introduction: Males with Klinefelter syndrome (non-mosaic karyotype 47,XXY), the most common sex chromosome aneuploidy, exhibit increased rates of ageing-related morbidities and mortality. Associated with that, a significantly reduced life span has been observed. The aim of this study was to investigate whether Klinefelter patients show molecular signs of premature ageing. Patients and Methods: We assessed age-associated DNA methylation patterns (by pyrosequencing) and relative telomere length (TL; by quantitative polymerase chain reaction) as molecular ageing biomarkers in blood from Klinefelter patients ( n = 178 and 266 for DNA methylation and TL, respectively) aged 18-71 years. Age-matched healthy males ( n = 184 and 196 for DNA methylation and TL, respectively) and females ( n = 50) served as controls. Results: Age-associated DNA methylation patterns did not indicate accelerated ageing/premature ageing in men with Klinefelter syndrome. In young Klinefelter individuals aged 18-24 years, we observed significantly longer telomeres compared to controls (mean = 1.51 vs. 1.09 and 1.26 in female and male controls, respectively). Telomere length in subsequent age groups, however, were not significantly different to controls. Conclusions: We did not find molecular hints for premature ageing in Klinefelter patients based on DNA methylation patterns. However, we observed higher baseline TL at adolescent age but comparable TL with progressive age in other age groups. Whether this is due to an accelerated TL attrition, putatively related to the observed co-morbidities in Klinefelter syndrome, remains to be addressed in a further longitudinal study. Funding: German Research Foundation (DFG No. GR1547/19-1; CRU 326 No. GR1547/25-1), IZKF Münster (Grant CRA03/09) E. Pohl: None. S. Muschal: None. S. Kliesch: None. M. Zitzmann: None. J. Rohayem: None. J. Gromoll: None. S. Laurentino: None. P01.055.A Maternally inherited copy-number variations may explain cases of severe idiopathic male infertility

F. K. Mastrorosa 1 , M. J. Xavier 1 , A. Mikulasova 1 , M. S. Oud 2 , R. M. Smith 3 , G. Astuti 2 , B. Alobaidi 1 , J. Greenwood 4 , S. J. Cockell 5 , C. Gilissen 6 , J. Coxhead 7 , H. Sheth 1 , G. van der Heijden 2,3 , K. McEleny 4 , L. Ramos 3 , J. A. Veltman 1,2

1 Biosciences Institute, Newcastle University, Newcastle upon Tyne, United Kingdom, 2 Department of Human Genetics, Donders Institute for Brain, Cognition and Behaviour, Radboudumc, Nijmegen, Netherlands, 3 Department of Obstetrics and Gynaecology, Radboudumc, Nijmegen, Netherlands, 4 Newcastle Fertility Centre, Newcastle University, Newcastle upon Tyne, United Kingdom, 5 Bioinformatics Support Unit, Newcastle University, Newcastle upon Tyne, United Kingdom, 6 Department of Human Genetics, Radboud Institute for Molecular Life Sciences, Radboudumc, Nijmegen, Netherlands, 7 Genomics Core Facility, Newcastle University, Newcastle upon Tyne, United Kingdom
Severe cases of infertility such as azoospermia and oligozoospermia often have genetic causes, with Klinefelter syndrome and chromosome Y microdeletions being the most common. However, of all infertile men, 40% have unknown aetiology after being tested for all known causes. In this study, we explored for the first time the role of maternally inherited copy-number variations (CNVs) on a large scale, sequencing the exome of 160 patient-parents trios, a methodology primarily used to detect single nucleotide variants (SNVs) but also robust for CNV detection. Among all rare maternally inherited CNVs detected, 3 were considered of potential pathogenicity. One is a large duplication (~1.7 Mb) involving, among others, VCX gene, an X-linked multi-copy gene that leads to apoptosis and cell growth inhibition when duplicated in murine germ cells in vitro. A second case carried a ~80 kb duplication involving two genes, one of which was FAS , a known regulator of testicular germ cell apoptosis. The third CNV, instead, was a deletion of ~200 kb affecting, among others, PSME4 , a gene required for normal fertility in male mice but dispensable in females. The results of this study, although preliminary, provide a first indication that maternally inherited CNVs may explain cases of severe spermatogenic failure. Currently, additional 35 patient-parents trios and 149 patients are being screened to detect variations affecting the same genes and additional rare variations. Genome sequencing data will soon be available for all the trios, which will improve CNV detection and clarify the contribution of these variations to the disorder. F.K. Mastrorosa: None. M.J. Xavier: None. A. Mikulasova: None. M.S. Oud: None. R.M. Smith: None. G. Astuti: None. B. Alobaidi: None. J. Greenwood: None. S.J. Cockell: None. C. Gilissen: None. J. Coxhead: None. H. Sheth: None. G. van der Heijden: None. K. McEleny: None. L. Ramos: None. J.A. Veltman: None. P01.056.B Improved classification of testicular histopathology to promote uniform diagnosis and discovery of genetic causes for male infertility

G. W. van der Heijden 1 , M. J. Wyrwoll 2 , L. Ramos 1 , J. Veltman 3 , F. Tüttelmann 4

1 1. Division of Reproductive Medicine, Department of Obstetrics and Gynecology, Radboud University Medical Center, Nijmegen, Netherlands, 2 Centre of Reproductive Medicine and Andrology, Department of Clinical and Surgical Andrology, University of Münster, Münster, Germany, 3 Institute of Genetic Medicine, Newcastle University, Newcastle, Netherlands, 4 Institute of Human Genetics, University of Münster, Münster, Germany
With the availability of Next Generation Sequencing the genetic causes of male infertility are increasingly being chartered. A genetic diagnosis combined with a clear unambiguous description of accompanying testis histology will hugely benefit interpretation of the function of identified genes. However, a universal ontology for testis histopathology is lacking. The Human Phenotype Ontology (HPO) is a formal ontology, which describes phenotypes encountered in human diseases. The HPO allows phenotype driven differential diagnostics in clinical routine and research. The current hierarchy of terms regarding azoospermia, is not a systematical one. Moreover, the majority of testicular phenotypes are not annotated yet. Building on what has been published previously and our own experience we propose a framework for testis histopathology that consists of five main categories. Four of these are subdivided to obtain information on the timing of a germ cell arrest or the severity of overall loss of spermatogenesis. To establish a lower threshold for complete spermatogenesis we determined the number of tubules containing elongating spermatids in a collection of samples of men with an obstructive azoospermia due to a previous vasectomy (N=117). This analysis suggested 50% to be a reasonable cut-off to distinguish hypospermatogenesis from complete spermatogenesis. The framework is easy to understand and straightforward in its application. It gathers most relevant information to study the impact of an identified mutation on spermatogenesis. We conclude that once this framework is incorporated in the HPO, the standardized vocabulary will facilitate communication and interpretation in both a clinical routine and research. G.W. van der Heijden: None. M.J. Wyrwoll: None. L. Ramos: None. J. Veltman: None. F. Tüttelmann: None. P01.057.C Identification of two homozygous mutations, in the male reproductive tract specific β-defensin 126/128 genes, potentially underlie a severe sperm dysfunction .

S. Kane 1 , D. Lester 1 , S. Cameron 1 , C. Barratt 2 , S. Martins da Silva 2 , S. Brown 1

1 School of Science, Engineering & Technology, Abertay University, Dundee, United Kingdom, 2 Reproductive and Developmental Biology, School of Medicine, Ninewells Hospital and Medical School, University of Dundee, Dundee, United Kingdom
Introduction: Idiopathic infertility affects 15% couples globally and recent evidence suggests genetic abnormalities are a causative factor in a significant number of cases. Hence, we conducted whole exome sequencing of men undergoing assisted conception to identify genetic lesions. The β-defensin 126 (DEFB126) and β-defensin 128 (DEFB128), are genes preferentially expressed in the epididymis, where these proteins may help protect the maturing sperm cells and play a vital role during sperm capacitation. Materials and Methods: DNA extracted from the blood of 26 unrelated, sub-fertile men were sequenced using whole exome sequencing. Pathogenic rare variants were identified using bioinformatics analysis and validated using Sanger sequencing. Results: A novel and rare homozygous pathogenic 1-bp frame-shift insertion causing a premature stop codon was identified in DEFB128 gene from one patient. Six patients showed a previously reported homozygous 2bp deletion, causing a frame-shift mutation in the DEFB126 gene (p.Pro106ArgfsTer?). All patients ascertained with the DEFB126/DEFB128 mutations were unable to naturally conceive and hence were sub-fertile. Conclusions: The DEFB128 mutation (rs11396059), is rare and has not been associated with any disease to date. As the patient with the DEFB128 mutation has no other medical condition we predict that it is the cause of infertility. The DEFB126 mutation (rs140685149), is a common variant, and this homozygous variant in sub-fertile men has previously been implicated in facilitating sperm movement in the female reproductive tract. These results suggest that, β-defensin genes play a significant role in male reproduction. This work was supported by the R-LINCS studentship, Abertay University. S. Kane: None. D. Lester: None. S. Cameron: None. C. Barratt: None. S. Martins da Silva: None. S. Brown: None. P01.058.A The sperm epigenome does not display recurrent epimutations in patients with severely impaired spermatogenesis

E. Leitao 1 , S. Di Persio 2 , S. Laurentino 2 , M. Wöste 3 , M. Dugas 3 , S. Kliesch 4 , N. Neuhaus 2 , B. Horsthemke 1,5

1 Institute of Human Genetics, University Hospital Essen, Essen, Germany, 2 Centre of Reproductive Medicine and Andrology, University Hospital Münster, Münster, Germany, 3 Institute of Medical Informatics, University Münster, Münster, Germany, 4 Centre of Reproductive Medicine and Andrology, Department of Clinical and Surgical Andrology, University Hospital Münster, Münster, Germany, 5 Institute of Human Genetics, University Hospital Münster, Münster, Germany
Introduction: Previous studies have identified aberrant DNA methylation of imprinted genes (e.g. MEST and H19 ) in the sperm of infertile patients, but the prevalence and genomic extent of these abnormal methylation patterns have remained unknown. Material and Methods: We collected swim-up sperm samples from 40 normozoospermic and 93 oligoasthenoteratozoospermic patients and determined H19 and MEST methylation levels by deep bisulfite sequencing. Based on this screening, we defined three groups: normal controls (NC), abnormally methylated infertile (AMI; n = 7) and normally methylated infertile (NMI; n = 86), and performed whole genome bisulfite sequencing of five NC, five AMI and six NMI samples. Results: Whole-genome comparison of NC and AMI samples revealed that each AMI sample had abnormal methylation levels of all 50 imprinting control regions and >2,000 germ cell-specific genes with distinct DNA methylation differences in blood and germ cells. This is consistent with the presence of somatic cell DNA in AMI samples. The comparison of NC and NMI samples revealed 19 differentially methylated regions, none of which could be validated in an independent cohort of 40 men. Analysis of the H19 CTCF-binding region 6 in samples without somatic cell DNA revealed an association between DNA methylation levels and genotype (rs2071094), but not fertility phenotype. Conclusions: Our results suggest that somatic DNA contamination and genetic variation confound methylation studies in sperm of infertile men and that the prevalence of aberrant methylation in infertile men has been overestimated, which is reassuring for patients undergoing assisted reproduction. Funded by DFG grants HO 949/23-1 and NE 2190/3-1. E. Leitao: None. S. Di Persio: None. S. Laurentino: None. M. Wöste: None. M. Dugas: None. S. Kliesch: None. N. Neuhaus: None. B. Horsthemke: None. P01.059.B miR-200 family inhibits trophoblast invasion through regulating epithelial-mesenchymal transition

M. Su 1 , H. Tsai 1 , C. Wang 2

1 Department of Obstetrics and Gynecology, National Cheng-Kung University Hospital, Tainan, Taiwan, 2 Department of Cell Biology and Anatomy, National Cheng-Kung University, Tainan, Taiwan
Epithelial-to-mesenchymal transition (EMT) is a biological process that epithelial cells lose cell polarity and cell-cell adhesion, and gain migratory and invasive abilities to become mesenchymal cells. In human placenta, the chorionic villi gradually transform from proliferative cytotrophoblasts and extravillous trophoblasts into a migratory and invasive trophoblast phenotype. Failure in altering the behavior and morphology of trophoblasts in placenta may result in insufficient trophoblast invasion, which leads to placental dysfunction and the diseased placenta. MiR-200 family, including miR-200a and -141, were highly expressed in human placenta and were upregulated in patients of preeclampsia. miR-200a and miR-141 were demonstrated to inhibit the formation of primary clila in trophoblasts and therefore have a less ability for cell invasion. In this study, miR-200a and miR-141 were shown to inhibit trophoblasts (HTR-8/SVneo) invasion and migration by a transfection assay. In western blotting and qRT-PCR, miR-200a and miR-141 stimulated epithelial markers, such as E-cadherin and ZO-1, whereas suppressed mesenchymal and transcriptional factors, such as ZEB1, MMP2, MMP9 and TGF-b1. These results suggest trophoblast migration and invasion mediated by miR-200 family may through the mechanism of EMT, and may therefore predispose to develop preeclampsia in human pregnancy. M. Su: None. H. Tsai: None. C. Wang: None. P01.060.C Identification of plasma miRNAs associated with endometriosis in Bulgarian patients

V. Spasova 1 , V. Karamisheva 1,2 , O. Antonova 1 , Z. Hammoudeh 1 , L. Koleva 3 , R. Staneva 1 , M. Ganev 1 , D. Nesheva 1 , A. Nachev 1,2 , D. Toncheva 1 , S. Hadzhidekova 1

1 Medical University of Sofia, Sofia, Bulgaria, 2 University obstetrics and gynecology hospital “Maichin dom, Sofia, Bulgaria, 3 University Hospital “Pirogov”, Sofia, Bulgaria
Introduction: Diagnosis of endometriosis still poses a significant challenge for it usually requires surgery. The need of invasive procedure significantly contributes to late diagnosis and less effective treatment. The aim of this study is to screen for changes in miRNAs expression in peripheral blood that could be further researched as potential non-invasive diagnostic tool for endometriosis. Materials and methods: 15 patients with surgically confirmed advanced endometriosis and 15 healthy controls were included in the study. Peripheral blood samples were collected after signed informed consent. Plasma RNA was isolated with miRNeasy-Serum/Plasma Kit in conjunction with Plasma Spike-in-Control (Qiagen®). Equimolar amounts of RNA were used to prepare two pools: 1)late stage endometriosis and 2)healthy controls. Reverse transcription of the pool samples was performed by miScript II RT Kit (Qiagen®). Expression spectra of 84 miRNAs (miScript miRNA-PCR-Array-Human Serum & Plasma, Qiagen®) by real-time PCR quantification via miScript-SYBRGreen PCR Kit (Qiagen®) were analyzed on Applied Biosystems™7500 instrument. Results: The preliminary results revealed 65 upregulated miRNAs and no downregulated miRNAs between cases and controls. The fold change between 5 and 10 was detected in 21 miRNAs and fold change above 10 was detected in 44 miRNAs. The most significantly overexpressed miRNAs were miR-106a and miR-17-3p which are associated with cell proliferation and apoptosis. Conclusions: MiRNA expression level differences were found in blood between endometriosis patients and healthy controls. This may lay the foundations for future in-depth research on novel non-invasive biomarker for endometriosis. Acknowledgements: Contract Д-212/12.12.2018 V. Spasova: None. V. Karamisheva: None. O. Antonova: None. Z. Hammoudeh: None. L. Koleva: None. R. Staneva: None. M. Ganev: None. D. Nesheva: None. A. Nachev: None. D. Toncheva: None. S. Hadzhidekova: None. P01.061.A Sequence variants in TBX6 are associated with disorders of the Müllerian ducts - an Update

A. Tewes 1 , J. Hucke 2 , T. Römer 3 , K. Kapczuk 4 , C. Schippert 5 , P. Hillemanns 5 , A. Bohring 1 , P. Wieacker 1 , S. Ledig 1

1 Institute of Human Genetics, Münster, Germany, 2 Department of Obstetrics and Gynecology, Wuppertal, Germany, 3 Department of Obstetrics and Gynecology, Köln-Weyertal, Germany, 4 Division of Gynecology, Poznan, Poland, 5 Department of Obstetrics and Gynecology, Hannover, Germany
Müllerian anomalies include formation, fusion or absorption defects of the Müllerian ducts such as unicornuate , didelphys, bicornuate or septate uterus and vaginal septa as well as the Mayer-Rokitansky-Küster-Hauser syndrome (MRKHS) which is characterized by the congenital absence of uterus and vagina. The incidence of MRKHS is estimated to be 1:4.500 and 1:250 in other Müllerian anomalies. Most cases of Müllerian anomalies are sporadic; familial cases seem to be autosomal dominant inherited with reduced penetrance. Until now the knowledge about the etiology of anomalies of the Müllerian ducts is very limited. In approximately 1% of patients with Müllerian anomalies recurrent deletions in 16p11.2 encompassing TBX6 were found as causative in these patients. Moreover, in previous studies, we and others found possibly pathogenic variants in patients with Müllerian anomalies in TBX6 . Since TBX6 encodes a conserved transcription factor playing an essential role in developmental processes like mesoderm formation and specification, we suggested TBX6 as strong candidate gene. Thus we performed analysis of the TBX6 gene (NM_004608) in additional 126 patients with Müllerian anomalies. We detected 4 possibly pathogenic variants (c.358A>G p.(Met120Val), c.704G>A p.(Gly235Asp), c.815G>A p.(Arg272Gln), c.1146C>A p.(Tyr382*)) in TBX6 in 5/126 patients and could exclude the previously reported variant c.484G>A p.(Gly162Ser) as being causative for disorders of the Müllerian ducts in the European non-Finnish population. In summary, we present increasing evidence for the association of variants in TBX6 with malformations of the Müllerian ducts. A. Tewes: None. J. Hucke: None. T. Römer: None. K. Kapczuk: None. C. Schippert: None. P. Hillemanns: None. A. Bohring: None. P. Wieacker: None. S. Ledig: None. P01.062.B Circulating fetal trophoblastic cell eligible for NIPD: the exception rather than the rule

L. Cayrefourcq 1 , M. C. Vincent 2 , S. Pierredon 2 , C. Moutou 3 , M. Imbert-Bouteille 2 , E. Haquet 4 , J. Puechberty 4 , M. Willems 4 , C. Liautard-Haag 2 , N. Molinari 5 , C. Zordan 6 , V. Dorian 6 , A. Chaussenot 7 , L. Monteil 8 , C. Miry 9 , Y. Petrov 10 , E. Le Boette 11 , M. Fradin 12 , C. Alix-Panabiere 1 , C. Guissart 2

1 Laboratoire de cellules circulantes rares humaines_University Medical Center of Montpellier, Montpellier, France, 2 Laboratoire de Genetique Moleculaire_University Medical Center of Montpellier, Montpellier cedex 5, France, 3 Laboratoire de diagnostic pré-implantatoire_CMCO_University Medical Center of Strasbourg, Schiltigheim, France, 4 Departement de Genetique Medicale_University Medical Center of Montpellier, Montpellier, France, 5 DIM_University Medical Center of Montpellier, Montpellier, France, 6 Service de Genetique Medicale_University Medical Center of Bordeaux, Bordeaux, France, 7 Service de Genetique Medicale_University Medical Center of Nice, Nice, France, 8 Service de Genetique Medicale_University Medical Center of Toulouse, Toulouse, France, 9 Department of Medical Medecinee_University Medical Center of Strasbourg, Schiltigheim, France, 10 Laboratoire de cytologie et cytogenetique_University Medical Center of Nimes, Nîmes, France, 11 Service de Genetique Medicale_ Medical Center of Saint Brieuc, Saint Brieuc, France, 12 Service de Genetique Medicale_Medical Center of Saint Brieuc, Saint Brieuc, France
Introduction: Non-Invasive Prenatal Diagnosis (NIPD), based on the analysis of circulating cell-free fetal DNA (cff-DNA), is successfully implemented for an increasing number of monogenic diseases. However, technical issues related to cff-DNA characteristics remain, and not all mutations can be screened with this method, particularly triplet expansion mutations that frequently concern prenatal diagnosis requests. The objective of this study was to develop an approach to isolate and analyze Circulating Trophoblastic Fetal Cells (CFTCs) for NIPD of monogenic diseases caused by triplet repeat expansion or point mutations. Materials and Methods: We developed a method for CFTC isolation based on DEPArray TM sorting and used Huntington’s disease as the clinical model for CFTC-based NIPD. Then, we investigated whether CFTC isolation and Whole Genome Amplification (WGA) could be used for NIPD in couples at risk of transmitting different monogenic diseases. Results: Our data showed that the allele drop-out rate was 3-fold higher in CFTCs than in maternal cells processed in the same way. Moreover, we gave new insights into CFTCs by compiling data obtained by extensive molecular testing by microsatellite multiplex PCR genotyping and by WGA followed by mini-exome sequencing. Conclusions: CFTCs appear to be often characterized by a random state of genomic degradation. This might challenge CFTC use for routine NIPD. This emphasizes the importance to have not a single diagnostic cell but multiple diagnostic cells. This research was funded by French Ministry of Health - DGOS, grant number PHRCI-16-068 (DIACCIMEX) and by Agence de la biomédecine, grant number 18AMP009 (DIAFEXOME) L. Cayrefourcq: None. M.C. Vincent: None. S. Pierredon: None. C. Moutou: None. M. Imbert-Bouteille: None. E. Haquet: None. J. Puechberty: None. M. Willems: None. C. Liautard-Haag: None. N. Molinari: None. C. Zordan: None. V. Dorian: None. A. Chaussenot: None. L. Monteil: None. C. Miry: None. Y. Petrov: None. E. Le Boette: None. M. Fradin: None. C. Alix-Panabiere: None. C. Guissart: None. P01.063.C Development of non-invasive prenatal diagnosis for monogenic diseases: the example of Marfan syndrome and GCK diabetes

T. Nouspikel 1 , A. Bottani 2 , V. Schwitzgebel 1

1 University Hospitals of Geneva, Geneva, Switzerland, 2 Hôpital du Valais, Sion, Switzerland
Introduction: The presence of foetal DNA in maternal plasma allows for non-invasive prenatal diagnosis of Mendelian diseases, through detection of known parental mutations. While identifying paternal mutations is straightforward, determining whether a maternal mutation is present in the foetus requires relative dosage of maternal haplotypes (RHDO), via heterozygous SNPs on either side of the mutation. Results: A custom library of 95 SNPs spread over a ~1 Mb region around the FBN1 gene was used for a posteriori analysis of two pregnancies in a couple with a paternal FBN1:c.5143delA mutation causing Marfan syndrome. In both cases, the mutation was detected in maternal plasma and confirmed in amniotic fluid or cord blood. We also verified that the method could determine which maternal haplotype each foetus had inherited. In another couple with a maternal GCK:p.Gly246Arg mutation causing MODY2, a panel of 99 SNPs over 0.7 Mb around GCK was used to analyse maternal plasma at 16 and 30 weeks gestation. In both samples, we correctly determined the maternal haplotype inherited by the foetus and diagnosed the presence of the mutation, confirmed by cord blood DNA analysis. The same results were obtained by quantifying the number of DNA molecules, obtained through molecular barcodes, rather than sequence reads. Bioinformatic reduction of foetal fractions showed that determination of maternal haplotypes remained possible down to 4.3% foetal DNA. Conclusions: Although preliminary, our results indicate that a molecular barcode library of ~100 SNPs is adequate to achieve non-invasive prenatal diagnosis of monogenic diseases, even at low foetal fractions. T. Nouspikel: None. A. Bottani: None. V. Schwitzgebel: None. P01.064.A NIPT revealed in a pregnant woman 5Mbp-13q duplication, proved as a familial CNV by array CGH

I. Dimova 1 , I. Bradinova 2 , V. Peycheva 3 , R. Bozhilova 3 , R. Raynova 2 , K. Kercheva 1 , A. Savov 2 , I. Kremensky 3 , R. Kaneva 3

1 Medical University Sofia, Sofia, Bulgaria, 2 SBALAG "Maichin dom", Medical University Sofia, Sofia, Bulgaria, 3 Molecular Medicine Center, Medical University Sofia, Sofia, Bulgaria
Subchromosomal copy number variations (CNVs), known as segmental deletions and duplications, are extensively distributed in human genome. A substantial proportion of these CNVs are associated with severe diseases, even with some newly identified microdeletion and microduplication syndromes. Recently it was showed that NIPT has a good performance in detecting fetal subchromosomal CNVs, especially for CNVs >10 Mb, and can be incorporated into the routine NIPT chromosomal aneuploidies screening with high sensitivity and specificity. The prenatal detection of CNVs with clinical significance would benefit all the pregnant women in genetic counselling and clinical management of the pregnant outcomes. We report here a result from NIPT performed in a first trimester of a pregnant woman with third normal pregnancy. The test revealed 5Mbp-13q duplication and array CGH was indicated for confirmation of the aberration after amniocentesis. Array CGH confirmed the aberration in a fetus with the following size: 13q21.2q21.31 (60,540,987-65,431,854)x3 (4.89 Mb). Such duplications in 13q are described in the literature to be associated with the developmental delay, intellectual deficits, behavioral problems, and facial dysmorphisms. We tested all family members by array CGH for the presence of the aberrations - the parents and two siblings (9 years old girl and 12 years old boy). The same aberration was detected in clinically healthy mother and sister, thus determining the familial character of the aberration. In conclusion, careful evaluation should be done for each submiscroscopic aberration detected by NIPT in order to make proper classification and give an adequate genetic counselling. I. Dimova: None. I. Bradinova: None. V. Peycheva: None. R. Bozhilova: None. R. Raynova: None. K. Kercheva: None. A. Savov: None. I. Kremensky: None. R. Kaneva: None. P01.067.A False positive trisomy 21 NIPT-result due to maternal inherited CNV

M. Blankenburg 1 , A. Weber 1 , S. Rauschke 1 , R. Becker 2 , K. Pötschick 3 , M. Stumm 1

1 Medicover Genetics GmbH, Berlin, Germany, 2 Zentrum für Pränataldiagnostik Kudamm 199, Berlin, Germany, 3 Medicover Humangenetik-Berlin-Mitte, Berlin, Germany
Introduction: Noninvasive prenatal tests (NIPT) are screening methods to detect fetal aneuploidies with high specificity and sensitivity. Nevertheless, maternal factors as copy number variations (CNV) are reasons for discrepancies between NIPT-results and fetal karyotypes. We report a case with a conspicuous trisomy 21 result after NIPT, an unsuspicious fetal ultrasound and a normal karyotype following conventional chromosome analysis (CA) of amniotic fluid cells. Material and Methods: Next Generation sequencing (NGS) of cfDNA, CA from peripheral blood, amniotic fluid and placental cells, Fluorescence-in-situ-Hybridization (FISH) on metaphases and interphases, array-CGH (aCGH) and quantitative PCR (qPCR). Results: NIPT indicated a fetal trisomy 21. In contrast, ultrasound examination and amniocentesis showed normal results. Reanalysis of NIPT by WISECONDOR algorithm showed a 10 Mb gain on chromosome 21. No comparable cases were described in literature or databases. The parents declined further diagnostics until birth. Postpartum CA and FISH from blood and placental cells resulted in normal karyotypes. In contrast, aCGH confirmed a 1,44 Mb gain in chromosomal region 21q21.3q22.11 and the aberration was classified as variant of unclear significance (VUS). Additional qPCR analyses confirmed the gain and showed it was maternal inherited. Therefore, we reclassified the aberration as a familial benign CNV. Conclusions: To best of our knowledge, this is the first report of a small benign maternal chromosome 21 CNV leading to a false positive trisomy 21 NIPT result. That case demonstrates again the importance of a critical reflection of genotype/phenotype correlations after NIPT. M. Blankenburg: None. A. Weber: None. S. Rauschke: None. R. Becker: None. K. Pötschick: None. M. Stumm: None. P01.068.B Circulating miRNAs as biomarkers to assess fetal fraction level

G. Santoro 1 , S. Caporilli 1 , C. Lapucci 1 , M. Rusin 2 , M. Giannoccaro 1 , A. Della Gatta 2 , S. Amodeo 2 , A. Farina 2

1 Synlab Italia, Castenedolo, Italy, 2 Università di Bologna, Dipartimento di Scienze Mediche e Chirurgiche (DIMEC), Bologna, Italy
Non-invasive prenatal testing (NIPT) methods are widely used as screening tool for fetal aneuploidies. They are based on the isolation of cell-free DNA (cfDNA) from maternal plasma which for the majority is maternal, but a small percentage of it comes from the placenta (cell-free fetal DNA or cffDNA). cffDNA percentage or fetal fraction (FF) ranges from < 4% to >30% and it influences NIPT outcome. According to neoBona® NIPT method used in our laboratory, invalidation occurs with a FF <4%. Recently, circulating microRNAs (miRNAs) have been investigated as potential biomarkers for screening in pregnancy. We aimed to investigate them as biomarkers in relation to FF level. Previously tested neoBona® samples were divided into one “control group” (FF >4%) and one “test group” (FF <4%). Samples were analysed in the nanostring nCounter® platform by using a pre-customised panel of 799 miRNAs. Each miRNA expression value was normalised to 1 in the “control group” and “test group” samples were analysed for expression defects. Data filtering was set using 1.5-fold of changes in signal as threshold. Ninety-three per cent of miRNAs showed no significant changes of expression whilst 4 miRNAs (named miR-579, miR-3144, miR-612 and miR-6721) were significantly over- or under-expressed. Bioinformatic analysis showed involvement of these miRNAs and their target genes in several biological pathways. Further investigations of these preliminary data could provide a new insight into the biological roles of the miRNAs in relation to FF level and in their potential use as biomarkers. G. Santoro: None. S. Caporilli: None. C. Lapucci: None. M. Rusin: None. M. Giannoccaro: None. A. Della Gatta: None. S. Amodeo: None. A. Farina: None. P01.071.B An Italian public regional healthcare system experience offering non invasive aneuploidy screening test based on cfDNA (NIPT)

C. Pescucci 1 , F. Gerundino 1 , C. Giuliani 1 , B. Minuti 1 , F. Sbernini 1 , S. Frusconi 1 , F. Marin 1 , L. Giulietti 1 , C. Berni 2 , L. Pasquini 3 , C. Luchi 4 , F. Severi 5 , A. Cordisco 6 , B. Melani 7 , E. Mainandi 8 , E. Pelo 1

1 SOD Diagnostica Genetica, Azienda Ospedaliero Universitaria Careggi, Firenze, Italy, 2 P.O. Rete integrata delle malattie rare e dei difetti congeniti e sviluppo della rete dei punti nascita e della pediatria specialistica, Settore Qualità dei servizi e reti cliniche, Regione Toscana, Firenze, Italy, 3 Medicina e Diagnosi Fetale, Azienda Ospedaliero Universitaria Careggi, Firenze, Italy, 4 U.O. Ostericia e Ginecologia, Azienda Ospedaliero-Universitaria Pisana, Pisa, Italy, 5 Dipartimento di Medicina Molecolare dello sviluppo, Azienda Ospedaliero-Universitaria Senese, Siena, Italy, 6 Centro Unico di Diagnosi Prenatale del P.O. Piero Palagi, Firenze, Italy, 7 UO ginecologia e ostetricia - ASL DI PISTOIA, Pistoia, Italy, 8 Consultori e Diagnosi Prenatale USL 4, Prato, Italy
Introduction: Non invasive prenatal testing (NIPT) to investigate the risk for fetal aneuploidies (chromosomes 13, 18 e 21 trisomies and X/Y aneuploidies) has been demonstrated to have high sensitivity and specificity and it is worldwide accepted both as contingent screening as primary test in first trimester. Materials and Methods: since March 2019, NIPT is offered by public healthcare system in Tuscany to pregnant women with conventional first trimester screening test (FTS) risk (nuchal translucency and biochemical measurements) ranging from 1:301-1:1000 or 1:2-1:300 if they opt-out invasive prenatal diagnosis. Women are counseled in different centers spread out over the regional area. Blood samples are collected in a single regional center authorized to perform the test for chromosomes 13, 18, 21, X and Y by CE-IVD VeriSeq™ NIPT Solution (Illumina Inc.). Results: Since March to December 2019, 2199 women have been screened of which 429 with a FTS risk ranging from 1:2-1:300 and 940 from 1:301-1:1000. 396 out of 429 NIPT results were at low risk for fetal aneuploidies whereas 7/429 resulted at high risk for T21 ( n = 4), T18 ( n = 1)and XXX ( n = 2). All these results have been confirmed by IPD. In the FTS 1:301-1:1000 risk group, 738 cases resulted at low risk for fetal aneuploidies and 5 at high risk for T21 ( n = 1), T18 ( n = 1), XXY ( n = 2) and XXX ( n = 1). Only sex chromosome aneuploidy results have been confirmed by IPD. Conclusions. Preliminary data show that of the Tuscany region protocol improve the pregnancy care pathway by reducing inappropriate invasive tests. C. Pescucci: None. F. Gerundino: None. C. Giuliani: None. B. Minuti: None. F. Sbernini: None. S. Frusconi: None. F. Marin: None. L. Giulietti: None. C. Berni: None. L. Pasquini: None. C. Luchi: None. F. Severi: None. A. Cordisco: None. B. Melani: None. E. Mainandi: None. E. Pelo: None. P01.075.C THE FREQUENCY OF RARE CHROMOSOMAL TRISOMIES USING GENOME WIDE NIPT

I. M. TEKIN 1 , H. OZDEMIR 2 , H. OZGUR 1 , Y. SAHIN 1

1 Genoks Genetic Diseases Diagnostic Center, Ankara, Turkey, 2 Department of Obstetrics and Gynecology, Division of Perinatology, Gazi University Faculty of Medicine, Ankara, Turkey
Objective: When non invasive prenatal testing (NIPT) incorporating whole genome sequencing (WGS) is performed, the autosomal aneuploidies other than trisomy 21, 18 and 13 has the potential to be detected. We present the frequency of rare chromosomal trisomies (RATs) that were identified between 1 July 2018 and 31 December 2019 Methods: Standard centrifugation methods were used for plasma isolation. Cell free DNA (cfDNA) was isolated and library construction was prepared according to BGI’s documented protocol. WGS of cfDNA was performed with BGISEQ-500 sequencer. Data was analysed using BGI’s proprietary bioinformatic programme, HALOS. Results: WGS data was generated from 13,802 NIPT cases. The mean maternal age was 33 years, fetal fraction was 10.7% and gestational age at testing was 14 weeks. Over an 18-month period, there were 30 RATs identified. The overall frequency of RATs was 0.21% in our study. Trisomies 16 and 22 were the most frequently observed RATs while other chromosomes were less frequently involved. Invasive prenatal diagnosis was performed in 37% (11/30) of cases with RATs. Three fetal aneuploidies were confirmed with amniocentesis; one was T16 and the others were T22. Conclusions: Rare autosomal aneuploidies are not uncommon in clinical practice when performing genome-wide NIPT. The review of the published reports show a significant risk for obstetric pathology and fetal anomalies such as early miscarriage, true fetal mosaicism, intrauterine growth restriction, in utero fetal demise, and uniparental disomy. We advise genetic counseling in all cases of rare chromosomal trisomies to improve pregnancy management. I.M. Tekin: None. H. Ozdemir: None. H. Ozgur: None. Y. Sahin: None. P01.076.A Next generation sequencing detects premeiotic errors in human oocytes and provides evidence of genetic influence

H. Ghevaria 1 , S. SenGupta 1 , R. Naja 2 , R. Odia 3 , P. Serhal 4 , X. Viñals Gonzalez 3 , X. Sun 1 , J. Delhanty 1

1 Preimplantation Genetics Group, University College London, London, United Kingdom, 2 Molecular Genetics Laboratory, Igenomix UK Ltd, London, London, United Kingdom, 3 Embryology Department, The Centre For Reproductive and Genetic Health, London, United Kingdom, 4 Clinical Department, The Centre For Reproductive and Genetic Health, London, United Kingdom
As women age the risk of an aneuploid oocyte increases with most errors affecting meiosis I/II. Application of next generation sequencing (NGS) has allowed us to confirm our finding, from molecular cytogenetics, that a significant proportion of apparently meiotic aneuploidy is in fact present in the early embryo, leading to a risk of oocyte aneuploidy irrespective of age. It has also provided evidence that genetic factors influence premeiotic oocyte aneuploidy. NGS was performed on oocytes using Ion ReproSeqPGS (ThermoFisher)/VeriSeqPGS (Illumina). Immature oocytes (84) included germinal vesicles & metaphase I oocytes. Mature oocytes (27) were metaphase II - 1st polar body complexes. Totally, 111 oocytes from 33 women, (average maternal age, 35.09 years) were tested. Overall 15/111 (13.5%) oocytes showed premeiotic (PM) errors. Eight of 33 patients had oocytes with PM errors. Aneuploidy analysis was based on the reproductive histories of female partners donating oocytes. Interestingly the first two groups of infertility patients showed almost identical aneuploidy rates in contrast to the social oocyte freezing group. Two young women preserving oocytes prior to breast cancer treatment showed complex aneuploidies. We conclude that the application of NGS has provided accurate information regarding the frequency of aneuploidy that is due to premeiotic errors compared with that caused by errors at MI of oogenesis and that on an individual basis this is influenced by genetic factors. Grant support: CRGH H. Ghevaria: None. S. SenGupta: None. R. Naja: None. R. Odia: None. P. Serhal: None. X. Viñals Gonzalez: None. X. Sun: None. J. Delhanty: None. P01.080.B Interrogating the parental and zygotic origin of de novo mutations in severe male infertility using long read sequencing

G. S. Holt 1 , B. Alobaidi 1 , L. Batty 1 , M. Oud 2 , H. Smith 1 , P. de Vries 2 , R. Smits 2 , L. Vissers 2 , L. Ramos 2 , M. Xavier 1 , J. Veltman 1

1 Biosciences Institute, Newcastle University, Newcastle Upon Tyne, United Kingdom, 2 Department of Human Genetics, Donders Institute for Brain Cognition and Behavior, Radboud University, Nijmegen, Netherlands
Male infertility affects approximately 7% of the male population and while genetic abnormalities are predicted to make up at least 15% of the cases, only ~4% are currently identified. In severe forms of male infertility, patients are found to have high numbers of genetic abnormalities and de novo mutations (DNMs). Interestingly DNMs are known to play an important role in severe genetic diseases with reduced reproductive fitness. Our research investigates the role of DNMs in severe male infertility by studying a unique cohort of azoospermic and severe oligozoospermic patients, and their fertile parents. Employing the Illumina’s Novaseq6000, 100 DNMs were identified in the exomes of 99 patients. While short-read sequencing data was used to collect the location and genotypes of all informative SNPs (iSNPs) surrounding the DNMs, limitations in fragment size prevented further investigation. In-depth examination of DNMs in context with their surrounding iSNPs requires targeted capture approaches for long-read sequencing that span the entire region of interest. Using PCR amplicon sequencing on the Oxford-Nanopore MinION platform we have been able to phase ~90% of the DNMs. Phasing analysis indicated that ~71% of DNMs occurred in the paternal gamete pre-fertilization, which is in concordance with current literature. A link between paternal DNMs and likely causation for disease has been noted, though not all data was available for this association at this time. Investigation of allele frequency identified 91% of the DNMs occurred pre-zygotically, why they failed to impact father spermatogenesis is of particular interest for further study. G.S. Holt: None. B. Alobaidi: None. L. Batty: None. M. Oud: None. H. Smith: None. P. de Vries: None. R. Smits: None. L. Vissers: None. L. Ramos: None. M. Xavier: None. J. Veltman: None. P01.082.A Polycystic ovary syndrome: Does it affect the gene expression patterns in human oocytes?

P. Tulay , Z. Al-Omar, B. Ozbakir

Near East University, Nicosia, Cyprus
Polycystic ovary syndrome is a common disorder among women of reproductive age. The aim of this study was to investigate the gene expression levels of a number of genes that is functioning in steroidogenesis and in the maintenance of telomeres. The spare oocytes were collected from IVF patients with polycystic ovaries and routine IVF patients as controls. DNA and RNA were extracted from each oocyte. Reverse transcription of RNA was performed and real time PCR was conducted to quantify the expression levels of eleven genes, TERF1, TRF2, TINF2, TPP1, POT1, RAP1, CYP11, CYP17, CYP19, HSD17B1 and HSD3B2 . The results of this study showed that the expression levels of TERT , TRF , TINF , TPP , RAP, CYP19 and HSD3B2 were similar between the two groups. However, the expression levels of POT and CYP11A1 genes were shown to be statistically different in the oocytes obtained from control patients (p < 0.05), whereas statistically significant expression levels were observed for CYP17 in the oocytes obtained from the study group (p < 0.05). The expression level of HSD17B1 was also shown to be statistically different in the oocytes (p < 0.05). The results of this study showed that different gene expression levels are observed in the oocytes obtained from females with polycystic ovaries compared to the controls. The extrapolation of the results indicates that the genes essential in maintaining DNA chromosome end integrity and steroidogenesis are crucial for the correct development of the oocytes. Any variations of the expression levels of these genes may result in anomalies of the oocyte maturation. P. Tulay: None. Z. Al-Omar: None. B. Ozbakir: None. P01.085.A Regulatory variants upstream of FLT1 as risk factors for preeclampsia

T. Kikas 1 , R. Inno 1 , K. Ratnik 1,2 , K. Rull 1,3,4 , M. Laan 1

1 University of Tartu, Institute of Bio- and Translational Medicine, Tartu, Estonia, 2 SYNLAB Estonia OÜ, Tartu, Estonia, 3 Women’s Clinic, Tartu University Hospital, Tartu, Estonia, 4 Department of Obstetrics and Gynecology, University of Tartu, Tartu, Estonia
Introduction: The first GWAS assessing the genetic risk to preeclampsia (PE) in placental samples by McGinnis et al., 2017 identified one statistically significant variant associated with PE (rs4769613 C>T; OR=1.2, P=5.4×10 −11 ). The variant is located near a well-known PE gene FLT1 encoding sFlt-1. The aim of this study was to further investigate the link between two variants highlighted in GWAS near FLT1 gene (rs4769613, rs12050029; 69kb/158kb, respectively) and PE in two independent cohorts. Materials and Methods: The study utilized placental samples from Estonian REPROMETA and HAPPY PREGNANCY (placentas n=329/1768, respectively) sample sets, including PE and non-PE controls (total n=96/2001). SNPs (rs4769613, rs12050029) were genotyped using Taqman assays. Association analyses and case-control testing were conducted in PLINK (linear or logistic regression, covariates gestational age, sex). Results: Case-control testing showed an increased risk for PE with rs4769613 C allele in both Estonian sample sets, concordant with the previous study. Meta-analysis across the two cohorts improved the significance (OR=1.75, P = 2.49×10 −3 ). As a novel finding, no associations between rs4769613 and other gestational complications (gestational diabetes, fetal growth, preterm birth) were detected, which suggests the variant is PE-specific. The gene expression of FLT1 was upregulated only in PE placentas carrying CC- and CT genotypes (t-test, P < 0.05), whereas in maternal serum, sFlt-1 was increased in PE cases in all genotype groups. Conclusions: The study emphasizes the placental rs4769613 as the only known robust and specific risk variant for PE, modulating placental FLT1 expression. Funding: IUT34-12 (Estonian Research Council); HAPPY PREGNANCY, 3.2.0701.12-0047 (European Regional Development Fund) T. Kikas: None. R. Inno: None. K. Ratnik: A. Employment (full or part-time); Significant; SYNLAB Estonia OÜ. K. Rull: None. M. Laan: None. P01.086.B The role of new genetic markers and natural selection in the formation of a hereditary predisposition to preeclampsia

V. Serebrova , E. Trifonova, V. Stepanov

Research Institute of Medical Genetics, Tomsk National Research Medical Center of the Russian Academy of Sciences, Tomsk, Russian Federation
Introduction: Preeclampsia (PE) is a pregnancy-specific complication with the poorly understood etiopathogenesis. There is a hypothesis that the risk to the development of PE may be a consequence of natural selection affecting on placental genes responsible for trophoblast invasion and remodeling of spiral arteries. The aim of this work was to characterize the genetic components of PE by analysis of regulatory single-nucleotide polymorphisms (rSNPs) of differentially expressed genes (DEGs) in placenta and to detect the role of natural selection in its formation. Materials and Methods: In this report we analyzed 46 rSNPs in 21 DEGs, associated with PE according to the analysis of the transcriptome in 551 patients with PE and 718 women with uncomplicated pregnancies of Buryat, Russian and Yakut populations using MassArray iPLEX (Sequenom). For detection of the signals of natural selection we used new INSIGHT method. Results: We have detected significant associations of PE with 14 rSNPs in 10 genes: BHLHE40, CORO2A , INHA , LHB , NDRG1 , PPP1R12C , PLIN2 , RDH13 , SASH1 , SYDE1 . The effect of weak negative selection has being shown for 4 rSNPs in 4 DEGs: rs10985257 in CORO2A gene, rs72959687 in INHA gene, rs34845949 in SASH1 gene, rs2227262 in NDRG1 gene. It should be noted that these results corresponds to number of evolutionary hypotheses of the occurrence of PE. Conclusions: Our results demonstrate the significant role of studied rSNPs of DEGs and their adaptive changes in the hereditary predisposition to PE. This work was supported by the Russian Foundation for Basic Research (grant №18-29-13045). V. Serebrova: None. E. Trifonova: None. V. Stepanov: None. P01.087.C Genetic risk factors for pre-eclampsia in the Finnish population

T. Kaartokallio 1 , S. Ruotsalainen 2 , T. Palviainen 2 , T. Hiekkalinna 3,4 , K. Auro 2,5 , M. Perola 5,6 , FINNPEC, K. Kivinen 2 , H. Laivuori 1,2,7

1 Medical and Clinical Genetics, University of Helsinki and Helsinki University Hospital, Helsinki, Finland, 2 Institute for Molecular Medicine Finland, Helsinki Institute of Life Science, University of Helsinki, Helsinki, Finland, 3 Finnish Institute for Health and Welfare, Helsinki, Finland, 4 Clinical and Molecular Metabolism Research Program, University of Helsinki, Helsinki, Finland, 5 National Institute for Health and Welfare, Helsinki, Finland, 6 Diabetes and Obesity Research Program, University of Helsinki, Helsinki, Finland, 7 Department of Obstetrics and Gynecology, Tampere University Hospital and Tampere University, Faculty of Medicine and Health Technology, Tampere, Finland
Introduction : Pre-eclampsia (PE) is a vascular pregnancy disorder with maternal and fetal genetic components. As the disease negatively affects reproductive success, the risk variants are assumed to be under purifying evolutionary selection. The amount of low-frequency pathogenic variants has shown to be increased in Finland due to recent bottlenecks, increasing statistical power in genetic studies. We aim to screen for genetic risk factors for PE in the families mainly originating from a northeastern Finland population isolate, and generalise the results for the whole population. Methods : The PE family cohort consists of 15 extended families. DNA is available for 174 family members (mothers: 62 PE+32 ctrl; babies: 41 PE+39 ctrl). Using genome-wide imputed data, we have performed linkage and family-based association analyses with PSEUDOMARKER and PLINK to study segregation of loci with both maternal and fetal phenotypes. We will seek replication of the findings by association analysis in two Finnish cohorts with genome-wide imputed data available, the FINRISK population survey (426 PE+8357 ctrl) and the FINNPEC case-control cohort (mothers:1533 PE+995 ctrl; babies:818 PE+925 ctrl). Results : In the linkage screen, we identified ~35 maternal and 40 fetal candidate loci when using a lenient p -value threshold p < 5x10 −5 . The FINRISK association analysis showed suggestive replication (p < 5x10 −6 ) of two of the loci, maternal signal in chr9 close to GAS1 and fetal signal in chr16 in CDH13 . Next steps include association analysis in FINNPEC and meta-analysis in both cohorts to verify these preliminary findings. Funding: Academy of Finland, Erkko, Kordelin and Sohlberg Foundations, and VTR funding. T. Kaartokallio: None. S. Ruotsalainen: None. T. Palviainen: None. T. Hiekkalinna: None. K. Auro: None. M. Perola: None. K. Kivinen: None. H. Laivuori: None. P01.089.B A Retrospective Study of In Vitro Fertilization and Preimplantation Genetic Testing for chromosomal aberrations (PGT-SR and PGT-A) combined with Clinical Outcome

M. Baetens 1 , I. De Croo 2 , A. Dheedene 1 , K. Tilleman 2 , B. Menten 1

1 Center for Medical Genetics, Ghent University Hospital, Ghent, Belgium, 2 Department for Reproductive Medicine, Ghent University Hospital, Ghent, Belgium
PGT for structural rearrangements (PGT-SR) can be offered to patients carrying an inversion, balanced reciprocal or Robertsonian translocation, and PGT for aneuploidy screening (PGT-A) to patients with fertility problems or recurrent miscarriages. Selecting against embryos without a chromosomal rearrangement could improve live birth rates while reducing miscarriage rates or avoiding birth of an affected child. PGT is an extended, stressful trajectory while precise outcome predictions are still hard to grasp. Counseling PGT couples remains delicate due to numerous parameters influencing outcome. Comparing success rates to (inter)national reference data is part of good clinical practice. PGT technologies evolve at high speed, going from different biopsy timepoints to more extensive genetic methodologies, making good comparisons difficult. By detailed evaluation of our in-house data, and comparison to published data from (inter)national clinics, we obtained additional insights and can provide couples with more patient-focused information. We performed a retrospective cohort study at the Department for Reproductive Medicine and the Center for Medical Genetics (Ghent University Hospital), including patients who underwent ICSI-IVF (January 2016-April 2019) followed by PGT-SR or PGT-A after trophectoderm biopsy (68 and 90 oocyte retrieval cycles respectively). Making use of comprehensive chromosome screening (shallow whole genome sequencing or sWGS), we detected chromosomal abnormalities in 65% of PGT-SR embryos. In the PGT-A group, 40% of embryos showed a numerical or structural rearrangement. For translocation carriers, pregnancy rate per transfer was 58% and live birth rate per transfer 44%. For PGT-A patients, pregnancy rate and live birth rate were 39% and 25% respectively. M. Baetens: None. I. De Croo: None. A. Dheedene: None. K. Tilleman: None. B. Menten: None. P01.090.C Preimplantation Genetic Testing (PGT) for cancer predisposition: A single center large cohort experience

S. Zuckerman 1 , O. Freireich 1 , T. Eldar-Geva 2 , S. Lieberman 1 , S. Shaviv 1 , A. Yadin 1 , E. Farhi 1 , E. Hakam-Spector 1 , M. Ben-Shlomo 1 , T. Azar 1 , E. Zivi 2 , E. Rubinstein 2 , O. Schonberger 2 , I. Ben-Ami 2 , G. Altarescu 1

1 Medical Genetics Institute, Shaare Zedek Medical Center, Jerusalem, Israel, 2 IVF unit, Shaare Zedek Medical Center, Jerusalem, Israel
In the last decade an increasing number of people carriers of cancer predisposition mutations (CCPM) are performing PGT-M. PGT-M for these late-onset and partial penetrant conditions implies that the procedure selects against embryos that might have been cancer-free. Moreover, preventive and therapeutic options for decreasing morbidity and mortality are available and ongoing cancer research might reveal treatments altering the natural history of cancer in the future. Most studies exploring perceptions of PGT-M among CCPM, were based on participants’ theoretical declarative intentions. Actual use has rarely been reported and analyzed. Between 2006-2019 overall 173 CCPM were counseled in our center: One couple between 2006-2008 and gradual increase until 102 CCPM between 2018-2019. Six were single women (3.4%) and 167 couples (96.5%). Among them 145(83.8%) were BRCA1/BRCA2 carriers (92/48 respectively; 5 BRCA1+BRCA2), 10(5.8%) Lynch carriers, 10(5.8%) APC carriers, 4(2.3%) TP53 carriers, 2(1.2%) MEN2 carriers, 1(0.6%) CHEK2 carrier and 1(0.6%) BMPR1A carrier. Overall, 150(86.7%) of the carriers were healthy and 23(13.2%) were diagnosed with cancer or related condition. In 120(69.4%) couples the carrier was the women partner; in 51(29.5%) couples the carrier was the men partner and in 2(1.2%) couples both partners were carriers. Five)2.8%) couples had additional reason for PGT-M/PGT-SR. Overall Live birth rate for embryo transfer was 32%. Reproductive decision-making regarding PGT-M for CCPM is a complex process. While this application for PGT-M becomes acceptable, health professionals involved in the process should be aware and address the specific issues and concerns of this group regarding medical, emotional and moral aspects. S. Zuckerman: None. O. Freireich: None. T. Eldar-Geva: None. S. Lieberman: None. S. Shaviv: None. A. Yadin: None. E. Farhi: None. E. Hakam-Spector: None. M. Ben-Shlomo: None. T. Azar: None. E. Zivi: None. E. Rubinstein: None. O. Schonberger: None. I. Ben-Ami: None. G. Altarescu: None. P01.091.A Obstetric risks associated with preimplantation genetic testing for monogenic disorders and structural chromosomal rearrangements (PGT-M/SR) compared to regular IVF/ICSI .

M. Heijligers 1 , Y. Arens 1 , R. van Golde 1 , M. van der Hoeven 1 , J. Dumoulin 1 , I. Homminga 2 , C. van Tilborg 3 , C. de Die-Smulders 1 , A. van Montfoort 1

1 Maastricht University Medical Centre+, Maastricht, Netherlands, 2 University Medical Center Groningen, Groningen, Netherlands, 3 University Medical Center Utrecht, Utrecht, Netherlands
Introduction: Earlier studies on the obstetric outcome in pregnancies following PGT are inconclusive since both an increased risk and no increased risk for adverse obstetric outcome are reported. Moreover, not all of these studies distinguish between PGT for monogenic disorders or structural chromosomal rearrangements (PGT-M/SR) and PGT for aneuploidies (PGT-A). Methods: This retrospective cohort study included all pregnancies resulting from PGT-M/SR ( n = 389) and IVF/ICSI treatments ( n = 1667) conducted between 2004 and 2014. Data were collected from the medical files and from questionnaires. These data included parental characteristics, characteristics of the treatment preceding the pregnancy and obstetric and neonatal characteristics. Results: Obstetric complications were reported in 18% and 19% of the singleton pregnancies in the PGT and regular IVF/ICSI group, respectively ( P = 0.103). The risk for stillbirth was lower in the PGT group ( P = 0.044). Birth weights of all singletons were converted into z-scores and showed a comparable birth weight for the two groups (0.09 vs. -0.02; P = 0.084). Within the multiple pregnancies, the risk for obstetric complications ( P = 0.535) and the incidence of fetal loss ( P = 0.617) was comparable between the PGT and IVF/ICSI group. The mean pregnancy duration was longer ( P = 0.037) and the mean birth weight was higher ( P = 0.010) in the PGT group. More monozygotic pregnancies were observed in the PGT group ( P = 0.009). Conclusions: The more invasive nature of the PGT-M/SR technique does not lead to an increased risk for obstetric complications as compared to a regular IVF/ICSI treatment. M. Heijligers: None. Y. Arens: None. R. van Golde: None. M. van der Hoeven: None. J. Dumoulin: None. I. Homminga: None. C. van Tilborg: None. C. de Die-Smulders: None. A. van Montfoort: None. P01.092.B Results gained from fetal exome sequencing inform clinical management. A report of 100 cases .

E. Dempsey 1 , L. Ive 2 , R. Dubis 2 , E. Serra 2 , H. Savage 2 , A. Haworth 2 , S. Mansour 1 , T. Homfray 1 , S. Drury 2

1 St George’s University of London, London, United Kingdom, 2 Congenica Ltd, Cambridge, United Kingdom
Introduction: Exome sequencing in cases of fetal structural malformation has been shown to increase diagnostic yield (over traditional diagnostic methods) by 8-80%, depending on phenotype. This technique is soon to be made available throughout England as part of a centralisation and standardisation of genetic laboratory services. Materials and Methods: We have undertaken exome sequencing in 100 cases of fetal anomaly as part of a service improvement project at St George’s Hospital. In half of cases sequencing was undertaken with the intention of informing the management of an ongoing pregnancy. In the remaining cases exome sequencing was undertaken post-mortem. Exome data was analysed via the application of patient-specific phenotype panels, reducing the risk of incidental findings. Results: Our diagnostic rate across all cases is ~ 40 % and higher in cases with skeletal and oedema phenotypes. Exome results have influenced the management of pregnancy and labour. They have also directed early neonatal care, including enabling the treatment of a newborn with enzyme replacement therapy. The results of exome sequencing have been used for accurate assessment of recurrence risk. In addition, results can be used for prenatal testing or preimplantation genetic diagnosis in a future pregnancy. Conclusions: Using specific case examples we will describe the way in which the results of fetal exome sequencing have influenced clinical management. We will also describe the challenges we have encountered in the implementation of a fetal exome sequencing service, including the analysis of variants of uncertain significance in phenotype-limited situations. E. Dempsey: None. L. Ive: A. Employment (full or part-time); Significant; Congenica Ltd. R. Dubis: A. Employment (full or part-time); Significant; Congenica Ltd. E. Serra: A. Employment (full or part-time); Significant; Congenica. H. Savage: A. Employment (full or part-time); Significant; Congenica Ltd. A. Haworth: A. Employment (full or part-time); Significant; Congenica Ltd. S. Mansour: None. T. Homfray: None. S. Drury: A. Employment (full or part-time); Significant; Congenica Ltd. P01.093.C Whole-exome sequencing in the prenatal setting: relating fetal phenotype to genotype

L. Leeuwen , K. Bouman, N. Corsten-Janssen

UMCG, Groningen, Netherlands
Introduction: Whole-exome sequencing (WES) is increasingly being used in the prenatal setting. However, interpretation of WES data is complicated due to various factors including limited knowledge on the prenatal fetal phenotype of rare monogenic disorders. We therefore aimed to describe the prenatal fetal phenotype of genetic variants found by WES. Methods: In this single-center study, we included fetuses in whom WES was indicated in a prenatal diagnostic setting between March 1, 2018 and January 1, 2020 during pregnancy or after termination of pregnancy. We performed trio WES using a phenotype-dependent filtering strategy. We documented prenatal and postnatal characteristics of fetuses and compared the phenotype with literature using PubMed. Results: WES was performed in 93 fetuses. A pathogenic or likely pathogenic variant was found in 29 (31.2%) fetuses (21 different genes). For eight genes, it was the first time a causative genetic variant was found in a prenatal setting ( C2CD3,CNOT1,GATA6, PLD1,POMK,SAMD9,SMC3,THOC6 ). In three fetuses, some detected structural anomalies have not previously been described ( PEX1-,POMK-,THOC6- variants). In three fetuses, diagnosis was filtered out in the prenatal setting due to various reasons, but could be established using additional postnatal phenotypic characteristics. In general, the prenatal phenotype on ultrasound corresponded well with postnatal phenotype although some features were not detected by the prenatal ultrasound examination. Conclusions: Increased knowledge about the prenatal fetal phenotype is important for improving the interpretation and classification of fetal genetic variants. This will lead to improvement of parental genetic counseling as well as prenatal and neonatal care. L. Leeuwen: None. K. Bouman: None. N. Corsten-Janssen: None. P01.098.B Lessons learned from rapid Exome Sequencing(rES)as a standard diagnostic test in a prenatal setting for fetuses with ultrasound abnormalities .

N. Corsten-Janssen 1 , K. Bouman 1 , A. J. Scheper 1 , M. T. Meems-Veldhuis 1 , J. B. G. M. Verheij 1 , R. Suijkerbuijk 1 , L. K. Duin 2 , G. T. R. Manten 3 , I. M. van Langen 1 , B. Sikkema-Raddatz 1 , H. Westers 1 , C. C. van Diemen 1

1 Department of Genetics, University of Groningen, University Medical Centre Groningen, Groningen, Netherlands, 2 Department of Obstetrics, Gynecology and Prenatal Diagnosis, University Medical Centre Groningen, Groningen, Netherlands, 3 Department of Obstetrics and Gynecology, Isala, Zwolle, Zwolle, Netherlands
Background: Adding rES to conventional genetic tests for fetuses with ultrasound abnormalities improves the diagnostic yield, but differs from performing postnatal rES for instance by its rapid turnaround time and the impact of a diagnosis. We will present the lessons we learned by performing rES as a diagnostic test in prenatal care from March 2018. Methods: We performed trio rES analysis using a custom virtual gene panel of ~4,300 OMIM genes during pregnancy. Inclusion criteria were: 1) two or more independent major fetal anomalies, or 2) hydrops fetalis or bilateral polycystic kidneys, or 3) one major fetal anomaly and a first-degree relative with the same anomaly. Results : We established a genetic rES-based diagnosis in 14 out of 54 (26%) eligible fetuses with a mean turnaround time of 14 days. We learned the following lessons: (1) We made unexpected diagnoses using our broad virtual panel, like Zellweger syndrome in a fetus suspected of a ciliopathy. (2) We missed four diagnoses that were made postnatally using additional phenotypic information and adapted our filter strategy. 3) We were confronted with incidental unsolicited findings and changed our filter strategy to minimize them. (4) There is a lack of knowledge about prenatal phenotype of known genetic conditions, which hinders diagnostics. Further, we experienced the impact of time pressure to generate a diagnosis in limited time and had to deal with changing fetal phenotypes. Conclusion : rES in the prenatal setting is feasible but challenging, and improves by adapting the process based on insights from previous experiences. N. Corsten-Janssen: None. K. Bouman: None. A.J. Scheper: None. M.T. Meems-Veldhuis: None. J.B.G.M. Verheij: None. R. Suijkerbuijk: None. L.K. Duin: None. G.T.R. Manten: None. I.M. van Langen: None. B. Sikkema-Raddatz: None. H. Westers: None. C.C. van Diemen: None. P01.099.C Improved fetal sex determination in twin pregnancies in low coverage whole-genome based non-invasive prenatal testing

T. Sedlackova 1 , M. Hyblova 2,3 , J. Budis 1,4,5 , M. Harsanyova 1,6 , M. Kucharik 1,5 , O. Pos 1,6 , M. Sekelska 2,3 , B. Nagy 7 , G. Minarik 2,3 , T. Szemes 1,5,6

1 Geneton Ltd., Bratislava, Slovakia, 2 Medirex Inc., Bratislava, Slovakia, 3 Trisomy Test Ltd., Bratislava, Slovakia, 4 Slovak Centre of Scientific and Technical Information, Bratislava, Slovakia, 5 Comenius University Science Park, Bratislava, Slovakia, 6 Department of Molecular Biology, Faculty of Natural Sciences, Comenius University, Bratislava, Slovakia, 7 Semmelweis University, Budapest, Hungary
Introduction: Fetal sex determination is already a common part of the standard non-invasive prenatal testing (NIPT). The current classification methods are almost entirely accurate for fetal sex prediction in singleton pregnancies, but they still show the limitations for multiple pregnancies, with 98% reported accuracy of fetal sex prediction in twins. Methods: We enrolled 99 maternal samples with twin pregnancies and confirmed fetus sex in this study. Since a precise estimation of the fetal fraction is a key for correct sex determination, we used a previously published improved predictor utilizing two different attributes of analyzed genomic material - length and genomic location of the sequenced DNA fragments. Subsequent fetal sex prediction was performed using an SVM classifier. Results: Our method has shown perfect prediction accuracy in distinguishing between pregnancies with two female fetuses and pregnancies with at least one male fetus. We observed one incorrect prediction between one and two male fetuses. Overall, the accuracy of fetal sex prediction was 98.99%. Conclusions: Here we described a novel bioinformatic algorithm to determine the sex of both fetuses in twin pregnancies, by the analysis of cell-free fetal DNA from the maternal plasma. Our algorithm achieved superior accuracy in fetal sex prediction in twins compared to known bioinformatics tools. Moreover, it provides supporting graphs for visual inspection and calculation of prediction confidence. Keywords: non-invasive prenatal testing, twin pregnancy, whole-genome sequencing, fetal sex prediction T. Sedlackova: A. Employment (full or part-time); Modest; Geneton Ltd.. M. Hyblova: None. J. Budis: None. M. Harsanyova: None. M. Kucharik: None. O. Pos: None. M. Sekelska: None. B. Nagy: None. G. Minarik: None. T. Szemes: None. P01.100.A The value of prenatal array to detect submicroscopic chromosomal imbalances in cases with abnormal fetal ultrasonographic findings; a retrospective study

L. OZER , M. Aydin, S. Aktuna, E. Unsal

Chromosomal microarray (CMA) is used in prenatal diagnosis for detecting CNVs which are too small to detect by karyotyping. Here we report our laboratory’s array data of prenatal samples and describe phenotype-genotype correlations. Methods: 180 specimens (149 amniotic fluid, 23 chorionic villi and 8 cord blood samples) were analyzed by array CGH. Detection of genomic CNVs was performed using Agilent, 8x60K CGH Microarray. Interpretation was done according to the ACMG guidelines. Results: %16.1 of patients (29/180), CNVs were detected. 24 of them had segmental duplication or deletion, 5 had trisomy and 1 had monosomy X. 3.3% of patients had aneuploidy, 4.4% of patients had VUS variants and %8.8 of patients had pathogenic segmental deletions/duplications. 61.1% of the segmental pathogenic variants were deletions and most of the VUS variants were duplications (62.5%). 50% of the segmental pathogenic variants were < 7 Mb and the smallest size was 0.5MB (deletion). 22.2% of pathogenic variants were well-defined CNV syndromes. The most prevalent indications were ultrasonographic anomalies, increased triple test risk and advanced maternal age. While 30% of patients with normal results have normal ultrasound, all patients with a pathogenic variant had an ultrasound anomaly. The most defined ultrasonographic findings in cases with pathogenic variants were cardiac anomalies, increased NT and brain anomalies. Conclusion: Detection of submicroscopic imbalances by CMA increases the power of prenatal diagnosis. Our data emphasize the importance of CMA in the prenatal diagnosis of the fetuses with abnormal ultrasonographic findings whereas conventional karyotyping resolution is inadequate to detect submicroscopic changes. L. Ozer: None. M. Aydin: None. S. Aktuna: None. E. Unsal: None. P01.102.C Targeted prenatal exome sequencing in a series of fetal structural anomalies detected by ultrasonography: first experience of the University Hospitals of Geneva

L. Quteineh 1 , J. Pellegrinelli 2 , M. Billieux 2 , M. Guipponi 1,3 , S. Laurent 1 , E. Hammar 1 , A. Murphy 1 , M. Carminho Amaro Rodrigues 1 , T. Ait-Mouhoub 1 , F. Sloan-Béna 1 , S. Unger 4 , F. Giuliano 4 , C. Rieubland 5 , J. Fluss 6 , P. Extermann 7 , J. Blouin 1,3 , M. Abramowicz 1 , S. Fokstuen 1

1 Service of Genetic Medicine, Geneva, Switzerland, 2 Department of gynecology and obstetrics, Geneva, Switzerland, 3 Department of Genetic Medicine and Development, Geneva, Switzerland, 4 Service of Genetic Medicine, Lausanne, Switzerland, 5 Department of Pediatrics, Division of Human Genetics, Bern, Switzerland, 6 Pediatric Neurology Unit, Geneva, Switzerland, 7 Dianecho echographic center, Geneva, Switzerland
Introduction: Diagnostic genome-wide sequencing approaches to investigate prenatally detected anomalies are increasingly used in fetal medicine. We aimed to evaluate the value of targeted whole exome sequencing (WES) in fetuses with prenatal sonographic abnormalities and normal microarray and karyotype. Materials and Methods: After informed consent was obtained, we sequenced the DNA from amniotic fluid cells of 17 fetuses by WES (Illumina NextSEQ500). We then performed bioinformatic analysis of a selected gene panel. All cases were ongoing pregnancies. The genomic results were obtained within 2 weeks. Results: The 17 fetuses presented with the following anomalies: isolated brain malformations ( n = 6), sustained increased nuchal translucency ( n = 3), multiple structural anomalies ( n = 3), polyhydramnios ( n = 1), situs inversus ( n = 1), renal cysts ( n = 1), hydrops fetalis ( n = 1) and skeletal dysplasia ( n = 1). Causing variants were identified in 4 cases (24%): two with sustained increased nuchal translucency presented with a de novo variant in RAF1 (p.Ser267Arg) and in BRAF (p.Thr241Arg) genes, compatible with a RASopathy, the fetus with skeletal dysplasia had a de novo variant in COL11A1 gene (p.Gln921_Pro926del) causing Stickler/Marshall syndrome and the fetus with hydrops fetalis had 2 variants in PIEZO1 gene (p.Ser182* and p.Phe1294del) causing autosomal recessive lymphatic dysplasia. Pregnancy was terminated in all 4 cases. Conclusions: targeted WES is an adequate approach of genome-wide sequencing for ongoing pregnancies with fetal structural abnormalities suspicious for a genetic etiology and normal microarray. It has a diagnostic utility and might facilitates genetic counseling. L. Quteineh: None. J. Pellegrinelli: None. M. Billieux: None. M. Guipponi: None. S. Laurent: None. E. Hammar: None. A. Murphy: None. M. Carminho Amaro Rodrigues: None. T. Ait-Mouhoub: None. F. Sloan-Béna: None. S. Unger: None. F. Giuliano: None. C. Rieubland: None. J. Fluss: None. P. Extermann: None. J. Blouin: None. M. Abramowicz: None. S. Fokstuen: None. P01.104.B Prenatal diagnosis of trisomies in CVS: are non-mosaic biallelic QF-PCR results prognostic?

A. R. Pendlebury-Watt 1 , K. Mann 1 , C. Ogilvie 2

1 Viapath analytics, London, United Kingdom, 2 Genetics Department, Guys and St. Thomas’ NHS Foundation Trust, London, United Kingdom
Placental mosaicism for chromosome aneuploidy complicates prenatal analysis of chorionic villus (CV) samples and has a reported incidence of up to 2%. Quantitative fluorescence-PCR (QF-PCR) analysis of microsatellite repeats is widely used to establish chromosome copy number of uncultured CV samples; if recommended preparation methods are employed, both cytotrophoblast and mesoderm should be represented. In the case of trisomy results, the presence of three different alleles is observed in ~90% of cases, confirming meiotic non-disjunction as the origin of the abnormal cell line. Where only biallelic 2:1 ratios are observed, the trisomy may have arisen by mitotic non-disjunction following a normal conception, in which case a normal cell line may be present; European and UK best practice guidelines recommend that non-mosaic biallelic (NMB) results should be reported more cautiously, given the increased risk of mosaicism. However, the likelihood of finding a normal cell line following an NMB result is not established. We have therefore audited 10 years of cases and compared NMB results to the follow-up karyotype and/or amniocentesis results. In total, 133 CVS showed NMB results for common trisomies, of which 131 were concordant with follow-up tests. Two trisomy 18 cases showed a normal result on amniocentesis, indicating confined placental mosaicism; neither case had ultrasound abnormalities present at referral. Therefore, in this cohort, ~1.5% of NMB QF-PCR cases subsequently showed evidence of mosaicism, this is reduced to 0% for cases with USS abnormalities. These figures may inform the reporting and pregnancy management of NMB cases. A.R. Pendlebury-Watt: None. K. Mann: None. C. Ogilvie: None. P01.105.C Parental experiences of rapid Exome Sequencing in case of serious ultrasound abnormalities during pregnancy

M. Plantinga 1 , L. Zwienenberg 1 , E. van Dijk 1 , H. Breet 1 , J. Diphoorn 1 , J. el Mecky 2 , K. Bouman 1 , J. Verheij 1 , A. Ranchor 3 , N. Corsten-Janssen 1 , I. van Langen 1

1 UMCG, Clinical Genetics, Groningen, Netherlands, 2 University of Southampton, CELS, Southampton, United Kingdom, 3 UMCG, Health Psychology, Groningen, Netherlands
Objective : Adding rapid Exome Sequencing (rES) to conventional genetic tests (QF-PCR and SNP-array) can improve the diagnostic yield of pregnancies showing ultrasound abnormalities but also includes a higher chance of unsolicited findings. rES was offered in the context of a prenatal rES implementation study with a turnaround time of 14 days. We evaluated how this offer was experienced in an explorative mixed-methods parental experience study. Methods: We investigated reasons for accepting and declining rES, the impact of rES on decision making, experiences of the counseling and test process, and understanding of test information and possible test outcomes. All couples were asked to fill two surveys, one after pre-test and one after post-test counseling. Part of the couples were approached for an interview until data saturation was reached. Results: 44 couples participated in the survey study, 11 in the interview study. In 22% of pregnancies, a diagnosis was found, in 31% an unsolicited finding. 95% wanted to be informed about unsolicited findings. The most important reason for accepting rES was to find a diagnosis. Most parents already decided to terminate the pregnancy on the basis of the ultrasound abnormalities, although rES results impacted decision in a few cases. The impact for future pregnancies was higher than with conventional testing and the fast turnaround was valued highly. The counseling and test process was experienced positive. Parents suggested to add information of other parents’ experiences and decisional process to the test information. Conclusion : Parents evaluate rES positively, despite the risk of unsolicited findings. M. Plantinga: None. L. Zwienenberg: None. E. van Dijk: None. H. Breet: None. J. Diphoorn: None. J. el Mecky: None. K. Bouman: None. J. Verheij: None. A. Ranchor: None. N. Corsten-Janssen: None. I. van Langen: None. P01.106.A When to test fetuses for RASopathies? Proposition from a systematic analysis of 353 multicenter cases and a postnatal cohort

A. Scott 1 , N. Di Giosaffatte 2 , V. Pinna 2 , P. Daniele 2 , S. Corno 3 , V. D’Ambrosio 3 , E. Andreucci 4 , A. Marozza 5 , F. Sirchia 6 , G. Tortora 7 , D. Mangiameli 8 , C. Di Marco 9 , I. Donati 10 , A. Zonta 11 , E. Grosso 11 , G. Mastromoro 12 , P. Versacci 13 , F. Pantaleoni 14 , F. C. Radio 14 , T. Mazza 15 , L. Papi 5 , T. Mattina 8 , A. Giancotti 3 , A. Pizzuti 12 , A. Laberge 1 , M. Tartaglia 14 , M. Delrue 1 , A. De Luca 2

1 Pediatrics Department, Medical Genetics Division, CHU Sainte-Justine, University of Montreal, Montreal, QC, Canada, 2 Medical Genetics Unit, Fondazione IRCCS-Casa Sollievo della Sofferenza, San Giovanni Rotondo, Italy, 3 Department of Maternal and Child Health and Urologic Science, "Sapienza" University, Policlinico Umberto I Hospital, Rome, Italy, 4 Medical Genetics Unit, Meyer Children’s University Hospital, Florence, Italy, 5 Section of Medical Genetics, Department of Experimental and Clinical Biomedical Sciences, University of Florence, Florence, Italy, 6 Institute for Maternal and Child Health, IRCCS "Burlo Garofolo", Trieste, Italy, 7 Azienda Ospedaliero-Universitaria Ospedali Riuniti di Ancona, Ancona, Italy, 8 Department of Pediatrics, Medical Genetics, University of Catania, Catania, Italy, 9 Ospedale Santa Maria Nuova, Florence, Italy, 10 Genetic Department, Infermi Hospital, Rimini, Italy, 11 Città Della Salute e Della Scienza University Hospital, Medical Genetics Unit, Torino, Italy, 12 Department of Experimental Medicine, Università Sapienza, Rome, Italy, 13 Department of Pediatrics, Università Sapienza, Rome, Italy, 14 Genetics and Rare Diseases Research Division, Ospedale Pediatrico Bambino Gesù, IRCCS, Rome, Italy, 15 Laboratory of Bioinformatics, IRCCS Casa Sollievo della Sofferenza, San Giovanni Rotondo, Italy
Introduction: Recent studies have identified some evocative prenatal features of RASopathies e.g. lymphatic dysplasia (cystic hygroma, hydrops, effusions), congenital heart disease, polyhydramnios, and renal anomalies. The aim of this study was to better define the prenatal indications for RASopathy testing and the genotype-phenotype correlations. Methods: 353 cases sent for prenatal RASopathy testing between 2012-2019, after a normal a-CGH, were collected. For most, 11 RASopathy genes were tested. Detailed ultrasound findings were available for 313 fetuses, whereas 40 cases had only clinical indications for testing collected. Twenty-five RASopathy patients diagnosed postnatally were added to widen the phenotypic spectrum. Results: See tables below for detailed results. The overall diagnostic yield was 14% (51/353). The largest contributors of mutations were PTPN11 (29%), RIT1 (15%), RAF1 (13%) and HRAS (12%). Severe postnatal phenotypes were in majority diagnosed prenatally and associated with mutations in HRAS , RIT1 and RAF1 . Conclusions: After normal a-CGH, RASopathies should be considered when any ultrasound finding of lymphatic dysplasia or suggestive congenital heart disease is found alone or in association. In the presence of persistent or associated hygroma, NT>6 mm, hypertrophic cardiomyopathy, or any finding combined with polyhydramnios or renal anomaly, the likelihood of RASopathy diagnosis is significantly increased. Table 1 Prenatal ultrasound findings in 313 patients with a normal array-CGH Table 2 Diagnostic yield of prenatal ultrasound findings N.B. Diagnostic yield of each major US finding was compared with the global diagnostic yield for p -value’s calculation A. Scott: None. N. Di Giosaffatte: None. V. Pinna: None. P. Daniele: None. S. Corno: None. V. D’Ambrosio: None. E. Andreucci: None. A. Marozza: None. F. Sirchia: None. G. Tortora: None. D. Mangiameli: None. C. Di Marco: None. I. Donati: None. A. Zonta: None. E. Grosso: None. G. Mastromoro: None. P. Versacci: None. F. Pantaleoni: None. F.C. Radio: None. T. Mazza: None. L. Papi: None. T. Mattina: None. A. Giancotti: None. A. Pizzuti: None. A. Laberge: D. Speakers Bureau/Honoraria (speakers bureau, symposia, and expert witness); Modest; Sobi. M. Tartaglia: None. M. Delrue: None. A. De Luca: None. P01.107.B Gene ontology enrichment analysis of congenital renal agenesis-associated genes

S. Kalantari 1,2,3 , I. Filges 1,2,3

1 Medical Genetics, Institute of Medical Genetics and Pathology, University Hospital Basel, Basel, Switzerland, 2 Department of Clinical Research, University Hospital Basel, Basel, Switzerland, 3 University of Basel, Basel, Switzerland
Introduction: Uni- or bilateral renal agenesis (RA) is a commonly occurring major congenital anomaly impacting fetal and neonatal outcomes. Although chromosomal abnormalities, CNVs and pathogenic variants in many genes have been associated to RA, the etiology is still poorly understood. Our aim was to better understand the biological pathways involved in RA and to identify interacting candidate pathways useful for further gene identification and molecular diagnosis in clinical care when applying genome-wide sequencing. Materials and Methods: We used Phenolyzer to obtain all genes known as causative for syndromic and non-syndromic uni- and bilateral RA. Using ClueGO gene enrichment analysis we classified the relationship between these genes and biological processes as defined by gene ontology. Results: We identified 289 genes described to be involved in the pathogenesis of RA when mutated. We obtained 69 groups of enriched biological processes. About 50% included pathways directly related to the development of urogenital organ tissues (e.g. metanephros, nephric duct, ureter). Furthermore, several ciliary, axis specification, hindgut development and endocrine pathways are enriched, which may relate to different clinical presentations of RA. The results also support the hypothesis that RA is part of the ciliopathy spectrum and developmentally related to dysplastic kidneys. Conclusions: Our gene ontology analysis shows that genes representing distinct biological pathways are significantly enriched. They may relate to different clinical presentations of RA, which will lead to an improved delineation of candidate genes and molecular diagnosis of this congenital anomaly in clinical practice. S. Kalantari: None. I. Filges: None. P01.108.C Is mitochondrial DNA (mtDNA) copy number in human cleavage-stage embryos a marker of embryonic quality and development, or modified by the presence of a mtDNA mutation?

J. Steffann 1 , K. Chatzovoulou 1 , N. Gigarel 1 , A. Mayeur 2 , F. Jabot-Hanin 3 , S. Monnot 1 , L. Hesters 2 , N. Frydman 2 , J. Bonnefont 1

1 Hopital Necker-Enfants Malades, Paris, France, 2 Hopital Antoine-Béclère, Clamart, France, 3 Université de Paris, Paris, France
Mitochondria play a critical role during the early stages of human embryo development by supplying sufficient levels of energy required for embryo survival. Because mtDNA replication is silenced during early embryonic development, the presence of a large number of mtDNA copies in the mature oocyte is mandatory. In human, several studies on blastocysts day-5 embryos have paradoxically evidenced a positive correlation between high quantities of mtDNA molecules and severe conditions, like aneuploidy, poor embryo quality, and implantation failure. However, data from day-3 embryos are much more limited and contradictory, making this point a highly controversial one. We have quantified the mtDNA copy number (CN) in residual DNA of 1 or 2 cells, sampled from day-3 embryos, in a context of preimplantation genetic analysis for a non-metabolic, non-mitochondrial DNA disorder ( n = 165, control group) and for a mtDNA mutation ( n = 16, mitochondrial group). A great dispersion of the mtDNA CN was found, among the different cells of a given embryo, and the different embryos from a given patient. A maternal effect regulating the mtDNA CN was observed, but remained of unknown origin. In particular, no correlation was detected between mtDNA CN and maternal age or ovarian reserve. mtDNA CN did not correlate with embryonic quality and ability of an embryo to implant and give a viable pregnancy. Finally, the mtDNA CN was not influenced by the presence of a mtDNA mutation. Based on these findings, mtDNA CN at day 3 of development cannot be used to select the best embryo to transfer. J. Steffann: None. K. Chatzovoulou: None. N. Gigarel: None. A. Mayeur: None. F. Jabot-Hanin: None. S. Monnot: None. L. Hesters: None. N. Frydman: None. J. Bonnefont: None. P01.109.A A FAST AND NON-INVASIVE METHOD TO DETERMINE FETAL RHD GENE STATUS

B. Schreiner , E. Lindström, B. Vanherberghen, A. Hedrum

Devyser, Hägersten, Sweden
Background: Hemolytic disease of the fetus and newborn (HDFN) is a serious complication of maternal and fetal blood group incompatibility. This occurs when a Rhesus-D negative woman, carrying a Rhesus-D positive fetus during her first pregnancy, is alloimmunized against RhD antigen and carries another Rhesus-positive fetus during a subsequent pregnancy. Nowadays, HDFN is rare since anti-D prophylaxis is provided to all RhD negative pregnant women. However, 40% of these women carry a Rhesus-negative fetus and are administered unnecessary treatment including all risks connected to blood-based products. Aims: We set out to develop a fast and reliable diagnostic test assessing the fetal RHD gene status from maternal blood at the end of the first pregnancy trimester to enable targeted anti-D prophylaxis. Methods: A real-time PCR-based assay was developed to detect cell-free fetal DNA (cffDNA) in maternal blood. The assay contains primer-probe combination specific for the RHD gene and the GAPDH house-keeping gene as internal control. CffDNA from clinical samples was automatically extracted from 1mL of plasma. Results: Devyser RHD exhibits high experiment repeatability between different reagent lots. The analytical sensitivity is down to DNA concentrations corresponding to less than 7 gene copies per reaction with 95% probability. Summary/conclusions: Devyser RHD provides a fast, non-invasive procedure to assess the fetal RHD status in Rhesus-D negative mothers. This product is validated together with an automated DNA extraction process and reaction set-up (QIASymphony AS/SP), enabling high-throughput sample analysis with low-risk for sample mix-up. Various kit sizes are available offering flexibility with sample throughput. B. Schreiner: A. Employment (full or part-time); Significant; Devyser. E. Lindström: A. Employment (full or part-time); Significant; Devyser. B. Vanherberghen: A. Employment (full or part-time); Significant; Devyser. A. Hedrum: A. Employment (full or part-time); Significant; Devyser. P01.110.B Diagnosis of Rothmund Thomson Syndrome by whole genome sequencing

Q. Zhou , J. Sun, T. Wang, Y. Xing, Z. Wan, X. Yang, Z. Liu, W. Wang

Genomics Institute of Beijing, Shenzhen, China
Introduction: Rothmund-Thomson Syndrome (RTS) is a rare disorder and has been described in all ethnicities, with several identified pathogenic variants. Since there is fewer than hundreds of individuals have been described, the population prevalence is unknown. The genetic features and follow-up data of individuals with RTS are limited. Methods: We performed a whole genome sequencing (WGS) using cord blood for an infant, who had RTS like clinical features in infancy. Results: The infant showed a developmental delay in infancy, with a small size in both height and weight. The erythema started appearing on her face at the age of three months and then spread to the entire body. Besides, she had neither eyelashes nor eyebrows, with sparse scalp hair. After analyzing the WGS data, we found a heterozygous nonsense mutation c.2752G>T (p.Glu918*) and a novel frameshift-insertion c.1547_1548insC (p.Ala516fs) in the gene RECQL4 , which is a known pathogenic gene of RTS. Both variants had potential effects on protein synthesis or function. Further validation indicated these were compound variants inheriting from parents. Conclusions: Taking together with the clinical characteristics and the identification of biallelic pathogenic variants in a recessive manner, we report a diagnosed infant with RTS. Our findings provide potential application of WGS for newborn to clinical diagnosis. The follow-up study can help to further understand the clinical features and the genotype-phenotype interactions of RTS. Grant: The Shenzhen Municipal Government of China (NO.JCYJ20170412152854656) Q. Zhou: None. J. Sun: None. T. Wang: None. Y. Xing: None. Z. Wan: None. X. Yang: None. Z. Liu: None. W. Wang: None. P01.111.C Simpson-Golabi-Behmel syndrome familial case series with an emphasis on prenatal diagnosis

N. Krasovskaja 1,2 , A. Matulevičienė 1,2 , K. Šiaurytė 2,3 , K. Grigalionienė 1,2 , L. Ambrozaitytė 1,2 , A. Utkus 2

1 Vilnius University Hospital Santaros Klinikos, Vilnius, Lithuania, 2 Department of Human and Medical Genetics, Institute of Biomedical Sciences, Faculty of Medicine, Vilnius University, Vilnius, Lithuania, 3 Faculty of Medicine, Vilnius University, Vilnius, Lithuania
Introduction: Simpson-Golabi-Behmel syndrome (SGBS) is a rare recessively inherited X-linked condition, with less than 300 reported cases. Characteristic features of SGBS include pre/postnatal overgrowth, distinctive craniofacial features (macrocephaly, coarse face with macroglossia), visceromegaly and increased risk of neoplasia. This condition is almost always diagnosed postnatally. Materials and methods: A 23-y/o woman with uninformative anamnesis on the 21 weeks’ gestation was referred due to abnormal fetal ultrasound findings (polyhydramnios, macrocephaly, corpus callosum agenesis, abdominal visceromegaly and abnormal genitalia). The results of fetal DNA qPCR and SNP-CGH tests from amniotic fluid revealed no chromosomal abnormalities. The precise evaluation of mother’s phenotype (tall stature, coarse facial features, macroglossia) and fetal scan were key to directly sequence GPC3 gene and make the correct SGBS diagnosis prenatally. Results: Sanger sequencing from amniotic fluid revealed hemizygous pathogenic variant NM_004484.3:c.1159C>T, (NP_004475.1:p.(Arg387Ter), rs122453121, CM073110) in exon 4 of the GPC3 gene, which determines SGBS. Mother was also found to be a carrier. Despite severe polyhydramnios progression on 33 weeks’ gestation, condition of the fetus was manageable and, pregnancy was carried to 38th weeks. The newborn’s phenotype presented with classical SGBS features. Conclusions: An accurate evaluation of maternal phenotype and fetal scan are essential pathognomonic signs for early suspicion of SGBS. Successful prenatal diagnosis enables to make decisions on time for family and physicians about the outcomes of pregnancy and delivery. N. Krasovskaja: None. A. Matulevičienė: None. K. Šiaurytė: None. K. Grigalionienė: None. L. Ambrozaitytė: None. A. Utkus: None. P01.112.A Prenatal diagnosis of skeletal dysplasias/anomalies: high diagnostic yield using microarray and sequencing data

I. Mademont-Soler 1 , A. Maroto 1 , J. Meléndez 2 , A. Cherino 1 , J. Perapoch 1 , N. Artigas 1 , B. Campos 3 , M. Segura 3 , C. Pérez 4 , E. Lloveras 4 , J. Hernando 1 , X. Queralt 1 , M. Obón 1

1 Hospital Universitari de Girona Dr. Josep Trueta, Salt, Spain, 2 Hospital Santa Caterina, Salt, Spain, 3 Quantitative Genomic Medicine Laboratories, Barcelona, Spain, 4 Synlab, Barcelona, Spain
Introduction: Skeletal dysplasias (SDs) are severe congenital disorders, mostly of genetic origin. Etiology remains unknown in ~50% of cases. Materials and Methods: Nine pregnancies with ultrasound findings compatible with SDs were recruited prospectively from 2017 to 2019. A diagnostic algorithm combining microarray, Sanger and Next-Generation Sequencing was used. Results: Definite diagnosis was achieved in 6 cases, uncertain results in 2 and negative results in 1. Conclusions: A combination of molecular cytogenetics and sequencing offers a high diagnostic yield (6/9), facilitating genetic counselling and clinical management. An individualized approach, multidisciplinary teams and the contribution of new sequencing technologies to diagnosis are recognized. I. Mademont-Soler: None. A. Maroto: None. J. Meléndez: None. A. Cherino: None. J. Perapoch: None. N. Artigas: None. B. Campos: None. M. Segura: None. C. Pérez: None. E. Lloveras: None. J. Hernando: None. X. Queralt: None. M. Obón: None. P01.114.C Embryo haplotype phasing based on non-invasive procedures

L. A. Alcaraz 1 , C. Pérez-Pelegrín 1 , S. González-Reig 1 , P. Brígido 1 , P. Piqueras 2 , A. Martí 2 , F. Sánchez-Martín 2 , V. Penacho 1

1 Bioarray, SL, Elche, Spain, 2 Ginemed, Sevilla, Spain
Introduction: Preimplantation genetic testing (PGT) represents a widely applied genetic procedure for many IVF centers. Nowadays, for these purposes embryo material is needed. The removal of three cells in a day 5 embryo, represents an invasive procedure that could concern the embryo viability and increase the cost of a PGT. In the past few years there have been an increasing interest in the possibility of obtaining reliable DNA in the spent culture media (SCM) where the embryo is growing (1, 2). Materials and Methods: For optimal PGT-M results, we aim to compare embryo biopsy and SCM. After that, we want to go further: discern whether the SCM is even a better choice to diagnose an embryo in comparison with the trophectoderm biopsy, comparing a complete embryo. All based on next generation sequencing (NGS) with PGDSeq TM (Journey Genomics S.L.U.) technology. Results: We can confirm a high coincidence of SCM with complete embryo and the embryo biopsy. Tis primary results are promising, even if there are less polymorphisms obtained from this kind of material, we were able to study the haplotypes and the inheritance of the embryos. Conclusions: A non-invasive perspective on the PGT world is certainly to be the next generation of samples. Bibliography:1. Huang, L. et al. Noninvasive preimplantation genetic testing for aneuploidy in spent medium may be more reliable than trophectoderm biopsy. Proc. Natl. Acad. Sci. 116, 14105-14112 (2019).2. Li, P. et al . Preimplantation Genetic Screening with Spent Culture Medium/Blastocoel Fluid for in Vitro Fertilization. Sci. Rep . 8 , 9275 (2018). L.A. Alcaraz: None. C. Pérez-Pelegrín: None. S. González-Reig: None. P. Brígido: None. P. Piqueras: None. A. Martí: None. F. Sánchez-Martín: None. V. Penacho: None. P01.115.A Sperm protamine mRNA ratio in relation to DNA fragmentation and semen analysis

M. A. Ishchuk 1,2 , E. M. Komarova 2 , O. V. Malysheva 2 , I. D. Mekina 2 , E. A. Lesik 2 , A. M. Gzgzyan 2,1 , V. S. Baranov 2,1 , I. Y. Kogan 2

1 St. Petersburg State University, Saint-Petersburg, Russian Federation, 2 The Research Institute of Obstetrics, Gynecology and Reproductology named after D.O.Ott, Saint-Petersburg, Russian Federation
Introduction: Sperm contain a complex population of RNA, and the utility of sperm RNA in fertility research are currently being explored. During mammalian spermatogenesis, the chromatin structure undergoes substantial condensation. The key role in this process is played by protamines 1 and 2 (PRM1, PRM2). Abnormal condensation could lead to DNA breaks, which is linked to pregnancy loss after assisted fertilization, disturbances in embryo development and increased risk of abortions. The aim of this study was to estimate the relations between protamine mRNA ration and semen quality and further DNA fragmentation. Finding sperm-quality markers would help to understand the causes of male infertility and to improve the male reproductive health. Materials and Methods: Human ejaculates were obtained from 33 patients and semen analyses were assessed by WHO criteria (2010). We evaluated the sperm DNA fragmentation measured by TUNEL assay. Patient’s ejaculates were purified by density-gradient centrifugation, sperm cells were lysed, mRNA extracted, reverse transcribed and subjected to real-time qPCR using specific primer pairs for protamine-1 and protamine-2. Results: The sperm protamine mRNA ratio in normozoospermic men ( n = 19; 2,86 ± 0,67) differed significantly from that of patozoospermic patients ( n = 14; 3,43 ± 0,79; p<0,05). A significant correlation was shown between sperm DNA fragmentation and the PRM2/PRM1 mRNA ratio (r=0,33; p <0,05). In the group of patients with an increased sperm DNA fragmentation ( n = 14; 3,26 ± 0,19) the PRM2/PRM1 ratio was significantly higher than in the group of patients with normal rates ( n = 19; 2,74 ± 0,18; p < 0,05). Conclusions: Abnormal sperm protamine ratio associated with poor semen quality and DNA fragmentation. M.A. Ishchuk: None. E.M. Komarova: None. O.V. Malysheva: None. I.D. Mekina: None. E.A. Lesik: None. A.M. Gzgzyan: None. V.S. Baranov: None. I.Y. Kogan: None. P01.116.B Investigating PHF13 induced infertility through single cell transcriptomics and non-vertebrate model organisms

E. Bosch 1 , S. Lukassen 2 , A. Gregor 1 , A. B. Ekici 1 , C. Zweier 1 , A. Winterpacht 1

1 Institute of Human Genetics, FAU Erlangen-Nürnberg, Erlangen, Germany, 2 Digital Health Center, Berlin Institute of Health (BIH) and Charité, Berlin, Germany
Infertility is a common medical problem worldwide. Of all infertility cases, approximately 40 - 50% are due to “male factor” infertility, mostly caused by spermatogenic failure. Despite extensive research, there is still limited knowledge of the genetic causes and underlying molecular mechanisms. We previously showed that deficiency for the epigenetic reader PHF13 in the mouse results in testis hypoplasia due to a spermatogenic defect. Here, we obtained single-cell RNA expression profiles from Phf13 −/− and Phf13 +/- testis and compared them with wild type data. The results revealed a loss of meiotic sex chromosome inactivation (MSCI) in pachytene spermatocytes of PHF13 deficient mice. This is remarkable, since canonical PHF13 is only expressed in spermatogonia. In combination with ChIPseq data demonstrating an enrichment of PHF13 binding sites on the X- and Y-chromosome, we hypothesize that PHF13 mediates epigenetic changes on the sex chromosomes in spermatogonia, rendering the chromosomes competent for later inactivation. PHF13 is evolutionarily conserved down to Bilateria including sea urchin, acorn worm and several insects like the flour beetle ( Tribolium ), but strikingly not in the evolutionary “young” genus Drosophila . Consequently, we ubiquitously overexpressed the Tribolium Phf13- homolog in D. melanogaster and compared these lines with wild type lines and lines overexpressing neuronal control genes. Phf13 -overexpressing strains but not the control strains show a high grade of infertility in seemingly normal flies. This strongly indicates a conserved and very specific function of this protein in germ cell development, making PHF13 an interesting infertility factor and a fundamental player in germ cell differentiation. E. Bosch: None. S. Lukassen: None. A. Gregor: None. A.B. Ekici: None. C. Zweier: None. A. Winterpacht: None. P01.118.A Telomere length in karyotypically normal and abnormal chorion in progressing pregnancies and embryonic losses

A. V. Tikhonov 1 , M. I. Krapivin 1 , A. V. Petrovskaia-Kaminskaia 1 , A. A. Smirnova 2 , Y. M. Sagurova 3 , O. A. Efimova 1 , A. A. Pendina 1 , O. G. Chiryaeva 1 , L. I. Petrova 1 , V. S. Dudkina 1 , V. S. Baranov 1,3

1 D.O. Ott Research Institute of Obstetrics, Gynecology and Reproductology, St. Petersburg, Russian Federation, 2 St. Petersburg State Pediatric Medical University, St. Petersburg, Russian Federation, 3 St. Petersburg State University, St. Petersburg, Russian Federation
Telomere length (TL) is considered to be an indicator of biological age. Its correct regulation during pregnancy is crucial for embryonic development. The possible contribution of TL to the developmental capacity of embryos with abnormal karyotype remains unknown so far. Here, we studied TL in karyotypically normal and abnormal chorion in progressing pregnancies and embryonic losses. The chorionic villus samples were obtained by chorion biopsy from 15 patients with progressing first trimester pregnancy referred to invasive prenatal diagnosis and by curettage from 15 patients with missed abortion. The karyotypically abnormal samples included trisomy 21 and trisomy 16 cases in both progressing pregnancies ( n = 7) and missed abortion ( n = 7). The telomeric regions were detected on direct metaphase preparations by qFISH. Relative TLs were measured by dividing telomeric by subtelomeric fluorescence intensity assessed in ImageJ1.49v. The results were compared by the Mann-Whitney U-test. In progressing pregnancies, the relative TL was higher in karyotypically abnormal samples compared to karyotypically normal ones (p < 0.0001). In missed abortions, we observed the same tendency although the difference did not reach statistical significance (p=0.4652). When comparing the relative TL between the samples with normal karyotype in progressing pregnancies and missed abortions, we revealed no difference (p=0.8890). However, the relative TL was higher in the samples with abnormal karyotype in progressing pregnancies compared to those in missed abortions (p=0.0013). Our results suggest that in the case of karyotype abnormality, longer telomeres in chorionic villi may contribute to pregnancy success. Supported by RSF №19-75-00023. A.V. Tikhonov: None. M.I. Krapivin: None. A.V. Petrovskaia-Kaminskaia: None. A.A. Smirnova: None. Y.M. Sagurova: None. O.A. Efimova: None. A.A. Pendina: None. O.G. Chiryaeva: None. L.I. Petrova: None. V.S. Dudkina: None. V.S. Baranov: None. P01.120.C Telomere length of maternal and paternal chromosomes in human triploid zygotes

M. I. Krapivin 1 , A. A. Pendina 1 , O. A. Efimova 1 , I. D. Mekina 1 , E. M. Komarova 1 , Y. M. Sagurova 2 , I. V. Mayorova 2 , A. M. Gzgzyan 1 , I. Y. Kogan 1 , V. S. Baranov 1,2

1 D.O. Ott Research Institute of Obstetrics, Gynecology and Reproductology, St. Petersburg, Russian Federation, 2 Saint Petersburg State University, St. Petersburg, Russian Federation
Telomeres are complex structures of tandem DNA repeats and proteins at chromosome ends. Telomere shortening due to cell divisions, oxidative stress or genetic effects may result in end-to-end chromosome joining, their degradation, and cell death. In contrast to short telomeres in oocytes, sperm telomeres become longer with age. Telomere length (TL) correlates with embryo developmental potential advocating the importance of TL analysis after fertilization. The study was performed on metaphase chromosomes of 23 triploid human zygotes from 20 couples (women aged 32.04 ± 0.8; men aged 34.15 ± 1.2) referred for in vitro fertilization. Paternal (sperm-derived) chromosomes were identified immunocytochemically by weak DNA methylation and strong hydroxymethylation which contrasted them with strongly methylated and poorly hydroxymethylated maternal (oocyte-derived) chromosomes. After qFISH, relative TL was measured by dividing telomeric by subtelomeric fluorescence assessed in ImageJ1.49v. The relative TL was significantly higher in the paternal compared to the maternal chromosomes (Wilcoxon signed-rank test, р < 0.0001). Rare zygotes demonstrated the opposite pattern: higher TL in the maternal chromosomes. However, they seem to be the exception rather than the rule suggesting that some cases of developmental arrest may be caused by altered TL pattern. Neither in sperm- nor in oocyte-derived chromosomes the relative TL correlated with paternal (r=-0.058;р=0.736) or maternal age (r=0.155;р=0.44), respectively. Thus, after fertilization, TL in paternal chromosomes is “reprogrammed”. Being hypomethylated, paternal chromosomes are prone to recombination and, thus, to alternative lengthening of telomeres, which may be part of developmental program and explains the absence of correlation of TL in sperm-derived chromosomes with paternal age. Supported by RSF№18-75-10046. M.I. Krapivin: None. A.A. Pendina: None. O.A. Efimova: None. I.D. Mekina: None. E.M. Komarova: None. Y.M. Sagurova: None. I.V. Mayorova: None. A.M. Gzgzyan: None. I.Y. Kogan: None. V.S. Baranov: None. P01.121.A Transcriptomics of placental tissue: identification of potential biomarkers and biological pathways in the great obstetrical syndromes

E. Trifonova 1,2 , A. Markov 1 , A. Zarubin 1 , T. Gabidulina 2 , V. Stepanov 1

1 Research Institute of Medical Genetics, Tomsk National Research Medical Center of the Russian Academy of Sciences, Tomsk, Russian Federation, 2 Siberian State Medical University, Tomsk, Russian Federation
The great obstetrical syndromes (GOS) are common pregnancy complications that can have detrimental effects on morbidity and mortality of the mother and fetus as well as long-term health outcomes. Although they are distinct conditions, they are often considered together as they share a common etiology of inadequate placental perfusion. The aim of the work was to search for new genetic markers of GOS on the basis of integrative analysis of transcriptomic data. Genome-wide expression profiling was performed on placental tissue from preeclamptic and normal ( n = 47) pregnancies. Thirty original datasets from patients with preterm birth, intrauterine growth restriction, preeclampsia ( n = 434) were downloaded from Gene Expression Omnibus and were further integrated and analyzed with our data. Subsequently, 5023 differentially expressed genes (DEGs) between GOS patients and healthy women were identified. We found that the transcriptional activity of 64 DEGs changes in at least two GOS diseases. Weighted gene co-expression network analysis identified distinct gene modules associated with preterm birth, intrauterine growth restriction or preeclampsia. Using Gene Set Enrichment Analysis we identified the significant role of disturbance of intercellular interactions and regulation of proteins modification in placental tissue during the development of the GOS. We applied upstream analysis approach implemented in geneXplain platform and identified master regulators ( MECOM, CDC34, UBE2E1, UBE2D4, STAT3 and UBE2R2 ) that are new therapeutic targets. These key genes may be potential biomarkers of diagnosis, therapy and prognosis for GOS. This work was supported by the Russian Foundation for Basic Research (grant No. 18-29-13045, No. 18-44-700007). E. Trifonova: None. A. Markov: None. A. Zarubin: None. T. Gabidulina: None. V. Stepanov: None. P01.123.C The importance of trio WES integration into prenatal diagnosis

C. Rodriguez-Solera 1 , M. Martinez-Garcia 1 , I. Diez 1 , I. Sanchez-Navarro 1 , E. Barroso 1 , D. Rodriguez 1 , P. Maietta 1 , G. Martin-Serrano 1 , S. Izquierdo 2 , M. Miramar 2 , A. Rodriguez 2 , F. Diaz-Flores 3 , M. Zeleniuc 4 , L. Martorell 5 , J. Bermejo 6 , C. Toledo 7 , S. Lopez 8 , M. Sanchez 8 , S. Alvarez 1

1 NIMGenetics S.L, Madrid, Spain, 2 Hospital Universitario Miguel Servet, Zaragoza, Spain, 3 Hospital Universitario de Canarias, Tenerife, Spain, 4 Personal Genetics, Bucharest, Romania, 5 Hospital Sant Joan de Déu, Barcelona, Spain, 6 Hospital General de Segovia, Segovia, Spain, 7 Hospital Virgen de la Salud, Toledo, Spain, 8 Hospital Clínico de Santiago, Santiago de Compostela, Spain
Background : Exome sequencing has proved to be a useful diagnostic tool when fetal structural anomalies suggest a genetic etiology, but other standard prenatal genetic tests did not provide a diagnosis. We describe a single laboratory experience with prenatal and fetal samples. Material and Methods : This study includes 13 samples with ultrasound images suggesting skeletal abnormalities or rasopathies, among others. The samples origin were amniotic fluid (69%) and fetal tissues (31%). In six cases a whole exome sequencing with a trio analysis was performed. The genomic libraries were generated using SureSelectXT and sequenced on a Novaseq 6000. Data alignment, calling and filtering was performed using an in-house developed pipeline. Informed consent was obtained from all participants. Results : A molecular diagnosis was obtained on 1 amniotic fluid prenatal sample and on 1 fetal specimen (15% global diagnostic yield). All positive cases were obtained after trio analysis. Two pathogenic variants in the ESCO2 gene associated with Roberts syndrome were identified in the prenatal sample and one homozygous variant in the ASPM gene, associated with a primary microcephaly 5, was identified in fetal tissue. Both variants were cosegregated with the disease. Ultrasound studies did not suggest the candidate genes due to the insufficient and nonspecific information about the prenatal clinical presentation of these syndromes. Conclusion : This study highlights the importance of trio WES integration into prenatal diagnosis. Compared to ultrasound studies alone, prenatal molecular diagnoses led to a refined recurrence risk estimation, altered medical management and allows an informed reproductive planning for families. C. Rodriguez-Solera: None. M. Martinez-Garcia: None. I. Diez: None. I. Sanchez-Navarro: None. E. Barroso: None. D. Rodriguez: None. P. Maietta: None. G. Martin-Serrano: None. S. Izquierdo: None. M. Miramar: None. A. Rodriguez: None. F. Diaz-Flores: None. M. Zeleniuc: None. L. Martorell: None. J. Bermejo: None. C. Toledo: None. S. Lopez: None. M. Sanchez: None. S. Alvarez: None. P01.125.B Pregnancy outcome after failure of emergency contraception with ulipristal acetate - an observational study

M. Hoeltzenbein , J. K. Wagner, C. Schaefer, K. Dathe

Pharmakovigilanzzentrum Embryonaltoxikologie, Institut für Klinische Pharmakologie und Toxikologie, Charité-Universitätsmedizin Berlin, Berlin, Germany
Introduction: Ulipristal acetate (UPA) is a selective progesterone receptor modulator used for emergency contraception (EC) after unprotected sexual intercourse. Data on pregnancy outcome after failure of EC is very limited. Material and Methods: Observational study of prospectively ascertained pregnancies after UPA exposure for EC. In addition, retrospectively reported pregnancy outcomes were evaluated separately. Results: Among 216 requests to the German Embryotox institute related to EC with UPA, 95 concerned prospectively ascertained pregnancies after failed EC. Follow-up data on pregnancy outcome was obtained in 56 pregnancies: 7 spontaneous abortions, 12 terminations of pregnancy (ETOP) and 37 live births. Major birth defects were not reported. Only 34% of women had taken UPA during the fertile window. Among the 4 retrospectively reported pregnancies there was one child diagnosed with Beckwith-Wiedemann-syndrome (BWS) due to hypomethylation of IC2. Conclusions: We provide a preliminary basis for reassuring women who wish to carry their pregnancy to term after failed EC with UPA. However, due to the retrospective case report of a child with BWS after maternal UPA exposure possible epigenetic effects could not be excluded and require further evaluation. As ovarian stimulation alone (independent of artificial reproductive techniques) was previously discussed as a predisposing factor for disturbance of imprinting maintenance, UPA might have influenced preimplantation development and disturbed epigenetic processes. This work was performed with financial support from the German Federal Institute for Drugs and Medical Devices (BfArM). M. Hoeltzenbein: None. J.K. Wagner: None. C. Schaefer: None. K. Dathe: None. P01.126.C Chromosomal microarray confirms a rare prenatal case of WAGR syndrome

M. Shetty , P. Venkatesh, S. Menezes, S. Hegde

Manipal Hospital, Bangalore, India
Introduction: WAGR syndrome is a rare genetic disorder with prevalence of 1 in 500,000 to 1 million. It affects many body systems and is named for its main features: wilms tumor, aniridia, genitourinary anomalies and range of developmental delays. Children with WAGR syndrome present with aniridia, genito-urinary defects in the new born period and are at an increased risk for developing malignancies, varying range of intellectual disability and obesity in childhood. The syndrome is a contiguous gene deletion syndrome involving chromosome 11p13 containing WT1 and PAX6 genes. Materials and Methods: We report a case of 29-year-old Asian (Indian ethnicity) primigravida with non-consanguineous marriage. Her mid trimester anomaly scan at 18 weeks gestation showed bilateral ventriculomegaly, small cavum septum pellucidum and partial agenesis of corpus callosum. Amniocentesis was performed for chromosomal microarray analysis which was performed using an Agilent 8x60K array. The cytogenomic microarray analysis showed 4.9MB deletion in chromosome 11 (11p13-p12). Discussion: The prenatal diagnosis of WAGR syndrome is associated with borderline ventriculomegaly, absent corpus callosum and absent cavum septum pellucidum. In order to diagnose rare syndrome, microarray should be the first choice of investigation in the presence of any structural abnormality. This would help in understanding the prenatal presentation, potential co-morbid condition, counseling and accordingly decision making. Conclusions: The clinical association of WAGR syndrome in postnatal period is well established but the antenatal presentation is not well-understood and reported twice in the literature earlier. Microarray study confirms these rare non-classical prenatal manifestations of WAGR syndrome. M. Shetty: None. P. Venkatesh: None. S. Menezes: None. S. Hegde: None. P01.127.A Whole Exome Sequencing in 53 fetuses with abnormal ultrasound: 37% of diagnostic yield

D. Trost 1 , A. Boughalem 1 , P. Blanchet 2 , C. Cenni 2 , R. Dard 3 , A. Benachi 4 , V. Ciorna-Monferrato 5 , R. Touraine 6 , P. Kleinfinger 1 , L. Lohmann 1 , A. Luscan 1 , M. Valduga 1 , J. Costa 1

1 Laboratoire Cerba, Saint Ouen l’Aumone, France, 2 Hôpital Arnaud de Villeneuve, Montpellier, France, 3 Hôpital Poissy, Poissy, France, 4 Hôpital Antoine Béclère, Clamart, France, 5 Hôpital Metz-Thionville, Metz, France, 6 Hôpital Nord, Saint Etienne, France
Introduction: Fetal structural abnormalities detected by ultrasonography, have a range of genetic causes. Chromosomal aneuploidy and structural abnormalities are explored by karyotype testing and chromosomal microarray (CGH). The utility of prenatal whole exome sequencing (WES) is discussed. Information on the interest of prenatal WES remains limited. Methods and results: In our cohort of 53 fetuses with normal karyotype and CGH, Trio WES was performed. All fetuses had ultrasound abnormalities of poor prognosis. Most fetuses (26) were analyzed after medical termination of pregnancy, 24 analyzes were performed on ongoing pregnancies and 3 analyzes after intrauterine fetal demise. Pathogenic or likely pathogenic variants were found in 20 cases resulting in a diagnostic yield of 37%. Of these, 55% (11) were autosomal dominant with de novo variants, 45% of the diagnoses followed an autosomal recessive inheritance pattern (5 homozygous variants, 4 compound heterozygous variants). In urgent cases turn-around-time was 2 weeks. Reporting results for an antenatal Trio WES varies between 2-4 weeks. Conclusions: Whole exome sequencing identified a genetic cause for the fetal anomalies in 20 of the 53 (37%) cases analyzed. This made it possible to carry out genetic counseling for families and to offer targeted prenatal diagnosis for recessive diseases in case of a new pregnancy. D. Trost: None. A. Boughalem: None. P. Blanchet: None. C. Cenni: None. R. Dard: None. A. Benachi: None. V. Ciorna-Monferrato: None. R. Touraine: None. P. Kleinfinger: None. L. Lohmann: None. A. Luscan: None. M. Valduga: None. J. Costa: None.

P02 Sensory Disorders (Eye, Ear, Pain)
P02.01.C Disruption of PAX6 translation by 5′ UTR variants causes Congenital aniridia

A. Filatova , T. Vasilyeva, A. Marakhonov, R. Zinchenko, M. Skoblov

Research Centre for Medical Genetics, Moscow, Russian Federation
Introduction: Congenital aniridia (AN) is a rare autosomal dominant panocular disorder caused by mutations in the PAX6 gene. Previously, we performed a molecular genetic study of a large cohort of Russian patients with AN and found six PAX6 nucleotide variants in 5ʹ-UTR. It is known that 5′-UTR variants could influence the translation efficiency and cause some diseases, but for AN such a mechanism has not been previously shown. Thus, to investigate the pathogenicity of 5′-UTR PAX6 variants the functional studies are required. Materials and Methods: To study 5ʹ-UTR variants we generated luciferase constructs with full-length PAX6 5ʹ-UTR (wt and mutants). The translation efficiency was measured by luciferase dual-assay, mRNA structure and expression level were determined by RT-PCR and qPCR respectively. Results: Six patient-derived 5ʹ-UTR-variants were found to lead to a significant decrease in the translation efficiency, while mRNA expression level was not significantly reduced. Three of these variants also affect splicing. Further bioinformatics analysis allows us to suggest the mechanism of 5ʹ-UTR variants pathogenicity through disruption of an upstream ORF which possibly exists in PAX6 5ʹ-UTR. To validate this hypothesis, we performed additional mutagenesis experiments and confirmed the essential role of uORF in the molecular pathogenesis of the disease. Moreover, we showed that this uORF normally affects the main protein translation. Conclusions: We have identified uORF in 5′-UTR of the PAX6 gene and found that mutations that disrupt this uORF cause the congenital aniridia. In addition, we hypothesized that this uORF may participate in the normal regulation of PAX6 expression. A. Filatova: None. T. Vasilyeva: None. A. Marakhonov: None. R. Zinchenko: None. M. Skoblov: None. P02.02.A Evaluating diagnostic challenges with ABCA4 -related retinal disease - experience with a 7500 IRD patient cohort sent for genetic diagnostics

J. Känsäkoski 1 , K. Kämpjärvi 1 , S. Tuupanen 1 , K. Wells 1 , L. Sarantaus 1 , P. von Nandelstadh 1 , K. Merkkiniemi 1 , H. Västinsalo 1 , E. Mårtensson 1 , R. Perez Carro 1 , E. Sankila 2 , J. W. Koskenvuo 1 , S. Myllykangas 1 , T. Alastalo 1

1 Blueprint Genetics, Espoo, Finland, 2 Helsinki University Eye Hospital, Helsinki, Finland
Introduction: ABCA4 variants cause autosomal recessive Stargardt disease (STGD1), the most prevalent hereditary macular disease. Major challenges in STGD1 molecular diagnostics include (i) non-coding variants typically not targeted by available genetic tests (ii) copy number variants (CNV) missed by standard testing, and (iii) interpretation challenges with common hypomorphic variants. We evaluated the significance of challenging ABCA4 variants and their contribution to STGD1 diagnostic yield in a cohort of inherited retinal disease (IRD) patients. Materials and Methods: We included 7500 IRD patients tested at a CLIA laboratory between 2017 and 2019. Sequencing analysis, including established non-coding ABCA4 variants and high resolution CNV detection, was done by using in-house developed and validated NGS platform. Results: Two disease-causing ABCA4 variants were identified in 663 patients, of which 20 (20/663; 3%) had an established non-coding variant and 11 (11/663; 1.5%) a CNV. In 192 patients with clinically diagnosed ABCA4 -related disease, only one disease-causing variant was identified. Among those, the hypomorphic allele ABCA4 c.5603A>T, p.(Asn1868Ile) was identified in 109 patients (109/192: 57%), with a significantly increased allele frequency (126/384=0.328) compared to controls in gnomAD (AF 11928/282712=0.042) (OR: 11.087, 95% CI: 8.953-13.73, p < 0.001). The median age at genetic testing among the 109 patients was 56 years, which was significantly higher compared to 37 years among the 663 patients with two disease-causing variants. Conclusions: Non-coding variants and CNVs contribute significantly to diagnostic yield in ABCA4 -related disease. Our large dataset supports the important role of the hypomorphic allele ABCA4 c.5603A>T, p.(Asn1868Ile) in diagnostics of late-onset STGD1. J. Känsäkoski: A. Employment (full or part-time); Significant; Blueprint Genetics. K. Kämpjärvi: A. Employment (full or part-time); Significant; Blueprint Genetics. S. Tuupanen: A. Employment (full or part-time); Significant; Blueprint Genetics. K. Wells: A. Employment (full or part-time); Significant; Blueprint Genetics. L. Sarantaus: A. Employment (full or part-time); Significant; Blueprint Genetics. P. von Nandelstadh: A. Employment (full or part-time); Significant; Blueprint Genetics. K. Merkkiniemi: A. Employment (full or part-time); Significant; Blueprint Genetics. H. Västinsalo: A. Employment (full or part-time); Significant; Blueprint Genetics. E. Mårtensson: A. Employment (full or part-time); Significant; Blueprint Genetics. R. Perez Carro: A. Employment (full or part-time); Significant; Blueprint Genetics. E. Sankila: A. Employment (full or part-time); Modest; Blueprint Genetics. J.W. Koskenvuo: A. Employment (full or part-time); Significant; Blueprint Genetics. E. Ownership Interest (stock, stock options, patent or other intellectual property); Significant; Blueprint Genetics. S. Myllykangas: A. Employment (full or part-time); Significant; Blueprint Genetics. E. Ownership Interest (stock, stock options, patent or other intellectual property); Significant; Blueprint Genetics. T. Alastalo: A. Employment (full or part-time); Significant; Blueprint Genetics. E. Ownership Interest (stock, stock options, patent or other intellectual property); Significant; Blueprint Genetics. P02.03.B A novel splicing variant confirms COL11A1 as a cause of autosomal dominant non-syndromic hearing loss in the DFNA37 locus

B. Vona 1 , A. Tropitzsch 1 , A. Rad 1 , F. Schneider 1 , M. Müller 1 , T. Schade-Mann 1 , S. Biskup 2 , H. Löwenheim 1

1 Dept of Otolaryngology—Head & Neck Surgery, Tuebingen Hearing Research Centre, University of Tuebingen, Tuebingen, Germany, 2 CeGaT GmbH and Praxis für Humangenetik Tuebingen, Tuebingen, Germany
Introduction: Hereditary hearing loss (HL) is a clinically and genetically heterogeneous disorder. Many genes exhibiting either non-syndromic or syndromic HL have also been identified as causing both, owing to extensive phenotypic manifestations in the form of pleiotropy. The gene COL11A1 has long been associated with autosomal dominant Marshall syndrome and Stickler syndrome type II, as well as autosomal recessive fibrochondrogenesis. Each of these syndromes have overlapping phenotypes including skeletal abnormalities, dysmorphic features, variable cleft palate, ocular and auditory phenotypes that can include mild-to-moderate HL, and outer ear malformations. Recently, COL11A1 has been associated with autosomal dominant non-syndromic HL (DFNA37) through the genetic analysis of a large European-American family presenting a novel splice-site altering variant. Materials and Methods: A proband from a four generation family with non-syndromic HL was ascertained. DNA from the proband was subjected to a custom-designed high-throughput sequencing panel consisting of 160 HL-associated genes. An in vitro splice assay was performed to study the RNA-level effect of the variant. Results: The patient was diagnosed with moderate sensorineural HL. Additional syndromic features were absent. A heterozygous variant (c.4338+2T>C, p.?, NM_080629.2) was uncovered in COL11A1 that is predicted to abolish the 5′ splice site in exon 58 out of the 68 exons comprising COL11A1 . Furthermore, this novel variant affects a highly conserved nucleotide in the alpha chain domain. In vitro splice testing confirmed abnormal splicing. Conclusions: We report on the second DFNA37-associated splice-altering variant, providing confirmatory evidence of COL11A1 as a bona fide autosomal dominant non-syndromic HL gene. B. Vona: None. A. Tropitzsch: None. A. Rad: None. F. Schneider: None. M. Müller: None. T. Schade-Mann: None. S. Biskup: None. H. Löwenheim: None. P02.05.A Non-Mendelian inheritance in Bardet-Biedl syndrome: triallelism

I. Perea-Romero 1,2 , F. Blanco-Kelly 1,2 , I. Lorda-Sanchez 1 , I. Sanchez-Navarro 3 , O. Zurita 1,2 , R. Riveiro-Alvarez 1,2 , M. Trujillo-Tiebas 1,2 , A. Avila-Fernandez 1,2 , M. Corton 1,2 , D. Valverde 4,5 , C. Ayuso 1,2

1 Department of Genetics, Instituto de Investigación Sanitaria – Fundación Jiménez Díaz University Hospital – Universidad Autónoma de Madrid (IIS-FJD, UAM), Madrid, Spain, 2 Center for Biomedical Network Research on Rare Diseases (CIBERER), ISCIII, Madrid, Spain, 3 Medical Department, NIMGenetics, Madrid, Spain, 4 Department of Biochemistry, Genetics and Immunology, Biology Faculty, Vigo University, Pontevedra, Spain, 5 Center for Biomedical Network Research on Rare Diseases (CIBERER), ISCIII, Pontevedra, Spain
Introduction: Bardet-Biedl syndrome (BBS) is a ciliopathy characterized by retinal dystrophy, obesity, polydactyly, cognitive impairment, renal failure and hypogonadism. Typically, it has an autosomal recessive mode of inheritance, but this heterogeneous disease has variable expressivity between and within families, which might be partially explained by oligogenic inheritance, like triallelism. Materials and methods: A total of 61 families (78 affected cases) with an “a priori” diagnosis of BBS or BBS-like were studied using different molecular approaches (commercial genotyping microarrays, Sanger sequencing of specific genes and/or NGS technologies). Results: The characterization rate in our cohort was 79% (48/61 families). In 5 of the families, we found a possible triallelic inheritance, involving 6 out of the 7 affected cases. Three families carried biallelic BBS1 variants together with one allele in MKKS , one family with biallelic MKKS variants and one allele in BBS5 and one additional family carried biallelic C8orf37 variants and one allele in WDPCP . The MKKS (c.724G>T; p.Ala242Ser) variant was found to be part in 3 families accompanying different biallelic variants in BBS1 . In one family, the triallelic case showed a more severe phenotype than the biallelic affected sibling. However, the phenotypic modifier effect of the triallelism could not be stablished in 4 of the families, as they were either unique cases or both affected showed triallelism. Conclusions: Our study presents triallelism as an option in BBS diagnosis, being important to consider it in genetic counselling. Funding: FIS (PI16/00425 and PI19/00321), University Chair UAM-IIS-FJD Genomic Medicine and FEDER (European Regional Development Fund) I. Perea-Romero: None. F. Blanco-Kelly: None. I. Lorda-Sanchez: None. I. Sanchez-Navarro: None. O. Zurita: None. R. Riveiro-Alvarez: None. M. Trujillo-Tiebas: None. A. Avila-Fernandez: None. M. Corton: None. D. Valverde: None. C. Ayuso: None. P02.07.C Molecular genetic cause of non-syndromic congenital and juvenile cataracts in the Czech population

J. Moravikova 1 , L. Dudakova 1 , M. Schwarz 2 , L. Hlavata 1 , F. Malinka 1,3 , P. Liskova 1,4

1 Department of Paediatrics and Adolescent Medicine, First Faculty of Medicine, Charles University and General University Hospital in Prague, Prague, Czech Republic, 2 Department of Biology and Medical Genetics, Second Faculty of Medicine, Charles University and University Hospital Motol, Prague, Czech Republic, 3 Department of Computer Science, Czech Technical University in Prague, Prague, Czech Republic, 4 Department of Ophthalmology, First Faculty of Medicine, Charles University and General University Hospital in Prague, Prague, Czech Republic
Introduction: Congenital and juvenile cataracts are phenotypically and genetically very heterogeneous group. In this study, we aimed to investigate the molecular genetic cause in Czech patients with bilateral congenital or juvenile cataracts not associated with other ocular or systemic clinical findings. Materials and Methods: Whole exome sequencing was performed in 10 probands of Czech origin. Variants with minor allele frequency less than 0.005 as per GnomAD v.2 were filtered and those located in genes known to be associated with cataract development as per Cat-Map database were given a priority for further evaluation. Conventional sequencing was used to follow segregation of the presumably pathogenic variants in available first degree relatives. Results: Out of the 10 probands 6 had a family history of congenital or infantile cataracts. In total 5 variants classified as pathogenic or likely pathogenic as per American College of Medical Genetics and Genomics guidelines were identified. Two variants were novel; c.299T>A in GJA3 (NM_021954) and c.2665G>A in EPHA2 (NM_001329090). No pathogenic variants were found in 5 probands. Conclusions: Herein we report for the first time mutational spectrum of non-syndromic cataracts in Czech patient population. The failure to identify disease-causing variants in half of the analysed cases suggests, that either the underlying mechanisms are not of genetic origin and/or that the pathogenic changes are not located in coding regions of the genome. Only two probands with family history of cataracts remained unsolved. Alternatively, larger structural variants or rearrangement could be also implicated. Supported by AZV 17-30500A. J. Moravikova: None. L. Dudakova: None. M. Schwarz: None. L. Hlavata: None. F. Malinka: None. P. Liskova: None. P02.08.A Insights into the retinal function of ciliopathy proteins gained through zebrafish models

M. Masek, R. Bachmann-Gagescu

University of Zurich- Medical Genetics, Zürich, Switzerland
Ciliopathies are disorders caused by dysfunction of primary cilia, ubiquitous organelles involved in signal transduction. In retinal photoreceptors (PRs), light sensation occurs in the outer segments (OSs), which represent highly specialized ciliary compartments. Consequently, retinal disease is a common manifestation in ciliopathy patients. Our efforts to elucidate the molecular functions of ciliopathy proteins rely on zebrafish mutants in genes associated with Joubert syndrome, an iconic ciliopathy associated with retinal disease in 25% of patients. We focus on genes that encode proteins participating in distinct ciliary complexes: Cc2d2a at the transition zone, representing the gate to the cilium, or Talpid3/KIAA0586 at the basal body (BB), anchoring the cilium in the cell. While both mutants display decreased visual function, we identify distinct ultra-structural phenotypes indicating specific roles for each protein in primary cilium function. We find that Cc2d2a organizes the vesicle fusion machinery at the periciliary margin and provides a docking point for incoming vesicles through a chain of interactions linking CC2D2A to NINL-MICAL3-RAB8. In contrast, Talpid3 is involved in BB docking and ciliogenesis upstream of Rab8a activation. Retina-specific transcriptomics identifies both shared and distinct effects on gene transcription: while transcription of phototransduction genes is decreased in both mutants, likely as a secondary effect, we observe downregulation of the BBSome, a multi-protein complex associated with the ciliopathy Bardet-Biedl syndrome, only in talpid3 mutants, while Nek8 is upregulated only in cc2d2a mutants. In conclusion, our findings demonstrate how mutations in genes causing the same human disorder result in similar phenotypes through distinct mechanisms. M. Masek: None. R. Bachmann-Gagescu: None. P02.09.B Analysis of copy number variants in a large cohort of individuals with anophthalmia, microphthalmia and coloboma

F. Ceroni 1 , D. A. Bax 1 , R. J. Holt 1 , Y. Kesim 1 , A. Cole 2 , C. Ogilvie 3 , D. Bunyan 4,5 , L. Cooper-Charles 2 , D. McMullan 2 , M. Griffiths 2 , N. K. Ragge 1,2

1 Faculty of Health and Life Sciences, Oxford Brookes University, Oxford, United Kingdom, 2 West Midlands Regional Genetics Service, Birmingham Women’s and Children’s NHS Foundation Trust, Birmingham, United Kingdom, 3 Guy’s and St Thomas’ NHS Foundation Trust, London, United Kingdom, 4 Wessex Regional Genetics Laboratory, Salisbury District Hospital, Salisbury, United Kingdom, 5 Faculty of Medicine, University of Southampton, Southampton, United Kingdom
Introduction: Anophthalmia, microphthalmia and coloboma (AMC) are a genetically heterogenous collection of developmental eye disorders, with variants in >100 genes implicated. However, only 25-60% of patients receive genetic diagnoses. Structural variants are an important source of genetic variation, and have led to the identification of numerous eye development genes, including SOX2 and OTX2 . However, few studies systematically address their contribution to AMC. Here, we investigate a large cohort of AMC families for known and novel copy number variants (CNVs). Materials and methods: A cohort of 320 unrelated probands was screened using either diagnostic genome-wide aCGH platforms ( n = 156), including the customised OGT CytoSure 4x180K eye array, or the Illumina Infinium Global Screening Array ( n = 164). SNP data were analysed using the algorithms QuantiSNP and PennCNV. Variants of interest were validated using qPCR and long-range PCR. Results: Our analysis identified multiple variants of interest, including likely pathogenic CNVs affecting known AMC genes and rare CNVs highlighting new candidates. We describe a novel homozygous 113kb deletion 19kb upstream of MAB21L2 , a developmental gene involved in eye morphogenesis. This deletion removes multiple conserved cis-regulatory elements. We also identified deletions in regions with recurrent CNVs (1q21.1, 6q16, 10q11, 16p11.2, 22q11.2). Conclusions: Our study supports the importance of CNV screening in routine diagnostic testing. It also highlights how CNVs affecting noncoding elements can provide insights into the role of regulatory regions of eye development genes in AMC. Further investigation of such variants will improve the genetic diagnostic yield and our understanding of the aetiology of these conditions. F. Ceroni: None. D.A. Bax: None. R.J. Holt: None. Y. Kesim: None. A. Cole: None. C. Ogilvie: None. D. Bunyan: None. L. Cooper-Charles: None. D. McMullan: None. M. Griffiths: None. N.K. Ragge: None. P02.10.C Cataract and retinal dystrophy in Vps13b ∆Ex3/∆Ex3 mice .

V. R. M. Lhussiez 1 , Q. Cesar 2 , E. Dubus 2,3 , M. Simonutti 2 , E. Lizé 1 , S. Nguyen 1 , A. Geissler 3 , A. Bouchot 3 , S. Picaud 2 , E. F. Nandrot 2 , N. Acar 4 , L. Faivre 1,5,6 , C. Thauvin 1,5,7 , L. Duplomb 1,5 , R. Da Costa 1,5

1 INSERM, UMR1231, Equipe GAD, Université de Bourgogne Franche Comté, Dijon, France, 2 Sorbonne Université, INSERM, CNRS, Institut de la Vision, Paris, France, 3 Plateforme d’Imagerie Cellulaire CellImaP/DimaCell, Inserm LNC UMR1231, Dijon, France, 4 Centre des Sciences du Goût et de l’Alimentation, AgroSup Dijon, CNRS, INRAE, Université Bourgogne Franche-Comté, Dijon, France, 5 FHU TRANSLAD, CHU Dijon, Dijon, France, 6 Centre de Référence Anomalies du Développement et Syndromes Malformatifs, CHU Dijon, Dijon, France, 7 Centre de Référence Déficiences Intellectuelles de Causes Rares, CHU Dijon, Dijon, France
Cohen syndrome (CS) is a rare genetic disorder due to variation in the VPS13B gene. It is characterized by a wide variety of clinical features that includes a typical facial dysmorphism, hypotonia, neutropenia, microcephaly, intellectual disability and severe visual impairments. In their early childhood, CS patients already suffer from myopia and a retinal dystrophy that affects both peripheral and central vision. In addition, long-term outcome studies showed that cataract reaches a high prevalence in adults with CS in their forties. To understand how VPS13B variants lead to these visual impairments and have the possibility to assess therapeutic approaches, we generated the Vps13b ∆Ex3/∆Ex3 mouse model. Cataract was almost systematic in 2-month-old animals. Eye fundi appeared normal until cataract development, but OCT, ERG and histological data suggest that rod homeostasis may be affected in the few Vps13b ∆Ex3/∆Ex3 mice without cataract after 5 months of age. Immunostaining of the lens revealed that cataract formation was associated with the appearance of large vacuoles in the cortical area, epithelial-mesenchymal transition and fibrosis. In later stages, cataracts became hypermature, lens capsules ruptured, and sclerotic nuclear parts dissociated. Altogether, our results show that Vps13b has a function in lens homeostasis in mice and that the Vps13b ∆Ex3/∆Ex3 mouse line is a useful model to study the pathomechanism leading to CS-related cataract. This work was supported with funds from the JED Fondation and from the Fondation Maladies Rares. V.R.M. Lhussiez: None. Q. Cesar: None. E. Dubus: None. M. Simonutti: None. E. Lizé: None. S. Nguyen: None. A. Geissler: None. A. Bouchot: None. S. Picaud: None. E.F. Nandrot: None. N. Acar: None. L. Faivre: None. C. Thauvin: None. L. Duplomb: None. R. Da Costa: None. P02.12.B Dominant cone rod dystrophy (CORD7) previously assigned to mutation of RIMS1 , is fully explained by co-inheritance of a dominant allele of PROM1

G. Arno 1,2 , E. Schiff 2 , G. Wright 2 , N. Waseem 1 , Genomics England Research Consortium, M. Michaelides 1,2 , A. T. Moore 1,2,3 , A. R. Webster 1,2

1 UCL Institute of Ophthalmology, London, United Kingdom, 2 Moorfields Eye Hospital, London, United Kingdom, 3 University of California, San Francisco, San Francisco, CA, United States
A single missense variant (p.Arg820His) in RIMS1 has been associated with autosomal dominant cone rod dystrophy 7 (CORD-7) and enhanced cognition. This was discovered in 2003 following linkage (LOD 3.61) to a 7cM region on chromosome 6q, in a four generation British family in 1998. The RIMS1 gene is often included in diagnostic panels for retinal dystrophy. However, despite this, no further families with CORD7 have been reported. Widespread availability of massively parallel sequencing has enabled detection of genomic variation at a scale not possible in the early 2000s. Access to vast datasets in gnomAD and Genomics England 100k genomes project (GE100KGP) now enables better understanding of the validity of previously accepted genetic associations. Interrogation of the gnomAD datasets reveal 10 carriers of RIMS1 p.Arg820His (Non Finnish European allele frequency 0.0001). This would be unusual given the severity of visual impairment in adulthood and absence of the variant in further patient cohorts. Four members of the original CORD7 family were recruited to the GE100KGP, where WGS showed the two affected members to be heterozygous for a well characterised dominant allele (p.Arg373Cys) of PROM1 located on chromosome 4p. All affected individuals subsequently were heterozygotes for this variant. Cytogenetic analysis excluded a balanced translocation that might explain co-segregation of a region on 6q and 4p. This finding suggests the rare occurrence of an alpha error despite robust linkage analysis in a large family. So far there is no evidence that perturbation of RIMS1 affects the retina in humans. G. Arno: None. E. Schiff: None. G. Wright: None. N. Waseem: None. M. Michaelides: None. A.T. Moore: None. A.R. Webster: None. P02.14.A CRB1-related retinopathy superimposed on a S-adenosylhomocysteine hydrolase deficiency phenotype .

M. Di Scipio 1 , M. K. Grudzinska Pechhacker 1,2 , A. Vig 1 , A. Anupreet 1 , E. Tavares 1 , A. Vincent 1,2,3 , E. Heon 1,2,3

1 Genetics and Genome Biology, The Hopsital for Sick Children, Toronto, ON, Canada, 2 Department of Ophthalmology and Vision Sciences, University of Toronto, Toronto, ON, Canada, 3 Department of Ophthalmology and Vision Sciences, The Hospital for Sick Children, Toronto, ON, Canada
Background: S-adenosylhomocysteine deficiency is a rare metabolic disorder for which no eye phenotype has been documented. CRB1 gene is known to cause wide spectrum of autosomal recessive retinal diseases such as Leber’s congenital amaurosis, early onset cone-rod dystrophy, retinitis pigmentosa, isolated maculopathy and familial foveal retinoschisis; or autosomal dominant pigmented paravenous chorioretinal atrophy. Materials and Methods: The present report describes a non-consanguineous family, where two children were affected with S-adenosylhomocysteine deficiency and one of them presented with reduced vision due to maculopathy. Comprehensive eye examination was performed in available family members together with color vision test, contrast sensitivity, visual fields, fundus images, OCT, full field electroretinogram, pattern electroretinogram and visual evoked potentials. Genetic testing included WES, retinal dystrophy panel and segregation analysis. Results: Clinical whole exome sequencing revealed homozygous missense mutations in AHCY gene (c.148G>A, p.Ala50Thr) as a cause of S-adenosylhomocysteine deficiency. Because of a maculopathy in the proband, retinal dystrophy gene panel sequencing revealed two heterozygous missense mutations in CRB1 gene (c.1831T>C, p.Ser611Pro) and (c.3955T>C, p.Phe1319Leu) in the proband (III-1) and her mother. Mutations segregated with disease phenotype in family members. Conclusions: Establishing an ocular genetic diagnosis may be complicated by the co-existence of a rare systemic genetic disease with previously unknown eye involvement. Extensive phenotyping and genotyping of available family members, and genotype-phenotype correlations helps to establish a diagnosis. Grant References: Henry Brent Research Chair; Canadian Foundation Fighting Blindness; Career Development Award (Foundation fighting Blindness, USA). M. Di Scipio: None. M.K. Grudzinska Pechhacker: None. A. Vig: None. A. Anupreet: None. E. Tavares: None. A. Vincent: None. E. Heon: None. P02.18.B Fundus albipunctatus in a pedigree with autosomal-dominant transmission of G116R mutation in RLBP1

K. Mihova 1 , K. Kamenarova 1 , S. Cherninkova 2 , I. Dimova 1 , F. Shakola 1 , V. Mitev 1 , R. Kaneva 1

1 Department of Medical Chemistry and Biochemistry, Molecular Medicine Center, Medical University of Sofia, Sofia, Bulgaria, 2 Department of Neurology, University Hospital Alexandrovska, Medical University of Sofia, Sofia, Bulgaria
Introduction: Fundus albipunctatus (FA) is a rare form of congenital stationary night blindness in which white or yellow dots can be seen scattered through the fundus. Individuals complain of night blindness early in childhood without progression. By next-generation sequencing of clinical exome including all inherited retinal degeneration causative genes, we aimed to identify the disease-causing mutation in a patient with clinical signs of FA. Materials and Methods : Using Illumina® platform and TruSight One, we identified heterozygous RLBP1 -mutation in a young patient, which results in phenotype consistent with FA. Results : A 5-years-old patient presenting ophthalmoscopic phenotype including delayed dark adaptation, aggregation of yellowish-white lesions of various sizes in the retina, and slight vascular abnormalities, was screened for mutations in all known genes previously associated with inherited retinal degeneration. We identified a heterozygous mutation, c.346G>C (p.G116R), in the gene for cellular retinaldehyde-binding protein 1 ( RLBP1 ), already described in a pedigree affected by autosomal-recessive FA. All other genetic variants detected in the patient’s DNA were excluded after applying commonly used filtering criteria. Pedigree analysis showed the presence of p.G116R mutation in the patient’s mother who was a heterozygous carrier. Мother’s ophthalmological examination subsequently confirmed pre-existing stationary night blindness, prolonged dark adaptation with preserved visual acuity strongly suggesting a phenotype consistent with FA. Conclusions : Most reported pedigrees are consistent with a recessive pattern of Mendelian inheritance of FA, although autosomal-dominant inheritance has been also reported. Our results suggest that mutation RLBP1- p.G116R may be considered as dominant-acting in pedigrees with autosomal-dominant FA. Acknowledgements: D01-285; КП-06-Н33/12. K. Mihova: None. K. Kamenarova: None. S. Cherninkova: None. I. Dimova: None. F. Shakola: None. V. Mitev: None. R. Kaneva: None. P02.19.C The G-EAR Consortium study: new candidate genes for normal hearing function (NHF) and age-related hearing loss (ARHL) in a large and deeply phenotyped cohort .

M. Brumat 1 , A. Morgan 1,2 , A. P. Nagtegaal 3 , B. C. Oosterloo 3 , S. Seshadri 4,5 , N. L. Heard-Costa 4,6 , G. Van Camp 7 , E. Fransen 8 , P. Gasparini 1,2 , G. Girotto 1,2

1 Department of Medicine, Surgery and Health Sciences, University of Trieste, Trieste, Italy, 2 Institute for Maternal and Child Health – IRCCS Burlo Garofolo, Trieste, Italy, 3 Department of Otorhinolaryngology, Erasmus Medical Center, Rotterdam, Netherlands, 4 Framingham Heart Study, Framingham, MA, United States, 5 Glenn Biggs Institute for Alzheimer’s and Neurodegenerative Diseases, University of Texas Health Sciences Center, San Antonio, TX, United States, 6 Department of Neurology, Boston University School of Medicine, Boston, MA, United States, 7 Center of Medical Genetics, University of Antwerp and Antwerp University Hospital, Antwerp, Belgium, 8 Department of Biomedical Sciences, University of Antwerp, Antwerp, Belgium
Understanding the genetic basis of hearing traits and diseases (e.g. NHF and ARHL) is central to the development of precision medicine for the hearing impaired; to pursue this aim, fully audiometrically characterised cohorts are fundamental. Through the international G-EAR Consortium, we united data from ten such cohorts from Europe, USA, Caucasus and Central Asia (overall >9000 individuals) and performed a GWAS meta-analysis on NHF and ARHL, analysing in the former case single hearing thresholds or their average value across specific frequencies, and in the latter a case-control phenotype (PMID:29725052). Analyses used linear mixed model/logistic regression; meta-analysis was performed with METAL. Suggestive association signals were detected on chromosomes 3, 8, 11, 16 and 18. In particular, three genes - CADM2 , CALB1 and LRRC4C - result to be potentially associated with NHF and two more - CDH13 and CTIF - with ARHL. CADM2 encodes a protein of the nectin-like family, members of which are fundamental for the development of the inner ear; CALB1 encodes a calcium-binding protein that regulates the development of murine cochlear hair cells; LRRC4C encodes a transmembrane protein abundantly expressed in mouse inner ear. Finally, CDH13 and CTIF have been associated with NHF in previous studies, suggesting their possible role also in ARHL. Replication is in progress in an independent cohort of ~1500 individuals, fully phenotyped; updated results will be presented. These findings provide a strong contribution to our understanding of the genetic bases of both NHF and ARHL, laying the foundation of future therapeutic targets. M. Brumat: None. A. Morgan: None. A.P. Nagtegaal: None. B.C. Oosterloo: None. S. Seshadri: None. N.L. Heard-Costa: None. G. Van Camp: None. E. Fransen: None. P. Gasparini: None. G. Girotto: None. P02.20.A Further evidence of involvement of CDC14A in autosomal recessive non-syndromic hearing loss

J. Doll 1 , S. Kolb 1 , L. Schnapp 1 , A. Rad 2,3 , F. Rüschendorf 4 , I. Khan 5 , A. Adli 2 , A. Hasanzadeh 2 , D. Liedtke 1 , S. Knaup 1 , M. A. H. Hofrichter 1 , T. Müller 6 , M. Dittrich 1,6 , I. Kong 7 , H. Kim 8 , T. Haaf 1 , B. Vona 1,3

1 Institute of Human Genetics, Julius Maximilians University Würzburg, Würzburg, Germany, 2 Cellular and Molecular Research Center, Sabzevar University of Medical Sciences, Sabzevar, Iran, Islamic Republic of, 3 Tübingen Hearing Research Centre, Department of Otorhinolaryngology, Head and Neck Surgery, Eberhard Karls University, Tübingen, Germany, 4 Max Delbrück Center for Molecular Medicine in the Helmholtz Association, Berlin, Germany, 5 Department of Chemistry, Bacha Khan University, Charsadda, Pakistan, 6 Institute of Bioinformatics, Julius Maximilians University, Würzburg, Germany, 7 Department of Animal Science, Division of Applied Life Science (BK21plus), Institute of Agriculture and Life Science, Gyeongsang National University, Jinju, Korea, Republic of, 8 Neurological Disorders Research Center, Qatar Biomedical Research Institute, Hamad Bin Khalifa University, Doha, Qatar
Introduction: Hearing loss (HL) is a highly heterogeneous sensory disorder with a prevalence of 1-3 affected in 1000 newborns. Since 2016, the gene CDC14A (Cell Division Cycle 14A) is associated with autosomal recessive non-syndromic HL (DFNB32) and hearing impairment and infertile male syndrome (HIIMS). Materials and Methods: In order to identify likely pathogenic variants of already known and novel HL genes in Iranian and Pakistani families with sensorineural HL, exome sequencing, gene mapping approaches and bioinformatics analysis was performed. Segregation testing of variants ensued. To investigate functional consequences on the RNA-level of both loss-of-function variants and to evaluate altered expression levels, we used a minigene assay for the splice variant (c.1421+2T>C, p.?) and RT-qPCR for the c.1041dup variant. Results: We identified a novel homozygous CDC14A splice site variant (c.1421+2T>C, p.?) responsible for the disruption of a splice donor site based on in silico prediction tools. Moreover, we identified a novel homozygous frameshift variant (c.1041dup, p.Ser348Glnfs*2) in the gene CDC14A . We observed that the c.1421+T>C variant is responsible for the activation of a cryptic splice site resulting in a truncated transcript (c.1414_1421del, p.Val472Leufs*20). Significantly reduced CDC14A mRNA levels indicated a defective transcript due to the c.1041dup variant, which is likely targeted by nonsense-mediated mRNA decay (NMD). Conclusions: In summary, we identified and assessed the pathogenicity of two novel variants on the RNA-level and expand the spectrum of clinically relevant mutations in the gene CDC14A . J. Doll: None. S. Kolb: None. L. Schnapp: None. A. Rad: None. F. Rüschendorf: None. I. Khan: None. A. Adli: None. A. Hasanzadeh: None. D. Liedtke: None. S. Knaup: None. M.A.H. Hofrichter: None. T. Müller: None. M. Dittrich: None. I. Kong: None. H. Kim: None. T. Haaf: None. B. Vona: None. P02.21.B Causes of congenital hearing loss and cochlear implant outcome

C. Landgraf 1 , S. von Hardenberg 1 , A. Lesinski-Schiedat 2 , A. Büchner 2 , B. Schlegelberger 1 , B. Auber 1

1 Department of Human Genetics, Hannover Medical School (MHH), Hannover, Germany, 2 Department of Otorhinolaryngology, Hannover Medical School (MHH), Hannover, Germany
Aim : Personalize prognosis and therapy through genetic screening of potential cochlear implant patients. Background : Hearing loss (HL) can be divided into syndromic and non-syndromic hearing loss (NSHL, approximately 70 %). While pathogenic variants affecting the gene GJB2 account for roughly 10-30 % of NSHL, the majority of NSHL is distributed among more than 100 other genes. For many patients who do not benefit sufficiently from hearing aids, cochlear implants (CIs) are the device of choice. Performance of CI recipients is expected to depend on genetic background and a favorable outcome has been associated with pathogenic variants in genes affecting the function of the cochlear sensory organ (CSO); a negative outcome was reported for patients with variants in genes associated with spiral ganglion neuron (SGN) function. Patients : 129 unrelated hearing impaired children and adults without pathogenic variants in GJB2 and without evidence for acquired HL. Methods : Whole exome sequencing was performed analyzing 148 genes associated with HL listed in the Deafness Variation Database and additional 681 genes listed in the Human Phenotype Ontology database based on hearing impairment. Results : In 33 of 129 patients we identified pathogenic or likely pathogenic variants in 30 different genes, confirming the genetic heterogeneity of hereditary HL. Among these was one patient with a (homozygous) pathogenic variant in a gene proven to affect SGN function ( DFNB59 ). Conclusions: For meaningful results, a significantly higher number of cases is required. In addition, a further functional subdivision of patients with gene alterations influencing CSO function should be considered. C. Landgraf: None. S. von Hardenberg: None. A. Lesinski-Schiedat: None. A. Büchner: None. B. Schlegelberger: None. B. Auber: None. P02.22.C In silico and in vivo analyses of novel variants identified by Whole Exome Sequencing in Argentinean deaf patients: to be or not be pathogenic

P. I. Buonfiglio 1 , C. D. Bruque 2 , E. Goldschmidt 3 , V. Lotersztein 4 , S. Menazzi 5 , B. Paoli 6 , P. Plazas 7 , A. B. Elgoyhen 1 , V. K. Dalamón 1

1 Instituto de Investigaciones en Ingeniería Genética y Biología Molecular “Dr. Héctor Torres” - INGEBI/CONICET, Ciudad Autónoma de Buenos Aires, Argentina, 2 Centro Nacional de Genética Médica “A.N.L.I.S. – Dr. Carlos G. Malbrán “, Ciudad Autónoma de Buenos Aires, Argentina, 3 Servicio de Genética del Hospital General de Agudos “Dr. Juan A. Fernández”, Ciudad Autónoma de Buenos Aires, Argentina, 4 Servicio de Genética del Hospital Militar Central Cirujano Mayor “Dr. Cosme Argerich”, Ciudad Autónoma de Buenos Aires, Argentina, 5 Servicio de Genética del Hospital de Clínicas “José de San Martín”., Ciudad Autónoma de Buenos Aires, Argentina, 6 Servicio de Otorrinolaringología Infantil del Hospital de Clínicas “José de San Martín”., Ciudad Autónoma de Buenos Aires, Argentina, 7 Tercera Cátedra de Farmacología, Facultad de Medicina, Universidad de Buenos Aires, Ciudad Autónoma de Buenos Aires, Argentina
Hereditary hearing loss (HHL) is the most common sensory disorder affecting 1 in 500 newborn children. Since HHL is related to more than 150 target genes, we designed a diagnosis strategy in order to identify pathogenic variants. A total of 1250 patients were analyzed for frequent mutations in GJB2 and GJB6 genes by Sanger Sequencing, genotyping 25% of them. From undiagnosed patients, 29 families were selected to perform Whole exome sequencing. After filtering and analysis process, 45% of patients were genotyped, identifying 23 causative mutations (11 novel, 12 reported) classified according to ACMG Standards. Some of the novel variants were further studied in silico by structural and stability studies of the mutated proteins. In addition, datasets from deafness and specific variant databases were correlated with different protein motifs in order to predict the theoretical pathogenicity effect of the aminoacid changes. Furthermore, knock-down phenotype rescue assays in zebrafish are underway to accomplish in vivo validation. In some cases, extensive analysis reinforced the pathogenicity prediction effect of variants and surprisingly, in one case, discouraged the deleterious effect of a genetic variant to the protein. Preliminary results in zebrafish confirmed the pathogenicity of one novel variant in the hair cell function and auditory system. This study shows that our algorithm is successful for the genetic diagnosis of deafness. Comprehensive analysis is crucial to strengthen prediction of variant pathogenicity. These findings highlight the importance of genetic studies followed by in silico and in vivo validation to better understand the genetic basis of HHL. P.I. Buonfiglio: None. C.D. Bruque: None. E. Goldschmidt: None. V. Lotersztein: None. S. Menazzi: None. B. Paoli: None. P. Plazas: None. A.B. Elgoyhen: None. V.K. Dalamón: None. P02.23.A Genetic spectrum of hereditary hearing loss in multigenerational families with autosomal dominant mode of inheritance

M. Oldak 1 , M. L. Leja 1,2 , D. Ozieblo 1,3 , A. Sarosiak 1,3 , H. Skarzynski 4

1 Department of Genetics, Institute of Physiology and Pathology of Hearing, Warsaw, Poland, 2 Postgraduate School of Molecular Medicine, Medical University of Warsaw, Warsaw, Poland, Warsaw, Poland, 3 Postgraduate School of Molecular Medicine, Medical University of Warsaw, Warsaw, Poland, Warsaw, Poland, Warsaw, Poland, 4 3. Oto-Rhino-Laryngology Surgery Clinic, Institute of Physiology and Pathology of Hearing, Warsaw, Poland, Warsaw, Poland
Introduction: Hearing loss (HL) is the most common disability of human senses and genetic factors play an important role in its development. Autosomal dominant HL (ADHL) is the second most common form of inherited HL, characterized by postlingual age of onset and progression. To date 63 loci with 47 different genes were causally involved in ADHL. Materials and Methods : Multigenerational families with ADHL were recruited for the study ( n = 44). Genomic DNA was isolated from whole blood and buccal swabs samples. A high-throughput sequencing with custom multigene panel was performed on probands’ DNA samples. Family segregation analysis of the identified variants was conducted using Sanger sequencing. All detected variants were analyzed in the context of population databases and available literature. Pathogenicity of the identified variants was predicted by different computational approaches. Results : Genetic testing revealed probably pathogenic variants in about 52% (23/44) of the analyzed families. The majority of identified variants were novel, previously not reported and hitherto not linked to the disease. The most frequent genes with identified causative variants were MYO6 , COCH , DFNA5 , KCNQ4 , TECTA and POU4F3 . Conclusions : Our study revealed a high involvement of novel probably pathogenic variants in the development of ADHL and confirmed a high heterogeneity of the identified genetic changes. High-throughput sequencing in HL patients generates large amount of data that should be interpreted carefully and confirmed by family studies. There is also a need for functional validation of the detected novel variants. Supported by : NCN Research Grant no. 2016/22/E/NZ5/00470 SONATA BIS6 M. Oldak: None. M.L. Leja: None. D. Ozieblo: None. A. Sarosiak: None. H. Skarzynski: None. P02.24.B Wide spectrum of genetic hearing loss causes and large number of novel variants in cochlea implanted children

D. Ozieblo 1,2 , M. L. Leja 1,2 , H. Skarzynski 3 , M. Oldak 1

1 Department of Genetics, Institute of Physiology and Pathology of Hearing, Warsaw, Poland, 2 Postgraduate School of Molecular Medicine, Medical University of Warsaw, Warsaw, Poland, 3 Oto-Rhino-Laryngology Surgery Clinic, Institute of Physiology and Pathology of Hearing, Warsaw, Poland
Introduction: Every year, approximately 1-6/1000 children are born with severe to profound hearing loss (HL). For this group of patients cochlear implantation (CI) is the treatment of choice. In the majority of deaf children HL is determined by DFNB1 locus pathogenic variants. Here, we aim to dissect the genetic background of HL in DFNB1-negative CI patients. Materials and Methods: Genomic DNA was isolated from blood samples of carefully selected DFNB1-negative patients with profound prelingual HL ( n = 50) and their family members. In all probands whole exome sequencing (WES) followed by bioinformatics and expert analysis were performed. Standard Sanger sequencing was used for validation of selected variants, family segregation analysis and detection of the CEVA haplotype. Copy number variants were confirmed with qPCR. Results: In every tested subject WES revealed at least 5 probably pathogenic variants in HL-related genes. Causative variants were identified in 33 patients and in 39% (13/33) of them they were inherited in an autosomal dominant manner. Among the detected 53 disease-causing alleles, 55% (29/53) contained novel genetic alterations. Conclusions: Obtained results show a high heterogeneity of genetic HL causes in the population of Polish DFNB1-negative cochlea-implanted patients. In children with no family history of HL, the possibility of identifying causative variants inherited in both autosomal recessive and dominant manner should be taken into account. In patients with an unknown genetic cause of HL, the tested area should be expanded with whole genome analysis. Supported by NCN grant: 2017/27/N/NZ5/02369 D. Ozieblo: None. M.L. Leja: None. H. Skarzynski: None. M. Oldak: None. P02.25.C Diagnostic Efficiency of Clinical Exome Solution Panel in patients with Hearing loss/Hereditary Deafness by using Next Generation Sequencing

S. G. Temel 1,2 , A. Alemdar 1 , M. Yılmaz 3 , L. Aliyeva 2 , S. Ozemri Sag 2

1 Department of Translational Medicine, Bursa Uludag University, Bursa, Turkey, 2 Department of Medical Genetics, Bursa Uludag University, Bursa, Turkey, 3 Faculty of Medicine, Bursa Uludag University, Bursa, Turkey
Hearing loss (HL)/Hereditary Deafness (HD) is a heterogeneous disease which multiple genes have been found to be implicated in the disease etiopathogenesis. Genetic diagnosis is highly important for HL/HD patients in order to ensure the etiology of the condition and counsel the patients properly. In this study, 41 patients (22M/19F) who admitted to our clinic between February 2018 and November 2019 with HL/HD were screened for 110 genes related with syndromic/non-syndromic HL/HD. NGS is considered to be the best practice for diagnosis. Large gene panels also can evaluate non-prevelant genes and candidate variants specific to populations. All exons and exon-intron boundaries were amplifed by using Clinical Exome (Sophia Genetics) Solution (CES) kit in MiSeq NGS instrument. Variant analyis (SNV, Indel and CNV) and annotations were performed by Sophia DDM platform, classified according to ACMG criteria with in-silico tools and also investigated by HGMD Professional 2019-4 and other available online bioinformatic tools. We identified CNV in three patients, 26 pathogenic and likely pathogenic variants in 19 of 41 HL/HD patients (46 %) in 10 different genes. Similarly, 23 variants of uncertain significance are determined in 18 of 41 HL/HD patients (43 %) in 18 distinct genes. Besides these variations, we found 31 novel variations that are not reported before in literature or databases. Our study affirms that genetic screening of patients with HL by using CES panel not only easily diagnoses but also help counseling the patients and their family for their future risk of developing the disease. S.G. Temel: None. A. Alemdar: None. M. Yılmaz: None. L. Aliyeva: None. S. Ozemri Sag: None. P02.26.A NGS approach for molecular diagnosis of hereditary hearing loss

F. Gerundino 1 , C. Pescucci 1 , C. Giuliani 1 , B. Bianchi 2 , G. Traficante 3 , S. Bargiacchi 3 , S. Romano 4 , A. Cecconi 5 , C. Di Marco 6 , A. Orrico 7,8 , M. Mencarelli 9 , L. Candita 1 , C. Deledda 1 , M. Trafeli 1 , B. Minuti 1 , A. Renieri 9 , E. Pelo 1

1 SOD Diagnostica Genetica, AOU Careggi, Firenze, Italy, 2 Audiologia SOC Otorinolaringoiatria AOU Meyer, Firenze, Italy, 3 SOC Genetica Medica AOU Meyer, Firenze, Italy, 4 Medicina 1, Genetica Medica AOU Pisana, Pisa, Italy, 5 Ambulatorio Integrato Genetica Medica, Usl Toscana Centro, Firenze, Italy, 6 Ambulatorio Integrato Genetica Medica, Usl Toscana Centro, Florence, Italy, 7 Interdipartimental Program for molecular diagnosis and characterization of pathogenic mechanisms of rare genetic diseases, Siena, Italy, 8 Clinical Genetics. ASL Toscana Sudest. Ospedale della Misericordia., Grosseto, Italy, 9 Genetica Medica, Azienda Ospedaliera Universitaria Senese, Siena, Italy
Introduction: hearing loss (HL) is one of the most genetically heterogeneous disorders. Although pathogenic variants in GJB2 account for a large proportion of cases, many patients remain undiagnosed after DFNB1 testing. NGS analysis of HL-genes to simultaneously search for nucleotide variants and copy number variants (CNVs) greatly improve the detection rate. Materials and methods: clinical exome sequencing followed by in silico analysis of HL-gene panel (106 genes) was carried out on 117 patients on which DFNB1 pathogenic variants were previously excluded. Eighty cases show prelingual or childhood deafness while in 37 patients HL was progressive with variable age of onset. Clinical interpretation of genetic variants was performed according to ACMG2015 by InterVar and Varsome tools and revised according to the specific guidelines for HL-genes. Results: Forty-eight pathogenic or likely pathogenic variants and eight pathogenic CNVs (6 STRC -CATSPER2 and 2 OTOA whole gene deletions) were identified with an overall detection rate of 26.5%. Specific detection rate was 32% (12/37) for adult cases and 24% in children. In particular, NGS identified 12 different pathogenic variants in dominant, 28 in recessive and 8 in dominant/recessive genes. The genes most involved were SLC26A4 and STRC. A single pathogenic variant in SLC26A4 gene was found in 7 cases. Conclusions: overall detection rate of the test is in line with available data. MLPA analysis in target genes such as SLC26A4 should be performed to exclude the presence of a deletion at exonic level in cases where a single variant as been identified. F. Gerundino: None. C. Pescucci: None. C. Giuliani: None. B. Bianchi: None. G. Traficante: None. S. Bargiacchi: None. S. Romano: None. A. Cecconi: None. C. Di Marco: None. A. Orrico: None. M. Mencarelli: None. L. Candita: None. C. Deledda: None. M. Trafeli: None. B. Minuti: None. A. Renieri: None. E. Pelo: None. P02.27.B Complex assessment of pathogenicity of novel variant c.516G>C (p.Trp172Cys) in the GJB2 gene associated with hearing loss in indigenous peoples of Southern Siberia (Russia)

E. A. Maslova 1,2 , M. V. Zytsar 1 , V. Y. Danilchenko 1 , K. E. Orishchenko 1,2 , O. L. Posukh 1,2

1 Institute of Cytology and Genetics, Novosibirsk, Russian Federation, 2 Novosibirsk State University, Novosibirsk, Russian Federation
Assessment of pathogenicity of novel variants is a primary task for molecular diagnostics of hereditary diseases. Mutations in gene GJB2 encoding transmembrane protein Cx26 are the common cause for hearing loss worldwide. We present a complex assessment of pathogenicity of novel non-synonymous variant c.516G>C (p.Trp172Cys) in GJB2 found with high frequency in deaf patients belonging to indigenous peoples of Southern Siberia (Tuvinians and Altaians). Materials & Methods: Genetic analysis was performed in group of deaf patients and ethnically matched controls. The GJB2 knockout HeLa cell line (obtained by CRISPR/Cas9 system) was used to establishment of transgenic HeLa cell lines with different pathogenic GJB2 -variants and GJB2- wt. Cellular Cx26 localization was investigated by immunocytochemistry (ICC). The Cx26-channels permeability was analyzed by dye (PI) loading assay. Results: Segregation of variant c.516G>C (p.Trp172Cys) with hearing loss was established in analysis of pedigrees of deaf patients and its frequency was significantly higher in patient’s group than in controls. Most of in silico programs predicted a likely damaging effect of p.Trp172Cys. ICC showed predominantly cytoplasmic localization of the Cx26-p.Trp172Cys protein in contrast to Cx26-wt which represented distinct conglomerates on cell membrane. Dye (PI) loading assay revealed lower PI loading efficiencies in cells expressing mutant variant Cx26-p.Trp172Cys compared to Cx26-wt while the absence of PI accumulation was shown for other mutant Cx26-variants with known pathogenic effects. Conclusions: Several lines of evidences support pathogenicity of novel variant c.516G>C (p.Trp172Cys) in gene GJB2 and its involving in hearing loss. Study was supported by Project #0324-2019-0041-C-01 and RFBR grant #17-29-06016_ofi-m. E.A. Maslova: None. M.V. Zytsar: None. V.Y. Danilchenko: None. K.E. Orishchenko: None. O.L. Posukh: None. P02.28.C Genetic heterogeneity underlying hearing loss and Usher syndrome in Saudi population

K. Ramzan , F. Imtiaz, M. Al-Owain, S. Alhazzaa

King Faisal Specialist Hospital and research centre, Riyadh, Saudi Arabia
Hearing loss is one of the most common sensory disorders in humans with both genetic and environmental etiologies. Usher syndrome (USH) is the most common cause of combined blindness and deafness. Molecular diagnosis is of great significance in revealing the molecular pathogenesis and aiding the clinical diagnosis of this disease. Our study aims to comprehensively delineate the genetic basis of hearing loss in the individuals of Saudi Arabian origin. The identification of the causative gene in affected families with hearing loss is difficult due to extreme genetic heterogeneity and lack of phenotypic variability. Consanguineous families are a powerful resource for genetic linkage studies/homozygosity mapping for recessively inherited hearing impairment. Homozygosity mapping, linkage analysis and next generation sequencing Deafness Gene-panel and Whole Exome sequencing were conducted. Using the combined approaches, so far mutations in 32 different deafness genes including novel genes first time identified in this population, have been identified. More than 300 familial/sporadic cases were solved, including novel variants in known HL genes. Using these innovative molecular approaches, we were able to document the most common forms of hereditary hearing loss, their incidence and distribution in the Saudi population. The overall results of this study are highly suggestive that the underlying molecular basis of hearing loss in Saudi Arabia is very genetically heterogeneous. The benefit of this study will hopefully provide the foundation for knowledge and awareness through screening of carrier status and genetic counselling, thereby having a major impact upon early intervention for and prevention of hereditary hearing loss. K. Ramzan: None. F. Imtiaz: None. M. Al-Owain: None. S. Alhazzaa: None. P02.29.A ENT- Genetics Genome Boards: Over 50% diagnostic yield

M. Carminho-Rodrigues 1 , P. Senn 2 , M. Abramowicz 1 , E. Hammar 1 , L. Quteineh 1 , A. Murphy 1 , S. Laurent 1 , M. Guipponi 1 , H. Cao-Van 2 , A. Paoloni-Giacobino 1

1 Medical Genetics Unit, Geneva University Hospitals, Geneva, Switzerland, 2 ENT Unit, Geneva University Hospitals, Geneva, Switzerland
Introduction: Hearing impairment (HI) is the most common sensory impairment. It can be isolated or syndromic. Molecular diagnosis through whole exome sequencing (WES) can allow an improved management and surveillance of HI and possible associated anomalies. Here we show the results of a 3 years’ experience of Ear-nose-and-throat ( ENT) Genetics Genome boards. Materials and Methods: Our cohort includes children with mild to profound, unilateral or bilateral HI referred by ENT clinic from 2017 to 2019. We performed WES with bioinformatics analysis of 189 genes associated with HI. The results are discussed during a genome board where geneticists and ENT specialists confront the genetic variants found by WES with the auditory phenotype of the patients. Results: We tested 38 children, most often with moderate, bilateral sensorineural hearing loss. The diagnostic rate was 52.6% (20/38). Among the results we found variants associated with: syndromic forms: Usher, Wolfram-like, Branchio-oto-renal, Alport, Perrault, GATA3 ; non-syndromic forms: Cx26, ACTG1 , OTOA, ILDR1, SLC17A8, LOXHD1, TBC1D24 and STRC related HI associated or not with infertility in males. Conclusions: The diagnostic yield of our study (52.6%) is comparable to recent data in the literature. A genetic assessment of HI in young children makes the identification of the molecular cause possible, and permits to anticipate possible complications particularly in the case of syndromic forms with medical problems involving other organs or systems. Collaboration between ENT specialists and geneticists is essential to make an accurate and precise diagnosis and to personalize the follow-up and treatment of these young patients. M. Carminho-Rodrigues: None. P. Senn: None. M. Abramowicz: None. E. Hammar: None. L. Quteineh: None. A. Murphy: None. S. Laurent: None. M. Guipponi: None. H. Cao-Van: None. A. Paoloni-Giacobino: None. P02.30.B The utility of exome sequencing for hereditary eye diseases: single-centre experience

M. Volk 1 , N. Teran 1 , A. Maver 1 , H. Jaklič 1 , A. Fakin 2 , M. Hawlina 2 , B. Peterlin 1

1 Institute of Genomic Medicine, UMC Ljubljana, Ljubljana, Slovenia, 2 Eye hospital, UMC Ljubljana, Ljubljana, Slovenia
Introduction: Over 650 heritable conditions with ocular involvement have been described to date. These include isolated eye diseases (retinal degeneration, cataract, corneal dystrophy, optic atrophy, developmental ocular anomalies), as well as complex genetic syndromes with ocular manifestations. This group of disorders is genetically heterogeneous and diagnostically challenging. The aim of our study was to determine the clinical diagnostic utility of exome sequencing in hereditary eye diseases. Materials and Methods: 243 Slovene unselected consecutive patients were referred to our institution from November 2013 to October 2019 because of suspected hereditary eye disease. We performed exome sequencing with the mitochondrial sequence analysis based on the off-target exome reads. Filtered variants were analyzed according to population frequency, characterization in the ClinVar database, the putative impact of the variant, and predicted pathogenicity. Variants were classified according to ACMG standards and guidelines for variant interpretation. Results: We found causative pathogenic or likely pathogenic variants in 102 patients. The diagnostic yield was 50% for retinal degeneration (78/156), 20% for optic atrophy (6/30), 75% for high myopia (3/4), 29% for cataract (2/7), 45% for retinoblastoma (4/9), 20% for developmental ocular anomalies (3/15), 67% for corneal dystrophy (2/3), and 56% for syndromic ocular disease (11/19). Conclusions: The overall diagnostic yield for ophtalmogenetic referral in a cohort of unselected consecutive patients was 42%. Therefore, the exome sequencing is a cost-effective and efficient comprehensive approach that should be considered as a first-tier investigation for diagnostics of hereditary eye diseases. M. Volk: None. N. Teran: None. A. Maver: None. H. Jaklič: None. A. Fakin: None. M. Hawlina: None. B. Peterlin: None. P02.31.C Analysis of recurrent mutations in different Iranian families with hearing loss

M. A. H. Hofrichter 1 , A. Rad 2,3 , S. Kolb 1 , J. Weingart 1 , J. Doll 1 , T. Müller 4 , C. Remmele 5,1 , M. Dittrich 1,4 , R. Maroofian 6 , T. Haaf 1 , B. Vona 1,3

1 Institute of Human Genetics, Julius Maximilians University, Würzburg, Germany, 2 Cellular and Molecular Research Center, Sabzevar University of Medical Sciences, Sabzevar, Iran, Islamic Republic of, 3 Department of Otorhinolaryngology, Head and Neck Surgery, Tübingen Hearing Research Centre (THRC), Eberhard Karls University, Tübingen, Germany, 4 Institute of Bioinformatics, Julius Maximilians University, Würzburg, Germany, 5 Center for Rare Diseases, University Hospital Würzburg, Würzburg, Germany, 6 Genetics and Molecular Cell Sciences Research Centre, St George’s, University of London, Cranmer Terrace, London, United Kingdom
Introduction: Hereditary hearing loss (HL) is one of the most genetically heterogeneous disorders. The genetic landscape of non-syndromic HL includes over 100 genes. In aggregate, these genes harbor several thousand causally implicated variants. The observation of recurrent variants raises the question about whether variants arose from a single ancestral allele or were the result of independent mutational events. In our study that includes 150 Iranian probands, we identified 15 families with recurrent likely pathogenic variants in the genes SMPX , SLC26A4 , TECTA and TMIE . Methods: DNA samples from index patients were exome sequenced following Illumina exome protocols. Libraries were sequenced with a NextSeq500 benchtop sequencer. Variant and copy number variation analysis were performed using GensearchNGS and an in-house exome analysis pipeline. Segregation analysis was performed for all families. Haplotype reconstruction allowed comparison across families. Results: Haplotype analysis of the c.99del variant in SMPX that was identified in three Iranian families suggested a common descent. Interestingly, this X-linked variant was described as a founder mutation in an isolated Newfoundland population. In addition, more than one recurrent mutation was identified in the genes SLC26A4 and TECTA in families with autosomal recessive HL that was consistent with a founder effect. In three additional families, the c.250C>T variant in TMIE was implicated in autosomal recessive HL without sharing further common variants, suggesting a mutational hotspot. Conclusions: The genetic origin of recurrent mutations could be interesting for further characterization of HL genes with respect to ethnic background and susceptibility of mutational events. M.A.H. Hofrichter: None. A. Rad: None. S. Kolb: None. J. Weingart: None. J. Doll: None. T. Müller: None. C. Remmele: None. M. Dittrich: None. R. Maroofian: None. T. Haaf: None. B. Vona: None. P02.32.A Genomic approaches for the analysis fo more than 700 Italian patients affected by Hereditary Hearing Loss (HHL) reveal new genes/alleles .

A. Morgan 1,2 , G. Pelliccione 1 , F. Faletra 1 , D. Dell’Orco 3 , M. Brumat 2 , P. Gasparini 4,2 , G. Girotto 1,2

1 Institute for Maternal and Child Health – IRCCS Burlo Garofolo, Trieste, Italy, 2 Department of Medicine, Surgery and Health Sciences, University of Trieste, Trieste, Italy, 3 Department of Neurosciences, Biomedicine and Movement Sciences, Section of Biological Chemistry, University of Verona, Verona, Italy, 4 Institute for Maternal and Child Health – IRCCS, Burlo Garofolo, Trieste, Italy
Discerning the complex genetic basis of HHL is fundamental to facilitate a correct genetic counselling and molecular diagnosis. The use of high-throughput genome technologies has made great strides in the discovery of new alleles/genes identification. Here, 714 deeply clinically characterized patients were screened with a Targeted Re-Sequencing (TRS) panel of 96 deafness genes and, in negative cases, SNP-arrays were applied. Afterwards, families negative to both approaches were analysed by Whole Exome Sequencing (WES) for new candidates identification. TRS and SNP-arrays characterized ~28% of the HL cases (42% familial and 20% sporadic cases) highlighting STRC and TECTA as the second major players in the Italian population after GJB2 . Then, WES allowed the identification of five new HHL genes, two of them ( SLC12A2 and USP48 ) just identified. As regards SLC12A 2, a heterozygous missense variation (NM_001046.2, exon25, c.3431C>A, p.(T1144N)) has been identified in a three-generation HHL family. The variant is predicted as deleterious by several bioinformatic prediction tools and is not reported in any public database. Interestingly, studies in mouse and zebrafish models demonstrated Slc12a2 expression in the inner ear and its involvement in hearing loss. Moreover, to better evaluate the pathogenic effect of the identified variant, an in silico protein modelling has been performed suggesting that the substitution identified might alter the correct folding of the protein. Thanks to this approach ~35% (60% familial cases and 20% sporadic ones) of cases have been characterized confirming the large mutation’s spectrum of HHL, and the importance of an accurate clinical diagnosis. A. Morgan: None. G. Pelliccione: None. F. Faletra: None. D. Dell’Orco: None. M. Brumat: None. P. Gasparini: None. G. Girotto: None. P02.33.B Genetic causes of paediatric hereditary retinal diseases in Polish population

K. H. Chrzanowska 1 , B. Kocyła-Karczmarewicz 2 , A. M. Tracewska 3 , E. Ciara 1 , M. Rydzanicz 4 , P. Stawiński 4 , R. Płoski 4

1 Department of Medical Genetics, The Children’s Memorial Health Institute, Warsaw, Poland, 2 Department of Ophthalmology, The Children’s Memorial Health Institute, Warsaw, Poland, 3 DNA Analysis Laboratory, PORT Polish Center for Research Technology, Wrocław, Poland, 4 Department of Medical Genetics, Medical University of Warsaw, Warsaw, Poland
Introduction: Every year, Children’s Memorial Health Institute admits approximately 100 children suffering from vision impairment with suspicion of inherited retinal degeneration (IRD), which is confirmed in half of the cases. In many paediatric patients, genetic diagnosis is crucial for prognostics and future planning. Methods: 76 probands and 22 affected family members were ascertained, and a detailed medical and family history was obtained. Ophthalmological assessment consisted, when feasible, of: best-corrected Snellen visual acuity (BCVA), colour vision, visual field test, dilated ophthalmoscopy, colour fundus photography, autofluorescence imaging, spectral-domain optical coherence tomography (SD-OCT), fluorescein angiography and electrophysiological assessment. DNA was isolated from blood samples, and proband samples underwent targeted next-generation sequencing for 108 known IRD genes. Results: In the vast majority of affected individuals the genetic analyses confirmed the initial diagnosis; some diagnoses, however, needed to be revised. Causal variants were found in 2/3 of the families. In five cases, monoallelic pathogenic alterations were found in autosomal recessive genes. Most commonly mutated genes in this cohort were ABCA4 (23 individuals), CNGA3 (5) and CEP290 (4). Conclusions: Genetic analysis is crucial to achieve accurate diagnosis and prognosis, which enables informed choices for the future of the affected children. Many probands were the first individual suffering from IRD in the family. An important role of genetic diagnostics is to inform the parents about their risk for having another child with the disease. Finally, emerging targeted gene therapies indicate a chance of slowing or stopping the disease progression. Funding: National Science Centre (Poland) grant no. UMO-2015/19/D/NZ2/03193. 1 K.H. Chrzanowska: None. B. Kocyła-Karczmarewicz: None. A.M. Tracewska: None. E. Ciara: None. M. Rydzanicz: None. P. Stawiński: None. R. Płoski: None. P02.35.A Diagnostic exome sequencing in 50 patients with high myopia

V. J. M. Verhoeven 1,2 , A. E. G. Haarman 2,3 , M. van Tienhoven 1 , A. A. H. J. Thiadens 2 , S. E. Loudon 2 , A. de Klein 1 , E. Brosens 1 , J. R. Polling 2,4 , A. Bouman 1 , C. C. W. Klaver 5,3,2 , A. J. A. Kievit 1 , L. H. Hoefsloot 1

1 Dept. of Clinical Genetics, Erasmus Medical Center, Rotterdam, the Netherlands, Rotterdam, Netherlands, 2 Dept. of Ophthalmology, Erasmus Medical Center, Rotterdam, the Netherlands, Rotterdam, Netherlands, 3 Dept. of Epidemiology, Erasmus Medical Center, Rotterdam, the Netherlands, Rotterdam, Netherlands, 4 Dept. of Orthoptics, School of Applied Science Utrecht, Utrecht, Netherlands, 5 Dept. of Ophthalmology, UMC St Radboud, Nijmegen, the Netherlands, Nijmegen, Netherlands
Introduction: High myopia (HM; ≤-6 diopters) is a heterogeneous condition, and without clear accompanying features it can be difficult to pinpoint a genetic cause. The aim of this study was to evaluate the diagnostic yield of whole exome sequencing (WES) in patients with HM. Materials and methods: Patients with HM were recruited by ophthalmologists and clinical geneticists. Clinical features were categorized into isolated HM, HM with other ocular features or syndromic HM with systemic involvement. WES was performed using a vision gene panel including 477 genes. Results: WES was performed in 50 patients with HM; the majority had isolated HM (75%). A genetic cause was identified in 11 patients (22%; mean age at diagnosis 9.4 years (SD 7.5); mean refractive error -12.60 diopters (SD 6.22)). Four patients had mutations in known retinal dystrophy genes ( GUCY2D ; FAM161A; PDE6H; CACNA1F ); 3 female patients had a ARR3 mutation (X-linked female limited high myopia); 2 patients had homozygous or compound heterozygous COL18A1 mutations (Knobloch syndrome); 1 patient had a COL2A1 mutation (Stickler syndrome); 1 patient had a PAX6 mutation. In 14 patients (28%) a variant of unknown significance was found; follow-up examinations are necessary. Conclusions: The diagnostic yield of our WES vision gene panel for HM was 22%. It enabled us to diagnose various causes of HM, i.e. retinal dystrophies, connective tissue diseases, and non-syndromic HM, allowing appropriate follow up of systemic and ocular features and genetic counselling. Horizon 2020 (grant 648268); NWO (grants 91815655, 91617076); Oogfonds; ODAS; Uitzicht (grant 2017-28). V.J.M. Verhoeven: None. A.E.G. Haarman: None. M. van Tienhoven: None. A.A.H.J. Thiadens: None. S.E. Loudon: None. A. de Klein: None. E. Brosens: None. J.R. Polling: None. A. Bouman: None. C.C.W. Klaver: None. A.J.A. Kievit: None. L.H. Hoefsloot: None. P02.37.C Genetic architecture of inherited retinal degeneration in Germany: A large cohort study from a single diagnostic center over a 9-year period

N. Weisschuh 1 , C. D. Obermaier 2,3 , A. Bernd 1 , L. Kühlewein 1,4 , F. Nasser 5 , D. Zobor 1 , F. Battke 3 , E. Zrenner 1,6 , E. Weber 1 , B. Wissinger 1 , S. Biskup 2,3 , K. Stingl 5 , S. Kohl 1

1 Institute for Ophthalmic Research, Centre for Ophthalmology, University of Tübingen, Tuebingen, Germany, 2 Praxis fuer Humangenetik Tuebingen, Tuebingen, Germany, 3 Center for Genomics and Transcriptomics, CeGaT GmbH, Tübingen, Germany, 4 University Eye Hospital, Center for Ophthalmology, University of Tübingen, Tübingen, Germany, 5 University Eye Hospital, Center for Ophthalmology, University of Tübingen, Tuebingen, Germany, 6 Werner Reichardt Centre for Integrative Neuroscience (CIN), University of Tübingen, Tübingen, Germany
Introduction: The aim of this cohort study was to unravel the molecular basis of inherited retinal degeneration (IRD) in a large cohort of 2,156 affected patients diagnosed and followed-up in the largest center for inherited retinal diseases in Germany. Our study cohort accounts for approximately 7% of the estimated 30,000 IRD patients in Germany, thereby providing representative data for the prevalence and the mutation spectrum of IRD genes and mutations for the population in Germany. Presently, more than 260 `disease genes’ are known to cause IRDs. The identification of the underlying disease-causing mutations has been accelerated by the introduction of next-generation-sequencing technologies (NGS). Within the field of NGS platforms, the targeted capture of known ‘disease genes’ with its strong optimization of coverage on relevant targets has been proven superior over whole exome sequencing with respect to read depth and on-target efficiency. Materials and Methods: DNA samples were analyzed by targeted NGS and, whenever indicated, multiplex ligation-dependent probe amplification (MLPA). Patients with single-gene disorders were analyzed by Sanger sequencing and MLPA. Results: We achieved an overall molecular diagnostic solving rate of 70.8%. A total of 1,161 distinct variants were identified, including 501 novel variants, reaffirming the known vast genetic heterogeneity of IRD in a mainly outbred European population. Conclusions: Our study demonstrates the clinical utility of panel-based NGS in a large and highly heterogeneous cohort from an outbred population and for the first time gives a comprehensive representation of the genetic landscape of IRDs in Germany. N. Weisschuh: None. C.D. Obermaier: None. A. Bernd: None. L. Kühlewein: None. F. Nasser: None. D. Zobor: None. F. Battke: None. E. Zrenner: None. E. Weber: None. B. Wissinger: None. S. Biskup: None. K. Stingl: None. S. Kohl: None. P02.38.A Retinal disease as the leading clinical manifestation in patients with unexpected inherited systemic disorders detected by whole genome sequencing

A. Liebmann 1,2 , P. Mazzola 1,2 , K. Stingl 3 , T. Heinrich 1,2 , K. Schäferhoff 1,2 , S. Ossowski 1,2 , M. Sturm 1,2 , S. Kohl 4 , N. Weisschuh 4 , B. Wissinger 4 , O. Riess 1,2 , T. Haack 1,2

1 Institute of Medical Genetics and Applied Genomics, University of Tübingen, Tübingen, Germany, 2 Centre for Rare Diseases, University of Tübingen, Tübingen, Germany, 3 University Eye Hospital, Center for Ophthalmology, University of Tübingen, Tübingen, Germany, 4 Institute for Ophthalmic Research, Centre for Ophthalmology, University of Tübingen, Tübingen, Germany
Introduction: Implementation of genome-wide sequencing approaches such as exome and genome sequencing in routine diagnostics implicates the possibility of incidental findings and unexpected diagnoses. Here we present two patients, a 9-year-old girl with cone dystrophy and a 43-year old man with rod-cone dystrophy, initially referred to the Department of Ophthalmology to confirm inherited retinal disease. Material and Methods: Genomic DNA was processed using the TruSeq DNA PCR-free Kit (Illumina) for library preparation and paired-end whole genome sequencing (WGS) was performed on a NovaSeq6000 System (Illumina). Data analysis was conducted using an in-house bioinformatics pipeline optimized for structural/copy number variant detection. Results: WGS of the girl identified a clinically relevant homozygous deletion in CLN3 . The detected deletion c.461-280_677+382del, p.? is the most frequent pathogenic deletion in CLN3 associated with autosomal recessive neuronal ceroid lipofuscinosis. This lysosomal storage disease is characterized by seizures, psychomotor degeneration and visual failure. Clinical course is progressive and unfavorable. WGS of the man identified a pathogenic homozygous missense variant c.635G>T, p.Gly212Val in TMEM126B . Variants of TMEM126B are associated with autosomal recessive complex 1 deficiency. This mitochondrial disease is characterized by a variable clinical phenotype including neuromuscular, cardiovascular and metabolic symptoms and visual impairment. The detected variant has been described before in another patient with rod-cone dystrophy. Conclusions: This study highlights the importance to inform patients about the possibility of retinal disease being the leading clinical manifestation of unexpected inherited systemic disorders. Thus, comprehensive genetic counseling and appropriate documentation of informed consent is crucial prior to performing WGS. A. Liebmann: None. P. Mazzola: None. K. Stingl: None. T. Heinrich: None. K. Schäferhoff: None. S. Ossowski: None. M. Sturm: None. S. Kohl: None. N. Weisschuh: None. B. Wissinger: None. O. Riess: None. T. Haack: None. P02.44.A Shift of T-cell repertoire in cornea of keratoconus patients

L. Skorodumova 1 , A. Kanygina 1 , A. Belodedova 2 , E. Sharova 1 , B. Malyugin 2

1 Federal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological Agency, Moscow, Russian Federation, 2 S. Fyodorov Eye Microsurgery Complex Federal State Institution, Ministry of Health of Russian Federation, Moscow, Russian Federation
Introduction: Keratoconus is a pathologic condition of the eye which results in thinning and cone-shaped deformation of cornea. While its precise cause is still unknown, keratoconus is considered to be a multifactorial disease, and immune processes are assumed to contribute to its development, yet little data is available so far on the immune microenvironment of cornea in affected patients. Here we present the assessment of T cell repertoire in cornea and blood samples from 3 patients with keratoconus who underwent cornea transplantation. Materials and Methods: RNA was extracted from PBMC and cornea samples with RNeasy Micro Kit (Qiagen). RNA libraries of TCR-alpha and TCR-beta transcripts were prepared using SMARTer Human TCR a/b Profiling Kit (Takara Bio USA Inc.) and sequenced using Illumina MiSeq platform. TRA and TRB clonotypes were assembled and quantified with MiXCR package. CDR3 sequences were annotated against VDJdb, PIRD and McPAS-TCR databases. Results: Cornea samples for all 3 patients demonstrated significantly lower clonotype diversity than corresponding PBMC samples. We observed a shift of T cell repertoire in cornea compared to PBMC samples. The top abundant TCR clones in cornea were poorly represented or absent in corresponding blood samples, which indicates that they represent a tissue-specific population. For most of the CDR3 variants detected in cornea, no specificities against known antigens were found, and further research is needed to identify possible functions of these T cell populations. This study was supported by RFBR as part of the research project № 17-29-06077. L. Skorodumova: None. A. Kanygina: None. A. Belodedova: None. E. Sharova: None. B. Malyugin: None. P02.45.B Genomic landscape in spanish patients with ocular malformations by next generation sequencing

A. Damián 1 , A. Tamayo 1 , J. Moya 1 , C. Villaverde 1 , A. Avila-Fernández 1 , A. Arteche 1 , V. López 2 , M. Ballesta 2 , E. Guillen 2 , S. Tahsin Swafiri 1 , I. Lorda 1 , F. Blanco-Kelly 1 , M. Trujillo 1 , C. Ayuso 1 , M. Corton 1

1 Instituto de Investigación Sanitaria Fundación Jiménez Díaz, Madrid, Spain, 2 University Hospital Virgen de la Arrixaca, Murcia, Spain
Human eye development is coordinated by complex developmental processes which involves several hundreds of genes. Any disruption in this regulation during the eye organogenesis can lead to ocular malformations such as Microphthalmia- Anophthalmia-Coloboma (MAC). These diseases are very clinically and genetically heterogeneous conditions with overlapping phenotypes; consequently the mutational load is not well known and the genotype-phenotype correlations can be difficult to define. Our aim was to perform a comprehensive molecular screening in a cohort of Spanish patients with ocular malformations using different approaches of next-generation sequencing (NGS). We have findings in around 52% of the patients. One-third carried likely causal mutations, 8% were partially characterized and 8% had VUS. Different casual mutations were found and the most prevalent in our cohort are transcription factors such as OTX2, SOX2, VSX2 and PAX6. In contrast, others variants have expanded the phenotypic spectrum of some genes classically associated with other phenotypes such as GJA8, KERA, or the lens crystallins family genes suggesting new roles of them in the early development of the eye. This results show that not only NGS is efficient for the genetic analysis of ocular malformations but also for achieving new candidate genes. Therefore, we have update our gene panels resulting in an increase in our diagnostic yield. In contrast, 34% of patients remain uncharacterized, mostly unilateral or non-syndromic cases. So that, our future purposes are to understand the mechanisms involved in this disease by identifying novel genetic causes and exploring their functional role. A. Damián: None. A. Tamayo: None. J. Moya: None. C. Villaverde: None. A. Avila-Fernández: None. A. Arteche: None. V. López: None. M. Ballesta: None. E. Guillen: None. S. Tahsin Swafiri: None. I. Lorda: None. F. Blanco-Kelly: None. M. Trujillo: None. C. Ayuso: None. M. Corton: None. P02.46.C Exome-based panel testing as an efficient method to diagnose the highly heterogeneous ocular disorder spectrum Microphthalmia, Anophthalmia, Coloboma and Anterior Segment Dysgenesis (MAC-ASD)

H. Verdin 1 , T. Rosseel 1 , S. Vermeer 2 , I. Balikova 3 , P. Kestelyn 4 , C. Meunier 5 , F. Meire 6 , J. Van De Velde 1 , O. Vanakker 1 , J. Van Den Ende 7 , M. De Rademaeker 7 , M. Meuwissen 7 , M. De Vries 8 , Y. Sznajer 9 , M. Desmet 1 , T. Sante 1 , F. Coppieters 1 , B. P. Leroy 1,4,10 , E. De Baere 1

1 Center for Medical Genetics, Ghent University Hospital, Ghent, Belgium, 2 Center for Human Genetics, University Hospitals Leuven, Leuven, Belgium, 3 Department of Ophthalmology, University Hospitals Leuven, Leuven, Belgium, 4 Department of Ophthalmology, Ghent University and Ghent University Hospital, Ghent, Belgium, 5 Center for Human Genetics, Institute of Pathology and Genetics, Gosselies, Belgium, 6 Department of Ophthalmology, Hôpital Universitaire des Enfants Reine Fabiola, Brussels, Belgium, 7 Center for Medical Genetics, University of Antwerp and Antwerp University Hospital, Antwerp, Belgium, 8 Department of Ophthalmology, Antwerp University Hospital, Antwerp, Belgium, 9 Center for Human Genetics, Cliniques Universitaires St-Luc, Universite Catholique de Louvain, Brussels, Belgium, 10 Division of Ophthalmology and Center for Cellular & Molecular Therapeutics, The Children’s Hospital of Philadelphia, Philadelphia, PA, United States
Introduction: Microphthalmia and anophthalmia are part of a spectrum with ocular coloboma, a structural malformation resulting from incomplete fusion of the optic fissure, which likely have a shared, although heterogeneous genetic basis. They are collectively named MAC (Microphthalmia-Anophthalmia-Coloboma), characterized by a tremendous clinical heterogeneity and often associated with other ocular abnormalities such as anterior segment dysgenesis (ASD). We aimed to set up an exome-based gene panel test for MAC-ASD. Materials and Methods: Sample preparation was done with the SureSelectXT Low Input Human All Exon V7 kit using Bravo (Agilent Technologies) and pooled libraries were sequenced on a HiSeq 3000 or NovaSeq 6000 (Illumina) aiming for a minimal coverage of 20x for at least 90% of the investigated regions. Data analysis was executed with a bcbio-based pipeline and variant filtering with in-house Seqplorer software. Copy number variant (CNV) analysis was done using ExomeDepth. Results: As of May 2018, 26 patients, 12 with MAC (46.2%) and 14 with ASD (53.8%), underwent MAC-ASD panel analysis. The overall diagnostic yield was 9/26 (34.6%), in line with the reported range (11-36%). ASD has the highest yield (50%), compared to MAC (16.7%). In seven out of the nine cases a (likely) pathogenic variant was identified in ALDH1A3 , CPAMD8 , FOXE3 , MFRP , PAX6 , PITX2 and PITX3 . In two of the nine patients a CNV encompassing FOXC1 was found. Conclusions: Exome-based MAC-ASD panel analysis revealed a molecular diagnosis in 34.6% of a MAC-ASD cohort, highlighting the efficacy of exome-based panel testing in heterogeneous conditions. Funding: FWO/12R3419N; FWO/1802220N; BOF15/GOA/011. H. Verdin: None. T. Rosseel: None. S. Vermeer: None. I. Balikova: None. P. Kestelyn: None. C. Meunier: None. F. Meire: None. J. Van De Velde: None. O. Vanakker: None. J. Van Den Ende: None. M. De Rademaeker: None. M. Meuwissen: None. M. De Vries: None. Y. Sznajer: None. M. Desmet: None. T. Sante: None. F. Coppieters: None. B.P. Leroy: None. E. De Baere: None. P02.47.A The enigma of Midfacial Toddler Excoriation Syndrome (MiTES), a second PRDM12 poly-alanine tract expansion disorder

N. Sarveswaran 1 , G. Woods 1 , C. Moss 2

1 Cambridge Institute for Medical Research, Cambridge, United Kingdom, 2 Birmingham Children’s hospital, Birmingham, United Kingdom
Mid-face toddler excoriation syndrome (MiTES) is a new sensory disease first described in 2017 (PMID: 28028858). We report 12 cases in 7 families. The phenotype is of intense itch in early childhood, which causes mutilating self-injury by scratching. The intense itch is localised only to the midface and no other body parts; is not associated with altered sweating; it spontaneously subsides by 4-6 years; cognition and pain sensing is normal; there is no excess of Staph Aureus infections (on the face or elsewhere). Inheritance appeared recessive, as parents were unaffected, males and females were equally affected, and most cases were consanguineous. Older individuals “grow out” of the condition, facial injuries heal, and have normal whole-body itch and pain sensing. In six of seven families, we found the same mutation in affected individuals - a homozygous poly-Alanine repeat expansion from the normal range (7-13) to 18. Parents were always carriers. This was also found in a non-Asian, Caucasian, non-consanguineous family. In the seventh family, we found only a heterozygous 7 base pair deletion in PRDM12, but no second mutation. What is extraordinary is that a PRDM12 homozygous 19 poly-Alanine repeat expansion causes Congenital Insensitivity to Pain; which is not associated with excess itch, leads to self-inflicted lip, tongue and corneal damage, and always cause excess S. Aureus infections. No other poly-alanine tract expansion disease has two phenotypes. We present our cellular studies to try and understand the disease mechanism whereby a single additional alanine can cause such different phenotypes. N. Sarveswaran: None. G. Woods: None. C. Moss: None. P02.49.C Mutational analysis of 256 Russian patients with congenital aniridia and overlapping phenotypes

T. A. Vasilyeva , A. V. Marakhonov, V. V. Kadyshev, R. A. Zinchenko

Research Centre for Medical Genetics, Moscow, Russian Federation
Congenital aniridia (OMIM#106210) is an autosomal dominant eye disorder mainly caused by heterozygous PAX6 mutations or 11p13 chromosome rearrangements. Pathogenic variants in some other genes could cause overlapping phenotypes, also, PAX6 missense mutations could be associated with other than aniridia phenotypes. A total of 208 unrelated families (256 patients) with clinical diagnoses of either congenital aniridia, counting WAGR syndrome, or overlapping ophthalmological diagnoses characterized by iris hypoplasia were included in the study. Familial to sporadic proportion counted for 44/164. A combination of Sanger PAX6 sequencing, whole-exome sequencing, followed by MLPA analysis of 11p13, 4q25, 6p25.3 loci was implemented. 204 out of 208 families (98%) were found to have either heterozygous pathogenic nucleotide sequence variant or chromosome deletion. Disease-causing variants in PAX6 and deletions in 11p13 were found in 92.7%. Four other genes ( FOXC1 , PITX2 , NDP , CRYAA ) explained 5.3% of the examined families. 30.3% of the cohort carried pathogenic CNVs (62 in 11p13 and 1 in 6p25.3). A frequent 11p13 deletion of PAX6 3′- cis -regulatory region was determined (16/208, 7.7%). The frequency of this deletion in the cohort was higher than PAX6 hotspot c.718C>T rate (9/208, 4.3%). In total, repeated PAX6 variants were determined in 39/208 (18,8%) families, together with 11p13 downstream deletion they explained 55/208 (26.4%) of the families. PAX6 missense variants counted for 6/208 (2.8%). Mutational spectrum peculiarities in a large cohort of Russian families with congenital aniridia were established. Supported by grant RFBR 19-015-00122 and state task of the Ministry of education and science of Russia. T.A. Vasilyeva: None. A.V. Marakhonov: None. V.V. Kadyshev: None. R.A. Zinchenko: None. P02.50.A Revealing the role of enhancers in myopia

J. Swierkowska 1,2,3 , M. S. Tedja 1,2 , A. E. G. Haarman 1,2 , M. Ghanbari 1 , S. J. Erkeland 4 , M. Meester-Smoor 1,2 , C. C. W. Klaver 5,6,7 , V. J. M. Verhoeven 1,2,8

1 Department of Epidemiology, Erasmus Medical Center, Rotterdam, Netherlands, 2 Department of Ophthalmology, Erasmus Medical Center, Rotterdam, Netherlands, 3 Institute of Human Genetics, Polish Academy of Sciences, Poznan, Poland, 4 Department of Immunology, Erasmus Medical Center, Rotterdam, Netherlands, 5 Epidemiology and Ophthalmology Departments, Erasmus Medical Center, Rotterdam, Netherlands, 6 Department of Ophthalmology, Radboud University Medical Center, Nijmegen, Netherlands, 7 Institute of Molecular and Clinical Ophthalmology, Basel, Switzerland, 8 Department of Clinical Genetics, Erasmus Medical Center, Rotterdam, Netherlands
Introduction: Previous studies have indicated a crucial role of the non-coding genome in myopia (nearsightedness), a complex genetic trait. We aimed to identify enhancers, cis-regulatory elements regulating gene expression, associated with myopia leveraging summary statistics of previous genome-wide association studies (GWAS). Materials and Methods: GWAS on myopia related phenotypes (148,485 European, 11,935 Asian individuals) from CREAM and 23andMe were used to examine the association between enhancer-related SNPs and myopia. We obtained enhancer regions (N total =12,244) active in eye-related human tissues from the FANTOM5 database and performed a look-up of these regions in the GWAS. We prioritized enhancers and ANNOVAR determined target genes by ranking system based on LD, eQTLs, enhancer histone marks (HaploReg v4.1) and the association with myopia/ocular phenotype (GWAS catalog). Results: We selected enhancers expressed in ≥3 eye-related tissue types (N=7,944), of which 4,610 enhancers (N SNPs =9,253) were available in the GWAS for analysis. The look-up of these regions yielded 15 significant SNPs clustering in 12 enhancers regions exceeding the Bonferroni significance threshold ( P = 0.05/4,610=1.08x10 −5 ). The highest ranked rs9535268 ( P = 1.04x10 −9 ) and rs4415919 ( P = 3.76x10 −8 ) were annotated to an intergenic enhancer region between RCBTB1 (~16 kb) and ARL11 (~27 kb) genes. Interestingly, SNPs within RCBTB1 were associated with myopia in the GWAS. Conclusions: We selected enhancers with putative target genes, as RCBTB1 (RCC1 and BTB domain containing protein 1), that could explain significant loci identified in GWAS and play a role in myopia pathogenesis. Support: Horizon 2020 (648268); NWO (91815655, 91617076); Uitzicht (2017-28; Oogfonds; Landelijke Stichting voor Blinden en Slechtzienden; MaculaFonds), EMBO (8529). J. Swierkowska: None. M.S. Tedja: None. A.E.G. Haarman: None. M. Ghanbari: None. S.J. Erkeland: None. M. Meester-Smoor: None. C.C.W. Klaver: None. V.J.M. Verhoeven: None. P02.53.A Functional analysis of a novel Myo6 variant in hereditary hearing loss family from Qatar

M. K. Al-Kowari 1,2 , S. Da’as 1 , M. Guarch 1 , D. Abdurahman 1 , W. Hasan 1 , N. Krishnamoorthy 1 , H. Horn 2 , X. Estivill 1,2

1 Sidra Medicine, Doha, Qatar, 2 Biological and Biomedical Program, Hamad Bin Khalifa University, Doha, Qatar
Introduction: Hereditary Hearing Loss (HHL) is a common genetic disorder accounting for at least 60% of pre-lingual deafness in children. The long tradition of consanguinity among Qatari population increases the prevalence of HHL. A novel missense variant in Myo6 was detected in two siblinges diagnosed with severe to profound HL (Alkowari et al. 2017). Prediction tools revealed that this variant is pathogenic and conserved. Materials and methods: We functionally validate the pathogenicity of the novel variant using both cellular and animal models. HeLa cells were transiently transfected with plasmids carrying wildtype and mutated human Myo6 . A zebrafish model was generated using morpholino knock down and co-injection with synthetic RNA ( Myo6 WT and Myo6 p.E60Q ) to further characterize the phenotype. Results: HeLa cells transfected with Myo6 WT and Myo6 p.E60Q , overexpressed the same amount of protein. Cells with Myo6 WT showed localization of the protein in both cytoplasm and the plasma membrane. In contrast, Myo6 p.E60Q is not expressed in the plasma membrane but it’s restricted to the cytoplasm. Zebrafish model resulted in severe otic defects. Myo6 p.E60Q RNA injection resulted in alterations in the saccule that is responsible for hearing in zebrafish ear. Additionally, we found that zebrafish with Myo6 p.E60Q displayed shorter, less abundant and disordered hair bundles in otic hair cells when compared to control and Myo6 WT injected zebrafish. Conclusions: In this study we functionally characterized a novel genetic variant associated with hearing loss; in vitro studies demonstrated that c.G178C variant affects Myo6 protein trafficking. A zebrafish model confirmed the pathogenicity and identified significant otic defects. M.K. Al-Kowari: None. S. Da’as: None. M. Guarch: None. D. Abdurahman: None. W. Hasan: None. N. Krishnamoorthy: None. H. Horn: None. X. Estivill: None. P02.55.C Characterizing a large Iranian cohort of non-syndromic hearing loss families using molecular inversion probe and exome sequencing

A. Rad 1 , R. Maroofian 2 , N. Mazaheri 3 , J. Oostrik 4 , S. Loum 1 , H. Löwenheim 1 , T. Haaf 5 , H. Galehdari 6 , H. Kremer 4 , B. Vona 1

1 Department of Otorhinolaryngology, Head and Neck Surgery, Tübingen, Germany, 2 Department of Neuromuscular Disorders, UCL Queen Square Institute of Neurology,, London, United Kingdom, 3 Department of Genetics, Faculty of Science, Shahid Chamran University of Ahvaz, Ahvaz, Iran, Islamic Republic of, 4 Department of Otorhinolaryngology, Radboud University Medical Center,, Nijmegen, Netherlands, 5 Institute of Human Genetics, Julius Maximilians University, Würzburg, Germany, 6 Department of Genetics, Faculty of Science, Shahid Chamran University of Ahvaz,, Ahvaz, Iran, Islamic Republic of
Introduction: Hearing loss (HL) is the most prevalent sensory deficit. With about 120 genes implicated, non-syndromic hearing loss (NSHL) is extremely genetically heterogeneous. In this study, we investigated extended Iranian families with hereditary NSHL to uncover the genetic landscape and characterize its genetic heterogeneity. Materials and Methods: Using molecular inversion probes (MIPs), we targeted 89 genes involved in NSHL in 20 patients. Exome sequencing (ES) was performed in 102 GJB2 -negative and unresolved MIP-screened NSHL probands. Sanger sequencing, homozygosity mapping, and in vitro functional assays were used to confirm selected variants. Results: Our analysis resolved 50% (10/20) of the MIP-screened patients and about 76% (78/102) of patients subjected to ES. The overall diagnostic rate is about 78.5% (88/112). Nearly half of the diagnostic yield was attributed to the genes SLC26A4 (13.4%), MYO15A (8.0%), LOXHD1 (7.0%), MYO7A (6.2%), CDH23 (5.3%) and TMC1 (4.5%). Our data also identified CLRN2 as a novel NSHL gene in a family. A novel variant in the SPATA5 gene, known to cause epilepsy, hearing loss, and mental retardation syndrome, was implicated in a family with HL as the sole clinical feature. Conclusions: Using two approaches, we could resolve roughly 78.5% of the patients undergoing genetic screening for NSHL. Our cohort also uncovered LOXHD1 as a highly prevalent gene in NSHL diagnoses that was not similarly observed in large cohort screenings of Iranian HL patients. Furthermore, we introduce CLRN2 and SPATA5 as novel autosomal recessive NSHL genes. Select unresolved families from this cohort have been followed-up with genome sequencing. A. Rad: None. R. Maroofian: None. N. Mazaheri: None. J. Oostrik: None. S. Loum: None. H. Löwenheim: None. T. Haaf: None. H. Galehdari: None. H. Kremer: None. B. Vona: None. P02.56.A Multitopographical effect of mutations in the gene PAX6 for pediatric ophthalmology

V. V. Kadyshev , T. A. Vasilyeva, A. V. Marakhonov, S. I. Kutsev, R. A. Zinchenko

FSBSI Research Centre for Medical Genetics, Moscow, Russian Federation
The expression of the PAX6 gene is realized in the formation of almost all structures of the eye - the cornea, iris, lens, optic nerve and central part of the retina. Accounting this fact, the clinical spectrum expands when there is a single pathogenetic process. Materials and methods. The sample of patients with hereditary ophthalmological pathology was 31 children (0-16 years old). The examination included clinical (ophthalmoscopy, biomicroscopy, gonyoscopy, visometry, tonometry), instrumental (electrophysiological, ultrasound, etc.), genealogical and molecular genetic (NGS, direct Sanger sequencing, MLPA) research methods. Results. Accounting the topographical principle, mutations in the PAX6 gene led to lesions in both the anterior and posterior segments of the eyeball. Structures such as the optic disc and/or macular zone (posterior segment) are affected in 100% of cases. The lesion of the anterior segment of the eye is characterized by the involvement of different structures in the process: 6 patients had corneal changes of different grades (ex.; c. 1184-2_1184delAGGinsGGA PAX6 het; c. 1184-2_1184delAGGinsGGA PAX6 het); in 5 cases - pathology of the lens (NM_000280. 4(PAX6_v001):c. 765G>A het synchronous splicing); 7 patients had different grades of iris pathology. One patient with mutation NM_000280.4(PAX6_v001):c.109dupG had a congenital malformation-colobomatous microphthalmos. Two patients had anterior segment dysgenesis (corneal, iris, anterior chamber angle, lens) (c.142-139T>C PAX6 IVS5a-3T>C het; c.718C>T, p.(Arg240*)). Mutations in the PAX6 gene have a multitopographic effect on the eye, with the development of pathology in the anterior and posterior segments of the eyeball. Supported by RFBR grant 18-015-00090. All authors haven’t conflict of interest. V.V. Kadyshev: None. T.A. Vasilyeva: None. A.V. Marakhonov: None. S.I. Kutsev: None. R.A. Zinchenko: None. P02.57.B Nine Tunisian patients with typicalPeters plus syndrome

M. Guirat , S. Guidara, H. Kanoun, I. Boujelbene, N. Gharbi, I. B. Ayed, F. Abdelhedi, H. Kamoun, N. Belguith

Service de génétique médicale, CHU Hedi Chaker, Sfax, Tunisia
Peters plus syndrome is an autosomal recessive rare disorder comprising ocular anterior segment dysgenesis, short stature, hand abnormalities, distinctive facial features. It was related only to mutations in the B3GALTL gene (13q12.3), leading to the inactivation of the B1, 3-glucosyltransferase. In this study, we report 9 patients with typical Peters plus syndrome. Ocular abnormality, facial dysmorphism, and rhizomelic dwarfism were present in all cases. We noted the presence of 2 new anomalies not previously described in Peters Plus syndrome, namely renal malformation and sexual ambiguity. Total sequencing of the B3GALTL gene showed the presence of the splicing mutation (c.597-2A> G) in the B3GALTL gene in 7 patients. This mutation harbours a novel splice site mutation in the B3GALTL gene that modulates the mRNA secondary structure. These data confirm a key role of the genetic study that provides diagnosis confirmation and improves genetic counseling for the families M. Guirat: None. S. Guidara: None. H. Kanoun: None. I. Boujelbene: None. N. Gharbi: None. I.B. Ayed: None. F. Abdelhedi: None. H. Kamoun: None. N. Belguith: None. P02.58.C Severe forms of presbycusis are caused by very rare variants in genes underlying early-onset forms of deafness

S. boucher 1 , F. Wong Jun Tai 2 , A. Lelli 3 , S. Delmaghani 3 , A. Singh-Estivalet 3 , T. Dupont 3 , V. Michel 3 , N. Wolff 4 , A. Bahloul 3 , M. Niasme-Grare 5 , A. Bouaita 3 , D. Oficjalska-Pham 3 , S. Chardenoux 3 , Y. Bouyacoub 6 , D. Bouccara 7 , O. Deguine 8 , B. Fraysse 8 , L. Collet 9 , H. Thai-Van 10 , E. Ionescu 9 , J. Kemeny 11 , F. Giraudet 12 , J. Lavieille 13 , A. Devèze 13 , A. Roudevitch-Pujol 14 , V. Christophe 15 , C. Renard 16 , V. Franco-Vidal 17 , C. Thibult-Apt 17 , V. Darrouzet 17 , E. Bizaguet 18 , A. Coez 19 , A. Aubois 14 , G. Lefèvre 3 , N. Michalski 3 , P. Avan 20 , C. Bonnet 3 , C. Petit 21

1 INSERM UMRS1120, Institut de l’Audition, Pasteur Institute and University Hospital of Angers, Paris, France, 2 INSERM UMRS1120 and Institut de l’Audition, Pasteur Institute, Paris, France, 3 INSERM UMRS1120, Institut de l’Audition, Pasteur Institute, Paris, France, 4 Institut Pasteur, Unité de RMN des Biomolécules, UMR 3528, CNRS, Paris, France, 5 Service de Biochimie et Biologie Moléculaire, Hôpital d’Enfants Armand-Trousseau, AP-HP, Paris, France, 6 INSERM UMRS1120, Pasteur Institute, Paris, France, 7 Hôpital Beaujon, Hôpitaux Universitaires Paris Nord val-de-Seine, APHP, Paris, France, 8 Centre Hospitalier Universitaire de Toulouse, Hôpital Larrey, Service d’Oto-Rhino-Laryngologie et Chirurgie Cervico-Faciale, Toulouse, France, 9 Centre Hospitalier Universitaire, Hôpital Lyon-Sud, Audiologie et Explorations Orofaciales, Lyon, France, 10 Centre Hospitalier Universitaire, Hôpital Lyon-Sud, Audiologie et Explorations Orofaciales and Institut de l’Audition, Lyon and Paris, France, 11 Centre Hospitalier Universitaire de Clermont Ferrand, Hôpital Gabriel Montpied, Service d’Anatomo-Pathologie, Clermont Ferrand, France, 12 Centre Hospitalier Universitaire de Clermont Ferrand, Hôpital Gabriel Montpied, Service d’Anatomo-Pathologie, UMR 1107, INSERM and Centre Jean Perrin, Clermont Ferrand, France, 13 Centre Hospitalier Universitaire de Marseille, Hôpital Nord, Service d’Oto-Rhino-Laryngologie, Marseille, France, 14 Centre Hospitalier National d’Ophtalmologie des Quinze-Vingts, Centre d’Investigation Clinique, Paris, France, 15 Centre Hospitalier Universitaire de Lille, Hôpital Roger Salengro, Service d’Otologie et Otoneurologie, Lille, France, 16 Laboratoire d’Audiologie Renard, Lille, France, 17 Centre Hospitalier Universitaire de Bordeaux, Hôpital Pellegrin, Service d’Oto-Rhino-Laryngologie et Chirurgie Cervico-Faciale, Bordeaux, France, 18 Laboratoire de Correction Auditive, Paris, France, 19 Laboratoire de Correction Auditive and CEA-Inserm U1000 Neuroimaging and Psychiatry and DRM, DSV, Service Hospitalier Frédéric-Joliot, Paris, France, 20 INSERM UMR 1107, Laboratoire de Biophysique Neurosensorielle, Université d’Auvergne, Facultés de Médecine et de Pharmacie, Clermont Ferrand, France, 21 INSERM UMRS1120, Institut de l’Audition, Pasteur Institute and Collège de France, Paris, France
Introduction: Presbycusis is a major public health issue world wide with half its variance due to genetic predisposition. The aim of this study was to identify presbycusis causative genes by whole exome sequencing (WES). Patients and methods: We analysed 105 families, 122 simplex cases and 120 normal-hearing controls. After an in silico bioinformatic analysis to select predicted pathogenic variants, and according to the autosomal dominant mode of inheritance in 95% of the families, we studied very rare heterozygous variants (allele frequency : AF≤0.0001). Results: We found a significative higher ratio of pathogenic variants in autosomal dominant deafness genes carried by 24.7% of mARHL and 21,8% of sARHL (aside from three individuals carrying GJB2 biallelic variants) than in the controls (6,7%) (p < 0,001). By studing a mouse model Tmc1 N321I , we confirmed the pathogenicity of the mutation leading to congenital moderate hearing loss in homozygous mice due to a reduced mechanotransduction current amplitude, and to a progressive hearing loss beginning at 7 months and affecting the high frequencies in heterozygous carriers, validating also the mutation as responsible for a monogenic form of presbycusis. Conclusions: WES unraveled that a large proportion of severe presbycusis is due to new mutations in autosomal dominant deafness genes arguing for a genetic continuum between early-onset deafness and presbycusis. We demonstrated also the existence of monogenic forms of presbycusis that could be targetted by new personnalized therapies like gene therapy. This study was supported by a grant from “Fondation pour l’Audition” and also by the LabEx Lifesenses (ANR-10-LABX-65). S. boucher: None. F. Wong Jun Tai: None. A. Lelli: None. S. Delmaghani: None. A. Singh-Estivalet: None. T. Dupont: None. V. Michel: None. N. Wolff: None. A. Bahloul: None. M. Niasme-Grare: None. A. Bouaita: None. D. Oficjalska-Pham: None. S. Chardenoux: None. Y. Bouyacoub: None. D. Bouccara: None. O. Deguine: None. B. Fraysse: None. L. Collet: None. H. Thai-Van: None. E. Ionescu: None. J. Kemeny: None. F. Giraudet: None. J. Lavieille: None. A. Devèze: None. A. Roudevitch-Pujol: None. V. Christophe: None. C. Renard: None. V. Franco-Vidal: None. C. Thibult-Apt: None. V. Darrouzet: None. E. Bizaguet: None. A. Coez: None. A. Aubois: None. G. Lefèvre: None. N. Michalski: None. P. Avan: None. C. Bonnet: None. C. Petit: None. P02.59.A Genetic analysis of a Dutch discovery cohort suggests an association between primary open-angle glaucoma and the mitochondrial variant m.11915G/A in the MT-ND4 gene

V. Lo Faro 1 , I. Nolte 1 , J. ten Brink 2 , H. Snieder 1 , N. Jansonius 1 , A. Bergen 2

1 UMCG, Groningen, Netherlands, 2 AMC, Amsterdam, Netherlands
Introduction: To determine a possible association between mitochondrial variations and primary open-angle glaucoma (POAG) in a Dutch discovery cohort. Materials and Methods: 140 mtDNA single nucleotide polymorphisms (SNPs) were genotyped using the Illumina Infinium Global Screening Array-24 (GSA) 700K array set. Genetic analysis was performed in a case-control discovery cohort of 734 patients with POAG and 1,417 controls. The variants were classified and analyzed both as single SNPs and as haplogroups. SNP association was performed using logistic regression. The bioinformatics software HaploGrep was used to determine the haplogroup. The Chi-square test and Fisher’s two-sided exact test were used for haplogroups association. Results: For discovery, a nominal association with POAG was found for the allele A of m.11915G/A (rs2853496, p=0.016), within the MT-ND4 gene. The odds ratio was 0.80 (95% confidence interval 0.67-0.95) per copy of the rare allele and adjusted for gender. No haplogroup was associated with POAG in our cohort. Conclusions: The present study reinforces the concept that mitochondrial variation contributes to POAG susceptibility. Further genetic and functional studies are required to highlight the pathophysiological mechanisms underlying primary open-angle glaucoma. V. Lo Faro: None. I. Nolte: None. J. ten Brink: None. H. Snieder: None. N. Jansonius: None. A. Bergen: None. P02.61.C Whole Exome analysis versus a targeted panel for Polish patients with retinal dystrophies

E. Matczyńska 1,2 , P. Łyszkiewicz 1 , A. Wasowska 1,2 , R. Szymańczak 1 , K. Stradomska 1 , E. Suchecka 1 , M. Jędrzejowska 1 , S. Teper 2 , M. Wiącek 3 , A. Machalińska 3 , A. Boguszewska-Chachulska 1

1 Genomed SA, Warsaw, Poland, 2 Chair and Clinical Dept. of Ophthalmology, Medical University of Silesia, Katowice, Poland, 3 Pomeranian Medical University, Szczecin, Poland
A Whole Exome Sequencing (WES) approach was adopted, as a part of the recently accomplished NeuStemGen project, for identifying genetic causes of inherited retinal dystrophies (IRD) in a cohort of Polish patients. Project outcomes prompted the development of a targeted retinal panel, covering most pathogenic variants occurring in the Polish population. WES was performed for 105 patients with clinical symptoms of retinal dystrophies. A targeted panel, consisting of 278 IRD genes (including deep intronic regions for ABCA4 ), was used to sequence 29 IRD samples, including 6 samples not resolved by WES. Bioinformatic analysis was performed using standard BWA-GATK best practice pipeline and several databases, including POLGENOM, for variant frequency filtration. Copy number variants (CNVs) were analysed using the CoNVaDING program. Over 50 pathogenic/potentially pathogenic variants were identified using the WES analysis, involving genes already known to cause IRD. The diagnostic yield of this approach, however, did not exceed 60%. The pilot sequencing of the targeted retinal panel resulted in a significantly higher coverage (389x vs 78x), 98-99% concordance of identified variants and allowed to develop a CNV analysis pipeline. Data obtained using these two approaches were compared and support the decision of applying the large targeted panel for routine diagnostics of retinal dystrophies. This approach, focused on the complete set of retinal genes, assuring a high quality coverage and allowing for reliable analysis of CNVs and deep intronic variants, should be most efficient and cost-effective for a large cohort of Polish patients. Partially supported by STRATEGMED1/234261/2/NCBR/2014 . E. Matczyńska: None. P. Łyszkiewicz: None. A. Wasowska: None. R. Szymańczak: None. K. Stradomska: None. E. Suchecka: None. M. Jędrzejowska: None. S. Teper: None. M. Wiącek: None. A. Machalińska: None. A. Boguszewska-Chachulska: None. P02.63.B A genetic and clinical study of families with non-syndromic retinopathy consequent upon sequence variants in HGSNAT , the gene causing Sanfilippo C mucopolysaccharidosis

E. R. Schiff 1,2 , G. Arno 1,2,3 , K. Pierpoint 1 , R. Ba-Abbad 1,2 , S. Nutan 4 , O. A. Mahroo 1,2,5 , M. Michaelides 1,2 , K. Harvey 6 , A. R. Webster 1,2

1 Genetics Service, Moorfields Eye Hospital, London, United Kingdom, 2 UCL Institute of Ophthalmology, London, United Kingdom, 3 North East Thames Regional Genetics Service, Great Ormond Street Institute of Child Health, London, United Kingdom, 4 London North Genomic Laboratory Hub, Great Ormond Street NHS Foundation Trust, London, United Kingdom, 5 Section of Ophthalmology, King’s College London, London, United Kingdom, 6 Enzyme Unit, Chemical Pathology, Paediatric Laboratory Medicine, Great Ormond Street Hospital for Children NHS Foundation Trust, London, United Kingdom
Distinct genotypes in several genes (e.g. USH2A , FLVCR1 , CEP290 , CLN3 , MFSD8 ) have been shown to be associated with either syndromic or non-syndromic forms of retinitis pigmentosa (RP), a progressive degenerative condition initially affecting rod photoreceptors with subsequent cone phototreceptor loss; RP is the most prevalent form of inherited retinal disease (IRD), which is the commonest cause of blindness in the working-age population of England and Wales. HGSNAT (heparan-α-glucosaminide N-acetyltransferase) sequence variants cause mucopolysaccharidosis type IIIC (MPS-IIIC) or Sanfilippo C syndrome - a severe childhood-onset lysosomal storage disorder. Specific genotypes have, however, been associated with isolated retinal disease in a few reported individuals, thereby further extending the genes that can result in both syndromic and non-syndromic IRD. We have identified likely pathogenic variants in 13 individuals (11 families) with late onset (30s to 60s) and slowly progressing RP. None showed any other symptoms of MPS-IIIC. 7/11 families were compound heterozygotes with the allele c.1843G>A, p.(Ala615Thr). We detected 6 novel sequence variants (p.(Ser296Leu); p.(Phe428Cys); p.(Gly248Ala); p.(Gly418Arg), c.1543-2A>C; c.1708delA), and 5 variants that were previously described in MPS-IIIC patients. In one family, two siblings, genetically identical for the HGSNAT locus, were discordant for retinal disease. A mild but significant reduction of HGSNAT enzyme activity in leukocytes was observed in affected individuals but urinary glucosaminoglycans were not elevated. This case series expands the phenotypic and genotypic spectrum of non-syndromic HGSNAT-associated retinopathy, and in one family suggests the influence of trans -acting modifying factors. Funding: NIHR BRC, Moorfields Eye Hospital and UCL Institute of Ophthalmology. E.R. Schiff: None. G. Arno: None. K. Pierpoint: None. R. Ba-Abbad: None. S. Nutan: None. O.A. Mahroo: None. M. Michaelides: None. K. Harvey: None. A.R. Webster: None. P02.64.C First systematic molecular genetic analysis using NGS analysis of 100 Greek patients with retinal dystrophy

S. Kamakari 1 , S. Koukoula 2 , V. Kokkinou 1 , L. Haer-Wigman 3 , I. Datseris 4 , M. Tsilimbaris 5

1 Ophthalmic Genetics Unit, Athens, Greece, 2 Ophthalmica Institute of Ophthalmology and Microsurgery, Thessaloniki, Greece, 3 Department of Human Genetics, Donders Centre for Neuroscience, Radboud University Nijmegen Medical Centre, Nijmegen, Netherlands, 4 Department of Retinal Disorders, OMMA Ophthalmological Institute of Athens, Athens, Greece, 5 Department of Ophthalmology, University of Crete School of Medicine, Heraklion, Greece
Purpose: Inherited Retinal Dystrophies (IRDs) are characterized by clinical variability and genetic heterogeneity. The aim of this study was to molecularly diagnose 100 Greek patients with different forms of IRDs. Materials and Methods: 100 unrelated Greek patients were analyzed by Next Generation Sequencing (NGS), 13 and 87 of them using a 105 retinal and a 287 ophthalmic gene panel, respectively as described (Ellingford JM et al. J Med Genet 2016, Haer-Wigman L et al. Eur J Hum Genet. 2017). Additional analysis methods were used (Sanger, MLPA, array-CGH) in 6 cases. Results: Potentially pathogenic mutations were detected in 45 retinal dystrophy genes including ABCA4, PRPF31, SPATA7, MERTK, FAM161A, CDHR1, USH2A, CNGB1, PROM1 and RP2 genes. The detection mutation rates were 46.15% (6/13) and 81.6% (71/87) for the 105 and 287 gene panels, respectively. These mutation rates were achieved using complementary methods in 6 cases. Final diagnoses included retinitis pigmentosa, Usher syndrome, cone-rod dystrophy and Leber congenital amaurosis and two rare cases of Knobloch and Oliver-McFarlane syndromes due to mutations in the COL18A1 and PNPLA6 genes, respectively. Conclusions: This is the first systematic investigation of the molecular identity of 100 Greek patients with various subforms of IRDs by NGS and complementary methods leading to an overall mutation rate of 77%. A plethora of novel mutations was documented further expanding the genetic heterogeneity. The molecular identification established the complete diagnosis of the patients thus contributing to family making decision, prognosis and candidacy to current and future treatments. S. Kamakari: None. S. Koukoula: None. V. Kokkinou: None. L. Haer-Wigman: None. I. Datseris: None. M. Tsilimbaris: None. P02.65.A A possible epistatic interaction of two variants in autosomal dominant retinitis pigmentosa

R. A. Zinchenko , T. A. Vasilyeva, V. V. Kadyshev, A. V. Marakhonov

Research Center for Medical Genetics, Moscow, Russian Federation
Retinitis pigmentosa (RP) refers to a heterogeneous group of inherited ocular diseases that result in a progressive retinal degeneration affecting 1 in 3,000 to 5,000 people. To date, more than 80 genetic forms of RP are described. Here we perform genetic analysis of the two-generation family with 5 affected members with the terminal severe stage of RP. Whole exome sequencing (WES) of one of the patients revealed two candidate variants. The first one is pathogenic variant in the exon 16 of SNRNP200 gene (chr2:96958829G>A), leading to the missense substitution (p.Arg681Cys, NM_014014.4) in the heterozygous state. Mutations in the SNRNP200 gene in heterozygous state were described in patients with RP33 (OMIM 610359). The second variant is a novel single nucleotide variant in exon 1 of the PRPH2 gene (chr6:42689991C>T) resulting in the missense substitution (p.Val28Met, NM_000322.4) in a heterozygous state. Mutations in the PRPH2 gene in heterozygous, compound heterozygous and homozygous states were described in patients with Leber congenital amaurosis 18 (OMIM:608133), RP7 (OMIM:608133), retinitis punctata albescens (OMIM:136880), in heterozygous state - in patients with choroidal dystrophy, central areolar 2 (OMIM:613105), macular dystrophy, patterned, 1 (OMIM:169150), macular dystrophy, vitelliform, 3 (OMIM:608161). Segregation analysis revealed SNRNP200 to be co-segregated with the disease while PRPH2 variant was not found in 2 out of 5 patients. Nevertheless, it could make an impact on the clinical presentation by aggravation of clinical picture in patients bearing two variants. Supported by grant RFBR № 18-015-00090 and state task of the Ministry of education and science of Russia. R.A. Zinchenko: None. T.A. Vasilyeva: None. V.V. Kadyshev: None. A.V. Marakhonov: None. P02.68.A Loss of function of RIMS2 , a synaptic membrane exocytosis gene, causes a new phenotype characterized by congenital cone-rod synaptic disease associated with neurodevelopmental and pancreatic involvement

S. Mechaussier 1 , B. Almoallem 2 , C. Zeitz 3 , K. Van Schil 2 , L. Jeddawi 4 , J. Van Dorpe 5 , C. Condroyer 3 , O. Pelle 6 , M. Polak 7 , N. Boddaert 8 , N. Bahi-Buisson 9 , M. Cavallin 10 , J. Bacquet 11 , A. Mouallem-Bézière 11 , O. Zambrowski 11,12 , J. Sahel 3,13,14 , I. Audo 3,14,15 , J. Kaplan 1,11 , J. Rozet 1 , E. De Baere 2 , I. Perrault 1

1 Laboratory of Genetics in Ophthalmology (LGO), INSERM UMR1163, Institute of Genetic Diseases, Imagine and Paris University, Paris, France, 2 Center for Medical Genetics, Ghent University and Ghent University Hospital, Ghent, Belgium, 3 Sorbonne Université, INSERM, CNRS, Institut de la Vision, Paris, France, 4 Pediatric Ophthalmology Division, Dhahran Eye Specialist Hospital, Dhahran, Saudi Arabia, 5 Department of Pathology, Ghent University and Ghent University Hospital, Ghent, Belgium, 6 Cell Sorting Facility, INSERM UMR1163, Institute of Genetic Diseases, Imagine and Paris University, Paris, France, 7 Endocrinology, Gynecology and Pediatric Diabetology Department, University Hospital Necker-Enfants Malades, Paris, France, 8 Department of Pediatric Radiology, Hôpital Necker-Enfants Malades, Paris University, Paris, France, 9 Pediatric Neurology Department, University Hospital Necker-Enfants Malades, Paris, France, 10 Laboratory of Embryology and genetics of human malformation, INSERM UMR1163, Institute of Genetic Diseases, Imagine and Paris University, Paris, France, 11 Service d’Ophtalmologie, Centre Hospitalier Intercommunal de Créteil (CHIC), Créteil, France, 12 Ophthalmology Department, University Hospital Necker-Enfants Malades, Paris, France, 13 Department of Ophthalmology, The University of Pittsburgh School of Medicine, Pittsburgh, PA, United States, 14 Centre Hospitalier National d’Ophtalmologie des Quinze-Vingts, INSERM-DHOS CIC 1423, Paris, France, 15 Institute of Ophthalmology, University College of London, London, United Kingdom
Introduction: Anomalies in pre- and post-synaptic transmission of visual information from photoreceptors to bipolar cells are responsible for stationary congenital night blindness (CSNB). Pre-synaptic disorders, characterized by congenital nystagmus and photophobia, night blindness, are known as cone-rod synaptic disorder (CRSD) can be mistaken for Leber congenital amaurosis (LCA). Full-field electroretinogram is crucial for the differential diagnosis, but it can be difficult to perform in young children. Here, we report (i) the identification of biallelic mutations in the RIMS2 gene encoding a pre-synaptic protein not yet associated with human diseases in 3/4 families initially referred for LCA and (ii) the diagnostic adjustment to CRSD with variable associated alteration including autistic behavior and insulinopathy. Methods: Exome sequencing combined with targeted RIMS2 testing were performed in 46 prescreened patients with CRSD and 94 with LCA. Biallelic RIMS2 -mutated cases underwent ophthalmologic, neurologic and metabolic assessment. RIMS2 immunostaining was performed on human retina, brain and pancreas. Nonsense variants were functionally investigated in mammalian cells. Results: Biallelic RIMS2 sequence variants were found in four unrelated families. RIMS2, a protein regulating synaptic membrane exocytosis localizes in the human retinal outer plexiform layer, Purkinje cells and pancreatic islets. Apart from CRSD, neurodevelopmental disease was observed in all cases, and abnormal glucose homeostasis in the eldest patient. Nonsense variants lead to truncated RIMS2 and decreased insulin secretion in mammalian cells. Conclusions: This is the first report of syndromic CRSD with neurodevelopmental or pancreatic involvement due to RIMS2 loss-of-function. The disease presentation is consistent with the localization and function of RIMS2. S. Mechaussier: None. B. Almoallem: None. C. Zeitz: None. K. Van Schil: None. L. Jeddawi: None. J. Van Dorpe: None. C. Condroyer: None. O. Pelle: None. M. Polak: None. N. Boddaert: None. N. Bahi-Buisson: None. M. Cavallin: None. J. Bacquet: None. A. Mouallem-Bézière: None. O. Zambrowski: None. J. Sahel: None. I. Audo: None. J. Kaplan: None. J. Rozet: None. E. De Baere: None. I. Perrault: None. P02.69.B Whole exome sequencing in Slovak patients with bilateral sensorineural hearing impairment

Z. Slobodova 1,2 , L. Varga 1,2 , S. Borecka 2 , I. Masindova 1 , D. Danis 1 , L. Langova 3 , M. Skopkova 1 , M. Profant 2 , D. Gasperikova 1

1 Biomedical Research Center, Slovak Academy of Sciences, Bratislava, Slovakia, 2 Department of Otorhinolaryngology–Head and Neck Surgery, Faculty of Medicine, Bratislava, Slovakia, 3 Pediatric ENT Department of Medical Faculty and National Institute of Child Diseases, Bratislava, Slovakia
Background : The genetic heterogeneity of sensorineural hearing loss (SNHL) is a major hurdle to the discovery of disease-causing variants. Whole exome sequencing (WES) is a method that allows an efficient analysis of all known genes associated with hereditary hearing loss. Aim : Using WES to identify new genetic causes of hearing impairment in the Caucasian and Roma populations in Slovakia, where standard genetic tests performed so far have not elucidated the etiology of deafness. Patients and methods : We collected 1,369 individuals from 870 families with bilateral SNHL. The previous analysis revealed that 30% of the families have mutations in the DFNB1 locus. From the DFNB1 negative patients we selected 52 families, 42 of Caucasian and 10 of Roma ethnicity, for WES. Results : Using WES, we identified the genetic etiology in 22 probands of Caucasian ethnicity and in 1 proband of Roma ethnicity. The detected variants were found in 18 genes ( COCH , COL4A5 , CREBBP , EDNRB , EYA4 , ILDR1 , LOXHD1 , MYO6 , MYO15A , OTOA , OTOG , P2RX2 , SIX1 , TECTA, TMC1 , TMPRSS3 , DIAPH1, USH1C) harboring 17 pathogenic variants and 11 likely pathogenic variants responsible for hearing impairment. 16 variants have not yet been described in the literature. In one Roma family the causal variants were found in two genes ( PTPRQ and MYO15A ) in one individual. Conclusion : Using WES, we detected the genetic etiology of hearing impairment in 52% probands of Caucasian ethnicity and 10% of Roma probands. WES dramatically increases the detection of pathogenic variants in known genes associated with HL. Supported by: VEGA 1/0214/16, APVV 15-067 Z. Slobodova: None. L. Varga: None. S. Borecka: None. I. Masindova: None. D. Danis: None. L. Langova: None. M. Skopkova: None. M. Profant: None. D. Gasperikova: None. P02.70.C Genotype-phenotype correlations in a Spanish cohort of 506 families with bi-allelic ABCA4 mutations

M. del Pozo-Valero 1 , R. Riveiro-Álvarez 1 , F. Blanco-Kelly 1 , J. Aguirre-Lamban 1 , I. Martín-Mérida 1 , I. Iancu 1 , S. Swafiri 1 , I. Lorda-Sanchez 1 , E. Rodriguez-Pinilla 1 , M. Trujillo-Tiebas 1 , B. Jiménez-Rolando 2 , E. Carreño 2 , I. Mahillo-Fernandez 3 , C. Rivolta 4 , M. Corton 1 , A. Avila-Fernandez 1 , B. García-Sandoval 2 , C. Ayuso 1

1 Department of Genetics, Instituto de Investigación Sanitaria–Fundación Jiménez Díaz University Hospi, Madrid, Spain, 2 Department of Ophthalmology, Instituto de Investigación Sanitaria–Fundación Jiménez Díaz University Hospi, Madrid, Spain, 3 Department of Epidemiology, Instituto de Investigación Sanitaria–Fundación Jiménez Díaz University Hospi, Madrid, Spain, 4 Institute of Molecular and Clinical Ophthalmology Basel (IOB), Basel, Switzerland, Basel, Switzerland
Introduction: ABCA4 is the most mutated gene in inherited retinal dystrophies. A genotype-phenotype correlation model was described in 1999 with few cases reported. Here, we define genotype-phenotype correlations in the largest cohort study worldwide of patients: 434 with Stargardt disease (STGD1) and 72 with cone-rod dystrophy (CRD), all carrying biallelic ABCA4 mutations. Methods: Conventional genetic tools and Next Generation Sequencing technologies. Medical history and ophthalmological data were obtained for 372 patients. Genotype-phenotype correlation studies were carried out for the following variables: variant type, age at onset of symptoms (AO), and clinical phenotype (STGD1 or CRD). Results: Genotype-phenotype correlations showed that patients with biallelic truncating variants represented 41% of CRD and 6.5% of STGD1 patients in our cohort; these cases had a significantly earlier AO than patients carrying two mutations, one of which is a missense variant. CRD patients presented earlier AO than STGD1 patients. Conclusions: Our study, conducted in the largest ABCA4 -associated disease cohort reported to date, supports the genotype-phenotype model established for ABCA4 variants, and broadens the mutational spectrum of the gene. ABCA4 truncating variants were mostly associated with earlier AO and a CRD phenotype. According to our observations, ABCA4 patients presenting with two truncating variants may first present features of STGD1 but eventually develop rod dysfunction, underscoring the importance of an accurate genetic diagnosis. Grants: Instituto de Salud Carlos III (ISCIII), CIBERER (06/07/0036), IIS-FJD Biobank PT13/0010/0012, and FIS (PI16/00425) RAREGenomics-CM (CAM, B2017/BMD-3721), Conchita Rábago Foundation, Autonomous Community of Madrid (CAM, PEJ-2017-AI/BMD7256), Miguel Servet Program (CP12/03256) from ISCIII M. del Pozo-Valero: None. R. Riveiro-Álvarez: None. F. Blanco-Kelly: None. J. Aguirre-Lamban: None. I. Martín-Mérida: None. I. Iancu: None. S. Swafiri: None. I. Lorda-Sanchez: None. E. Rodriguez-Pinilla: None. M. Trujillo-Tiebas: None. B. Jiménez-Rolando: None. E. Carreño: None. I. Mahillo-Fernandez: None. C. Rivolta: None. M. Corton: None. A. Avila-Fernandez: None. B. García-Sandoval: None. C. Ayuso: None. P02.73.C Functional characterization of a Xenopus tropicalis knockout and a human cellular model of RCBTB1 -associated inherited retinal disease shows involvement of RCBTB1 in the cellular response to oxidative stress

M. Carron 1,2 , T. Naert 1 , G. Ascari 2 , S. Demuynck 1 , T. Van Nieuwenhuyzen 1 , T. Rosseel 2 , D. Priem 3,4 , A. Kremer 4 , H. Van Landeghem 1 , S. Hoogstoel 2 , F. Coppieters 2 , C. Guérin 5 , K. Vleminckx 1 , E. De Baere 6

1 Ghent University, Biomedical Molecular Biology, Ghent, Belgium, 2 Ghent University, Center for Medical Genetics and Dept of Biomolecular Medicine, Ghent, Belgium, 3 Ghent University, Ghent, Belgium, 4 VIB-UGent Center for Inflammation Research, Ghent, Belgium, 5 VIB Bioimaging Core, Ghent, Belgium, 6 Ghent University and Ghent University Hospital, Center for Medical Genetics and Dept of Biomolecular Medicine, Ghent, Belgium
Introduction: The function of RCBTB1 , recently implicated in syndromic and non-syndromic inherited retinal disease (IRD), remains unknown so far. Patients with biallelic missense variants in RCBTB1 display diverse IRD phenotypes such as retinitis pigmentosa. Here, we tested the hypothesis that RCBTB1 is involved in NRF2-regulated protection against reactive oxygen species in the eye, more specifically in the retinal pigment epithelium (RPE). Materials and Methods: A Xenopus tropicalis rcbtb1 −/− knockout (KO) was generated using CRISPR/Cas9 editing. Histology and three-dimensional electron microscopy was performed on retinas of rcbtb1 −/− frogs. RNA-seq analysis was performed on RCBTB1 -mutated patients’ lymphocytes, treated with H 2 O 2 , as well as on embryos from the rcbtb1 −/− KOs treated with CdCl 2 . An RCBTB1 knockdown cell line was generated in ARPE-19 cells and functional assays (flow cytometry, MTT-assay, cell death kinetics) assessed the consequences of RCBTB1 loss-of-function. Results: Rcbtb1 −/− animals showed changes in the RPE, similar to observations in human cases, including loss of apical-basal cell polarity, cuboidal cell morphology, spreading of the pigment granules and vacuolisation. NRF2 downstream targets and several metallothioneins were found to be differentially expressed, both in the KO and cellular models. Functional assays in ARPE-19 cells revealed that RCBTB1 depletion affects cellular responses to external insults of oxidative stress. Conclusions: We showed that the Xenopus tropicalis rcbtb1 −/− KO recapitulates the human IRD phenotype. Both in vivo and in vitro functional data show involvement of RCBTB1 in the cellular response to oxidative stress. This provides insight into the mechanism underlying RCBTB1 -associated IRD and uncovers potential therapeutic opportunities. Funding: FWO/1802220N; FWO/1S52818N; BOF15/GOA/011; BOF20/GOA/023. M. Carron: None. T. Naert: None. G. Ascari: None. S. Demuynck: None. T. Van Nieuwenhuyzen: None. T. Rosseel: None. D. Priem: None. A. Kremer: None. H. Van Landeghem: None. S. Hoogstoel: None. F. Coppieters: None. C. Guérin: None. K. Vleminckx: None. E. De Baere: None.

P03 Internal Organs & Endocrinology (Lung, Kidney, Liver, Gastrointestinal)
P03.01.A A cost effective experience in diagnosis of autosomal-dominant polycystic disease by custom-target NGS panel in a reference center .

J. NEVADO 1,2 , R. Mena 1 , R. Peces 3 , M. Melgosa 4 , A. Alonso 4 , R. Selgas 3 , L. Espinosa 4 , P. Barruz 1 , P. D. Lapunzina 1,2

1 INGEMM-IdiPaz, Madrid, Spain, 2 CIBERER, Madrid, Spain, 3 Sº Nefrología Adultos, HULP, Madrid, Spain, 4 Sº Nefrología Infantil, HULP, Madrid, Spain
ADPKD is the most common inherited kidney disease that is caused by mutations in the PKD1 / PKD2 . Most mutations take place in PKD1 . Its molecular diagnosis is a challenge by the high allelic heterogeneity and overlap of pseudogenes between 1-32 exons. So far, there are few data on PKD1 / PKD2 variants in the Spanish population. The main objective of this study has been to establish and know the mutations in ADPKD in our cohort. Mutational analysis of both PKD1 and PKD2 was performed on 158 unrelated families, by NGS using a custom exon-target panel from inherited nephropathies, Nephroseq®. To confirm SNVs, Long-Range-PCR and Sanger sequencing were performed on PKD1 or PCR, and Sanger sequencing for PKD2 . In addition, MLPA was used to rule out CNVs. SNVs were identified in 122 families (77.23%): 96 families in PKD1 (78.7%) and 26 families in PKD2 (22.3%). Globally: 64 frameshift and nonsense; 11 splicing, 36 missense, 8 in frame ins/del, and 4 large-deletions). Overall, 15% of variants were de novo and 50% were not previously described. This study demonstrates that NGS is a cost-effective experimental approach for simultaneous analysis of the PKD1 and PKD2 . The analysis of mutations in the ADPKD-Spanish population expands the understanding of the genetic diversity of the different ethnic groups, enriches the databases with the new mutations, and contributes to their genetic counselling. As well as, reproductive (six pregnancies were currently successful) and/or therapeutic decision-making (a significant number of patients with highly pathogenic variants are currently under treatment with Tolvaptan). J. Nevado: None. R. Mena: None. R. Peces: None. M. Melgosa: None. A. Alonso: None. R. Selgas: None. L. Espinosa: None. P. Barruz: None. P.D. Lapunzina: None. P03.04.A Clinical and molecular characteristics of young Russian patients with homo- and heterozygous AIRE mutations

E. N. Suspitsin 1,2 , L. V. Ditkovskaya 1 , T. V. Gabrusskaya 1 , N. B. Ulanova 1 , D. L. Strekalov 1 , M. N. Guseva 1

1 St.-Petersburg State Pediatric Medical University, St.-Petersburg, Russian Federation, 2 N.N. Petrov Institute of Oncology, St.-Petersburg, Russian Federation
Introduction: Hereditary defects of the AIRE gene are associated with a rare autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy syndrome (APECED). The disorder has been found in many populations worldwide. The clinical manifestations are quite variable even within families, but major features include hypoparathyroidism, Addison disease, and mucocutaneous candidiasis. Although the condition usually follows autosomal-recessive inheritance there is an increasing body of evidence that some cases may be caused by monoallelic gain-of-function (GOF) mutations. Materials and methods: 10 Russian patients (age 3-18 years) were subjected to the AIRE gene analysis. Of those, 5 had clinical signs of APECED syndrome suspected by immunologist, gastroenterologist or endocrinologist. In these patients Sanger sequencing of AIRE was performed. Another 5 patients underwent targeted sequencing of primary immunodeficiency (PID) genes including AIRE because of suspected PID condition. Results: 6 patients had biallelic AIRE mutations with the common c.769C>T (p.R257*) allele being detected in 4 individuals. One patient was homozygous for c.173C>T (p.A58V) variant recently described in Russian patients (Orlova et al, 2017). Rare c.32T>C (p.L11P) mutation was reported in Russian population for the first time. All monoallelic mutations ( n = 4) were represented by the c.901G>A (p.V301M) allele. This variant altering functional properties of the protein was once reported in a Russian female with late-onset classical APECED (Oftedal et al, 2015). Conclusions: Our study indicates that some GOF variants of AIRE are common in young Russian patients with manifestations of autoimmune disease. This work was supported by RFBR grant 17-29-06069 E.N. Suspitsin: None. L.V. Ditkovskaya: None. T.V. Gabrusskaya: None. N.B. Ulanova: None. D.L. Strekalov: None. M.N. Guseva: None. P03.05.B Genetic and molecular analysis of urinary magnesium concentration in Scottish and Croatian populations

C. B. Joseph 1 , C. M. Stanton 1 , C. Drake 1 , T. S. Boutin 1 , J. F. Wilson 2 , O. Polasek 3 , O. Devuyst 4 , T. Hurd 1 , C. Hayward 1

1 MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, United Kingdom, 2 Centre for Global Health Research, Usher Institute for Population Health Sciences and Informatics, University of Edinburgh, Edinburgh, United Kingdom, 3 Split Medical School, Split, Croatia, 4 Institute of Physiology, University of Zurich, Zurich, Switzerland
Magnesium is the second most abundant bivalent cation in the body and is essential for many cellular processes. Renal magnesium handling plays an important role in maintaining magnesium homeostasis, however the exact biological mechanisms remain unclear. A recent genome-wide association study (GWAS) identified an association between urinary magnesium concentration (uMg) and variants in the ARL15 gene on chromosome 5. ARL15 encodes a GTP-binding protein that regulates the magnesium transporter channel TRPM6, and other proteins involved in magnesium homeostasis in physiologically relevant cell lines. We conducted meta-analyses for urinary magnesium in 11, 617 individuals from Scottish and Croatian populations to determine possible genes involved in these traits. Mass spectrometry analysis was conducted to determine interactors of Arl15 and in-vitro knockout models of Arl15 was established using CRISPR-Cas9. The top SNP associated with uMg in our meta-analyses lies within a transcription factor binding site in an enhancer region of ARL15. Individuals homozygous for the alternate allele of rs35931 has lower urinary magnesium levels compared to individuals with the homozygous reference allele. Arl15 interacts with magnesium transporters within the distal convoluted tubule segment of the kidneys. Furthermore, we find that homozygous deletion of Arl15 is lethal at the organismal level. We hypothesise that the variant found affects the expression of ARL15, which, in turn modulates magnesium transport by regulating magnesium transporters. We are performing further functional studies to elucidate the role ARL15 plays in magnesium homeostasis both in vitro and in vivo. C.B. Joseph: None. C.M. Stanton: None. C. Drake: None. T.S. Boutin: None. J.F. Wilson: None. O. Polasek: None. O. Devuyst: None. T. Hurd: None. C. Hayward: None. P03.08.B Blood chimerism in monochorionic dizygotic twins (MCDZT) identified at molecular genetic testing of permanent neonatal diabetes mellitus

B. Lorenz-Depiereux 1 , U. Ahting 2 , G. Eckstein 1 , K. Warncke 3 , T. Meitinger 1,2 , S. Andres 2

1 Helmholtz Zentrum München German Research Center for Environmental Health, Institute of Human Genetics, Neuherberg, Germany, 2 Technical University of Munich, Institute of Human Genetics, Munich, Germany, 3 Technical University of Munich School of Medicine, Kinderklinik München Schwabing, Department of Pediatrics, Munich, Germany
In most cases dizygotic (DZ) twins have a dichorionic placenta, but in some cases - especially in the context of assisted reproduction - blood chimerism in DZ twins due to placental sharing has been shown, identifying them as monochorionic dizygotic twins (MCDZT). Here we report the first case of blood chimerism in MCDZT detected as an additional finding at molecular genetic testing. Dizygotic twin brothers were born to a female couple after assisted reproduction (IVF, in vitro fertilization). One presented with permanent neonatal diabetes mellitus. Molecular genetic analysis identified a heterozygous pathogenic variant within the insulin gene ( INS , dominant inheritance). Interestingly, the donor of the oocytes for IVF had been diagnosed with type 1 diabetes as an infant. Screening for the INS variant in her blood DNA was positive and altered the diagnosis to permanent neonatal diabetes mellitus. Testing of the unaffected brother for the INS variant surprisingly showed that he also carried the INS variant in blood DNA but not in DNA from buccal membrane cells. Additional microsatellite studies confirmed blood chimerism in both twins but ruled out chimerism in buccal membrane cells. Whether our findings should be classified as confined blood chimerism which is relatively common, or as an exceedingly rare reported tissue chimerism has to be elucidated by studies of other tissues. B. Lorenz-Depiereux: None. U. Ahting: None. G. Eckstein: None. K. Warncke: None. T. Meitinger: None. S. Andres: None. P03.10.A Ultra-rare variants in the TRPV6 gene cause autosomal dominant chronic pancreatitis

E. Masson 1,2 , J. M. Chen 1 , V. Rebours 3 , M. Le Rhun 4 , H. Audin 5 , A. Lachaux 6 , B. Caumont 7 , D. Lorenzo 3 , M. Fron 8 , K. Billiemaz 9 , R. Besnard 10 , L. Buscail 11 , GREPAN (Genetic REsearch on PANcreatitis) Study Group, C. Férec 1

1 Univ Brest, Inserm, EFS, UMR 1078, GGB, Brest, France, 2 CHRU Brest, Service de Génétique Médicale et de Biologie de la Reproduction, Brest, France, 3 Department of Gastroenterology and Pancreatology, Beaujon Hospital, APHP, Clichy, and Paris-Diderot University, Paris, France, 4 Service d’Hépato-Gastro-Entérologie, Cancérologie Digestive et Assistance Nutritionnelle, CHU Nantes, Nantes, France, 5 Médecine ‘Chauvet’ à Orientation Gastro-Entérologique, CH Gabriel Martin, Saint Paul, France, 6 Service de Gastroentérologie, Hépatologie et Nutrition Pédiatriques, HCL, Bron, France, 7 Service de Médecine à Orientation Hépato-–Gastro- Entérologique, CH Sud Gironde, Langon, France, 8 Service d’Hépato-Gastro-Entérologie, CHD Vendée, La Roche-Sur-Yon, France, 9 Service de Pédiatrie, CHU Saint-Etienne, Saint-Etienne, France, 10 Service d’Hépato-Gastro-Entérologie et Oncologie Digestive, CHR Orléans, Orléans, France, 11 Service de Gastroentérologie et Pancréatologie, CHU Toulouse, Toulouse, France
Until very recently, variants in the so far reported chronic pancreatitis (CP) genes such as PRSS1 have been found to cause/predispose to the disease through increasing trypsin activity or eliciting endoplasmic reticulum stress. In January 2020, an international collaboration reported association of functionally deficient variants in the TRPV6 gene with CP, identifying a novel mechanism of pancreatitis due to Ca 2+ dysregulation. This discovery was made essentially by analyzing young patients with idiopathic CP. Whether TRPV6 variants are responsible for autosomal dominantly inherited CP remains unknown. Herein, we analyzed the TRPV6 gene in probands of 235 such families by NGS. All families had at least two affected members and had remained genetically unexplained prior to the current analysis. We identified 11 ultra-rare variants (all were absent in 570 French controls and had an allele frequency of 0-0.000008 in gnomAD) in 11 families. Of the 11 variants, 5 were previously reported to be pathogenic. All the remaining six variants were novel, comprising a clear loss-of-function variant, a single amino acid deletion variant, and four missense variants. All these four missense variants were predicted to be of pathogenic relevance by Polyphen and/or SIFT and had a CADD score comparable to known pathogenic missense variants. In summary, we identified ultra-rare TRPV6 variants in ~5% of the studied families. This establishes TRPV6 as a relatively frequent autosomal dominant CP gene for which genetic testing is warranted. Our results also suggest that haploinsufficiency of the TRPV6 gene is sufficient to cause CP. (Work supported by INSERM). E. Masson: None. J.M. Chen: None. V. Rebours: None. M. Le Rhun: None. H. Audin: None. A. Lachaux: None. B. Caumont: None. D. Lorenzo: None. M. Fron: None. K. Billiemaz: None. R. Besnard: None. L. Buscail: None. C. Férec: None. P03.12.C Truncating LBX1 mutation in Congenital Central Hypoventilation Syndrome

M. Elbracht , M. Begemann, I. Kurth

Institute of Human Genetics, Aachen, Germany
Undine syndrome, a congenital central hypoventilation syndrome (CCHS), derives its name from the legend of the water nymph Ondine/Undine who punished her mortal husband for his unfaithfulness with a curse of stopping breathing and dying when he fell asleep. Normally, increasing CO 2 blood levels induce the hypercapnic reflex which accelerates breathing via specialized neurons of the pre-Bötzinger complex in the ventral hindbrain. Polyalanine expansions in PHOX2B and rarely other genes have been described to cause genetic forms of CCHS by disturbing the breathing reflex. Recently, a homozygous mutation in the homeodomain transcription factor LBX1 was reported in two brothers with neonatal hypoventilation and complex autonomic symptoms. The mutation affects the C-terminus of the protein and impairs cooperativity with PHOX2B, without disrupting the DNA-binding domain. We here report a female patient with severe hypoventilation who required continuous mechanical ventilation immediately after birth. During her first 11 years of life she was supplied with a tracheostomy and mask ventilation at night because of episodes of apnea. In childhood, she had severe constipation and, developed a paralytic ileus like Hirschsprung disease. Since the age of 11 she used a respiratory mask for sleeping and showed normocapnia. With consequent breathing control starting immediately after birth the now 23 years old woman developed excellently and completes a university education. Trio exome sequencing identified a homozygous frameshift-mutation in LBX1 (hg19: chr10:102987166del, NM_006562.4:c.707del, p.(Val236Alafs*59)). Our patient corroborates the role of C-terminal LBX1 mutations in CCHS and emphasizes the favorable clinical outcome in case of consequent treatment. M. Elbracht: None. M. Begemann: None. I. Kurth: None. P03.13.A Deletion and duplication: unusual case of diagnostics for patient with cystic fibrosis

S. Deryabina 1,2 , O. Lagutina 1 , N. Nikitina 1 , E. Nikolaeva 1

1 Medical Center “Health Care of Mother and Child”, Yekaterinburg, Russian Federation, 2 Federal State Autonomous Educational Institution of Higher Education «Ural Federal University named after the first President of Russia B.N. Yeltsin, Yekaterinburg, Russian Federation
Background: Cystic fibrosis (CF) is an autosomal recessive disease caused by a mutation in the CFTR (cystic fibrosis transmembrane conductance regulator) gene. Newborn screening for CF was introduced in the Russian Federation in 2006. The incidence of CF varies among different regions, in Sverdlovsk region it is 1: 9,500. The vast majority of recurrent CFTR variants are point mutations, but several populations have larger deletions and duplications, involving one or more complete exons. Here we want to report a case of CF with simultaneously existing deletion and duplication of different regions of 11 exon CFTR . Case presentation: The patient (a 1-month-old girl) was born at term to non-consanguineous parents. For newborn’s diagnostics CF we use a three-step IRT/IRT1↑/DNA algorithm. Our patient had elevated levels of immunoreactive trypsinogen both times (328.6 ng/ml and 299.8 ng/ml respectively). Moreover, the girl had pathological sweat chlorides - 110 mmol/L. Results: The trio genetic sequencing of the CFTR gene showed heterozygous delF508 mutation in exon 11 for the child and her father. For searching another variant we were done Multiplex Ligation-mediated Probe Amplification (MLPA) of the CFTR gene. We have found heterozygous deletion of exon 9 for the girl and her mother and have proved the deletion F508 for father, but this analysis has shown also duplication of exon 11 and 2 copies F508 for proband. Conclusions: We assume the proband has 3 copies of exon 11: two normal and one with del508F, and it is the defect copy, that is crucial for protein synthesis. S. Deryabina: None. O. Lagutina: None. N. Nikitina: None. E. Nikolaeva: None. P03.14.B Genotypic spectrum of cystic fibrosis in Cyprus with emphasis on the endemic L346P and the novel CFTRdup2 cases

A. Matthaiou 1 , P. Anagnostopoulou 1,2 , P. Kouis 1 , V. Neocleous 3 , T. Adamidi 4 , P. Ioannou 5 , P. Fanis 3 , C. Costi 3 , A. Georgiou 4 , L. A. Phylactou 3 , P. Yiallouros 1,5

1 Medical School, University of Cyprus, Nicosia, Cyprus, 2 Institute of Anatomy, University of Bern, Bern, Switzerland, 3 Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus, 4 Department of Pulmonology, Nicosia General Hospital, Nicosia, Cyprus, 5 Pediatric Pulmonology Unit, Archbishop Makarios III Hospital, Nicosia, Cyprus
Introduction: The aim of this study was to describe the epidemiology of CFTR mutations in the cystic fibrosis population in Cyprus with particular emphasis on the endemic L346P (p.Leu346Pro) cases and a novel case which bears the poorly described CFTRdup2 in homozygosity. Materials and Methods: The national cystic fibrosis patients’ registry in Cyprus was searched for the demographic, diagnostic, and genotypic data of all cases whose diagnosis was genetically confirmed via the identification of two mutant CFTR alleles. In all cases, genotyping was done by Sanger sequencing for the detection of common mutations and was followed by next-generation sequencing for the identification of uncommon mutations. Results: From an overall of 47 cases, the most common mutation was p.Phe508del with an allelic frequency of 46.9%, which is compatible with its northwest-to-southeast decreasing frequency gradient in Europe. Most mutations were classified as CFTR protein processing (class II, 50%) or conductance (class IV, 12.3%) defects. Multiple mutations were detected in 3 cases. Seven patients with mild clinical manifestations, mostly presenting with dehydration and/or electrolyte imbalance, carried p.Leu346Pro (7.1%), a class IV mutation detected solely in individuals of Cypriot descent. A novel case of a patient homozygous for the CFTRdup2 mutation was associated with a severe phenotype with hepatic involvement, biliary cirrhosis and eventually hepatic failure leading to death. Conclusions: The small population of cystic fibrosis patients in Cyprus follows the regional genotypic trends, although several cases bear unique mutational profiles, which contribute to the wide genotypic and phenotypic spectrum of the disease. A. Matthaiou: None. P. Anagnostopoulou: None. P. Kouis: None. V. Neocleous: None. T. Adamidi: None. P. Ioannou: None. P. Fanis: None. C. Costi: None. A. Georgiou: None. L.A. Phylactou: None. P. Yiallouros: None. P03.16.A Distinct regulatory elements enhance SLC6A14 expression modifying cystic fibrosis pancreas and lung phenotypes

M. Esmaeili 1 , N. Panjwani 1 , G. He 1,2 , F. Lin 1 , J. M. Rommens 1,3 , L. J. Strug 1,2

1 Program in Genetics and Genome Biology, The Hospital for Sick Children, Toronto, ON, Canada, 2 Division of Biostatistics, Dalla Lana School of Public Health, University of Toronto, Toronto, ON, Canada, 3 Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada
Introduction: Cystic Fibrosis (CF) individuals with the same CFTR mutations have variable lung and pancreatic disease severity. Genome wide association studies (GWAS) have indicated that SNP clusters in the immediate promoter region and far upstream of SLC6A14 modify meconium ileus (MI) occurrence at birth and progressive lung disease, respectively. SLC6A14 is a sodium- and chloride-dependent neutral and cationic amino acid transporter that is expressed in several tissues including lung and pancreatic duct. Materials and Methods: We are using public data and in vitro transcriptional reporter studies to understand how the SLC6A14 promoter and upstream SNPs contribute to modification of CF. Results: Colocalizing SLC6A14 promoter SNPs with expression quantitative trait loci (eQTL) of multiple organs from Genotype-Tissue Expression (GTEx) suggests that the pancreas drives MI susceptibility and indicates that the eQTL effect is driven primarily by males. Reporter assays in the CFPAC-1 pancreas cell line with a 2.4kb segment containing MI-associated SNPs display increased gene expression with the risk haplotypes, and exhibit transrepression with a steroid hormone receptor. The core promoter activity and haplotype effects were also evident in the lung cell lines Calu-3 and HBE. Analyses of ReMap and JASPAR databases pinpoint a series of transcription binding elements in the upstream region for ongoing investigations to understand how these segments crosstalk with the core promoter of SLC6A14 in the lung. Conclusions: Our studies implicate differences in gene regulation of SLC6A14 in the lung versus the pancreas, with increased expression level being detrimental to CF phenotypes in both organs. M. Esmaeili: None. N. Panjwani: None. G. He: None. F. Lin: None. J.M. Rommens: None. L.J. Strug: None. P03.17.B Impact of next-generation sequencing on diagnosis of cystic kidney diseases

L. Obeidová 1 , T. Seeman 2 , V. Elisakova 1 , K. Hirschfeldova 1 , I. Zednikova 1 , B. Janosikova 1 , M. Urbanova 1 , F. Liska 1 , O. Seda 1 , B. Chylikova 1 , J. Stekrova 1

1 Institute of Biology and Medical Genetics of the First Faculty of Medicine, Charles University and General University Hospital in Prague, Prague, Czech Republic, Prague, Czech Republic, 2 Department of Pediatrics, 2nd Faculty of Medicine, Charles University in Prague and Motol University Hospital, Prague, Czech Republic, Prague, Czech Republic
Introduction: Cystic kidney diseases are a very heterogeneous group of chronic kidney diseases with sometimes ambiguous phenotype. Thus, the clinical diagnosis is often difficult to be made. The aim of our study was to test the ability of next-generation sequencing (NGS) to improve the diagnosis precision in a heterogeneous group of children with clinically suspected cystic kidney diseases. Materials and Methods: Next-generation sequencing of genes responsible for the formation of cystic kidneys was performed in 32 unrelated patients with various clinically diagnosed cystic kidney diseases without a definitive genetic diagnosis. Results: The underlying pathogenic variants were detected in 69% of patients ( n = 22). The result of NGS correlated with the clinical diagnosis made before the NGS in 53% of patients ( n = 17), in the remaining 15 children (47%) the result of NGS revealed another type of cystic kidney disease than clinically suspected or did not find any causal mutation. The most common unexpected findings were variants in nephronophthisis (NPHP) genes in children with clinically suspected autosomal recessive polycystic kidney disease ( n = 4). Overall, we found 25 (probably) pathogenic variants in the PKHD1 gene, 8 variants in the TMEM67 gene, 4 variants in the PKD1 gene, 2 variants in the HNF1B gene and 2 variants in BBS1 and NPHP1 genes, respectively. Conclusions: NGS is a valuable tool in the diagnostics of various forms of cystic kidney diseases. Its results changed the clinically based diagnoses in 15% of the children. Supported by the ESIF - Operational programme Prague CZ.07.1.02/0.0/0.0/17_049/0000828, GAUK 1015 and PROGRES- Q25/LF1 L. Obeidová: None. T. Seeman: None. V. Elisakova: None. K. Hirschfeldova: None. I. Zednikova: None. B. Janosikova: None. M. Urbanova: None. F. Liska: None. O. Seda: None. B. Chylikova: None. J. Stekrova: None. P03.18.C Circulating miRNA as a predictive biomarker of type 2 diabetes in prediabetic patients-outcomes of 5-year prospective observational study

I. Sidorkiewicz , M. Niemira, K. Maliszewska, A. Bielska, A. Szałkowska, J. Raczkowska, G. Sokołowska, M. Górska, A. Krętowski

Medical University of Bialystok, Bialystok, Poland
Introduction: Type 2 Diabetes Mellitus (T2DM) remains the most prevalent form of diabetes. The detection of current T2DM diagnostic markers is generally late and occurs when displaying metabolic imbalance thus, novel biomarkers that could be useful as a new tool for predicting the progression from prediabetes to T2DM are needed. The goal of this study was to conduct a baseline comparison of serum-circulating miRNA in prediabetic individuals with distinction between those who later progressed to T2DM and those who did not. Materials and methods: The study group was divided – patients with newly diagnosed T2DM over 5 year observational period ( n = 18) and control group ( n = 12) with no known history of dysglycemia. The expression level of 798 miRNAs in serum samples using the NanoString technology was examined. Results: The study revealed that 18 miRNAs were deregulated in serum samples of patients who developed T2DM after 5 years versus non T2DM patients. Pathway analysis showed that miRNA target genes were mainly significantly enriched in homeostasis, neuronal signaling, protein complex oligomerization and core matrisome. ROC analysis demonstrated that miR-491-5p, miR-1307-3p, miR-298 and miR-1290 can be introduced as a diagnostic tool for the prediction of T2DM (AUC=0.940; 0.894; 0.877; 0.722, respectively). Conclusions: The results suggest that circulating miRNAs could potentially be used as predictive biomarkers of T2DM in prediabetic patients. I. Sidorkiewicz: None. M. Niemira: None. K. Maliszewska: None. A. Bielska: None. A. Szałkowska: None. J. Raczkowska: None. G. Sokołowska: None. M. Górska: None. A. Krętowski: None. P03.20.B Identification of a germline interstitial deletion in14q32.13 in a child and mother with DICER1 syndrome

I. Wieland 1 , I. Schanze 1 , A. Gnekow 2 , M. Kuhlen 2 , F. Maier 3 , D. Wahl 3 , M. Frühwald 2 , M. Zenker 1

1 Institute of Human Genetics, University Hospital, Otto-von-Guericke University, Magdeburg, Germany, 2 Swabian Children’s Cancer Center, University Children’s Hospital, Augsburg, Germany, 3 Praxis für Humangenetik, Augsburg, Germany
Introduction: DICER1 syndrome (MIM 601200) is a rare tumor predisposition syndrome with autosomal dominant inheritance usually manifesting in childhood and adolescence. Tumor development follows Knudson’s two-hit model involving the gene DICER1 located on chromosome 14q32.13. Predisposing germline pathogenic variants (first hit) are mainly small loss-of-function mutations. Tumor-related driver mutations are specific hot-spot missense mutations in trans (second hit) located in the RNAse IIIb domain of the DICER1 protein. Patients: We report on a female child with cystic nephroma of the right kidney and developmental delay at age 1 year. Facial features included frontal bossing, flat nasal bridge, hypertelorism and small mouth with thin lips. Body measurements were unremarkable. Her mother was reported with Sertoli-Leydig cell tumor (SLCT) and multinodular goiter at age 12 to 16 years. Clinical presentation of mother and child suggested familial inheritance of DICER1 syndrome. Results: No typical DICER1 loss-of-function germline mutation was detected in the child by standard Sanger sequencing. The child’s tumor tissue of the right kidney revealed DICER1 hot-spot mutation c.5437G>A p.Glu1813Lys. Subsequent molecular karyotyping of DNA from blood of the child using Affymetrix CytoScan HD SNP-Array showed a 2.31MB interstitial contiguous gene deletion in 14q32 (arr[hg19] 14q32.12q32.13(93835222_96155830)x1). The interval harbors 23 OMIM-listed genes including DICER1 . The array result was finally confirmed in the blood of child and mother by MLPA. Conclusions: Patients with DICER1 syndrome and germline 14q32 contiguous gene deletions infrequently have been detected. In the few patients reported to date various deletion intervals were identified suggesting no consistent chromosomal breakpoints. I. Wieland: None. I. Schanze: None. A. Gnekow: None. M. Kuhlen: None. F. Maier: None. D. Wahl: None. M. Frühwald: None. M. Zenker: None. P03.22.A Two cases with central precocious puberty caused by paternally inherited novel variants in DLK1gene

V. Karaman 1 , E. Karakılıç Özturan 2 , F. Baş 2 , S. Başaran 1 , Z. Uyguner 1

1 Department of Medical Genetics, Istanbul Faculty of Medicine, Istanbul University, Istanbul, Turkey, 2 Department of Pediatric Endocrinology, Istanbul Faculty of Medicine, Istanbul University, Istanbul, Turkey
Introduction: Gonadotropin-dependent or central precocious puberty (CPP) is caused by premature activation of the hypothalamic-pituitary-gonadal (HPG) axis. There are several genes playing roles in this pathway and two of them are maternally imprinted; 1) MKRN3 gene, located at 15q11.2, upstream suppressor of HPG axis, 2) DLK1 gene, located at 14q32, affecting kisspeptin signaling pathway hence important role for regulating pubertal timing. Materials and Methods: Coding regions of the MKRN3 (NM_005664.3) and DLK1 (NM_003836.6) genes were Sanger sequenced in 17 patients (15 female, 2 male). In presence of pathogenic variant, segregation analysis were performed in families. Results: We identified two novel heterozygous variants in two unrelated cases in DLK1 gene. The first variant was in exon 4 (c.357C>G (p.Tyr119Ter)) and the second was in intron 1 (c.67+78C>T), both were classified under probably pathogenic by in silico analysis. Segregation analysis presented the paternal inheritance of both, complying with the inheritance model of the genes. No pathogenic variant identified in MKRN3 gene in our cases. Conclusions: Previously, three small and one gross deletions of DLK1 were associated with CPP. Nonsense and splicing variant have first time presented in our study. The alteration identified in intron one needs further investigation on transcript level to show whether it causes splicing defect. Nevertheless, inheritance pattern complies with imprinted gene model of DLK1 . These results suggest that investigation of DLK1 gene may contribute to the elucidation of the genetic assosiation of familial CPP. This study was supported by Scientific Research Projects Coordination Unit of Istanbul University (TDK-2018-32682) V. Karaman: None. E. Karakılıç Özturan: None. F. Baş: None. S. Başaran: None. Z. Uyguner: None. P03.23.B Elucidating the molecular mechanism underlying laterality defects

D. Antony 1,2,3 , A. Maver 4 , M. A. Deardorff 5,6 , L. Slagter 2 , K. Wu 2 , Z. Bakey 1,2 , E. Y. Gulec 7 , A. Gezdirici 7 , H. Brunner 2,3 , M. Schmidts 1,2,3

1 Center for Pediatrics and Adolescent Medicine, University Hospital Freiburg, Freiburg, Germany, 2 Genome Research Division, Department of Human Genetics, Radboud UMC, Nijmegen, Netherlands, 3 Radboud Institute of Molecular Life Sciences, Nijmegen, Netherlands, 4 Centre for Mendelian Genomics, Clinical Institute of Medical Genetics, UMC Ljubljana, Ljubljana, Slovenia, 5 Department of Pediatrics and Pathology, The Perelman School of Medicine, University of Pennsylvania, Pennsylvania, PA, United States, 6 Laboratory Medicine, The Perelman School of Medicine, University of Pennsylvania, Pennsylvania, PA, United States, 7 University of Health Sciences, Kanuni Sultan Suleyman training and research Hospital, Department of Medical Genetics, Istanbul, Turkey
Introduction: Laterality defects are rare developmental disorders, occurring isolated or as part of complex syndromes. Underlying cause is frequently dysfunction of motile cilia causing Primary Ciliary Dyskinesia (PCD) and rarely, non-motile cilia defects or non-ciliary causes. In addition to randomization of the left right body axis, PCD is characterized by frequent respiratory infections and infertility. Methods: We used Whole Exome Sequencing (WES) to delineate the underlying molecular cause in 37 mainly consanguineous families with laterality defects. Results: We identified causative variants in 32% with majority of mutations detected in genes previously associated with PCD, including 2 small homozygous CNVs. Variants in non PCD genes, PKD1l1 and GJA1 were also detected. We further identified homozygous null mutations in two genes not previously associated with human disease, MNS1 and DNAH9 , both in cases with situs inversus but little or no respiratory symptoms. Gene matcher database revealed two additional families with biallelic DNAH9 mutation and immunofluorescence analysis revealed complete absence of DNAH9 from respiratory cilia of one affected while DNAH9 localises to the distal half of cilia in controls. Further, we found direct interaction of DNAH9 with the ODA docking complex protein CCDC114 using Y2H screening also interaction with DNAH5 and DNAI2 using co-Immunoprecipitation. Conclusions: We could only solve a third of the laterality cases genetically using WES, pointing towards non coding variations, genetic mosaicism or epigenetic effects while we were able to identify two novel human disease causing genes, MNS1 and DNAH9 , both causing laterality defects with no or little respiratory disease.ERC: TreatCilia D. Antony: None. A. Maver: None. M.A. Deardorff: None. L. Slagter: None. K. Wu: None. Z. Bakey: None. E.Y. Gulec: None. A. Gezdirici: None. H. Brunner: None. M. Schmidts: None. P03.24.C Insights into the proteomics of renal function: a trans-ethnic meta-analysis and Mendelian randomization study

P. R. Matias-Garcia 1,2,3 , R. Wilson 1,2 , Q. Guo 4 , S. B. Zaghlool 5 , S. Sharma 1,2 , P. Schlosser 6 , A. Köttgen 6,7 , A. Peters 2,8,9 , D. O. Mook-Kanamori 10 , J. Graumann 11,12 , W. König 8,13,14 , K. Hveem 15,16 , C. Jonasson 15,16 , A. Butterworth 4 , K. Suhre 5 , C. Gieger 1,2,8 , A. Teumer 17,18 , M. Waldenberger 1,2,8

1 Research Unit of Molecular Epidemiology, Helmholtz Zentrum München, German Research Center for Environmental Health, Neuherberg, Germany, 2 Institute of Epidemiology, Helmholtz Zentrum München, German Research Center for Environmental Health, Neuherberg, Germany, 3 School of Medicine, Technical University of Munich, Munich, Germany, 4 Cardiovascular Epidemiology Unit, Department of Public Health and Primary Care, University of Cambridge, Strangeways Research Laboratory, Wort’s Causeway, Cambridge, United Kingdom, 5 Department of Physiology and Biophysics, Weill Cornell Medicine-Qatar, Doha, Qatar, 6 Institute of Genetic Epidemiology, Faculty of Medicine and Medical Center-University of Freiburg, Freiburg, Germany, 7 Department of Epidemiology, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, United States, 8 German Research Center for Cardiovascular Research (DZHK), partner site Munich Heart Alliance, Munich, Germany, 9 German Center for Diabetes Research (DZD), München-Neuherberg, Neuherberg, Germany, 10 Department of Clinical Epidemiology, Leiden University Medical Centre, Leiden, Netherlands, 11 Scientific Service Group Biomolecular Mass Spectrometry, Max Planck Institute for Heart and Lung Research, W.G. Kerckhoff Institute, Bad Nauheim, Germany, 12 German Centre for Cardiovascular Research (DZHK), partner site Rhine-Main, Max Planck Institute of Heart and Lung Research, Bad Nauheim, Germany, 13 Deutsches Herzzentrum München, Technical University of Munich, Munich, Germany, 14 Institute of Epidemiology and Medical Biometry, University of Ulm, Ulm, Germany, 15 K.G. Jebsen Center for Genetic Epidemiology, Department of Public Health, Norwegian University of Science and Technology, Trondheim, Norway, 16 HUNT Research Center, Department of Public Health, Norwegian University of Science and Technology, Levanger, Norway, 17 Institute for Community Medicine, University Medicine Greifswald, Greifswald, Germany, 18 German Center for Cardiovascular Research (DZHK), partner site Greifswald, Greifswald, Germany
Introduction: Renal health is reflected in the kidneys’ ability to filter blood, clinically measured as the estimated glomerular filtration rate (eGFR). A number of plasma proteins have been found to be associated with renal phenotypes, but prior studies have been limited by smaller sample sizes and fewer proteins. Moreover, the causal mechanisms underlying these associations have not been investigated. Methods: We conducted the largest observational study of plasma proteins and renal function in terms of sample size (up to N=5,285 across four studies: KORA, INTERVAL, HUNT, QMDiab) and number of proteins (k=1,100). Associations with eGFR and chronic kidney disease (CKD) were tested using regression models in a trans-ethnic discovery-replication approach. We further examined the causal nature of these associations using two-sample bidirectional Mendelian randomization. Results: 57 proteins were found to be consistently associated with eGFR in population samples of European and admixed ancestry; of these, 18 were associated with CKD. Many well-known biomarkers of renal function (e.g. cystatin-C) were identified, as were JAM-B and contactin-4 as novel biomarkers. The strongest evidence of causality was observed in the positive effect of eGFR on testican-2 (p=2.84E-04), a finding consistent with the biological role of testican-2 in glomeruli remodeling and kidney injury repair. Conclusions: Our study uncovers novel plasma proteomic biomarkers for renal function and presents evidence for selected causal relationships. Plasma levels of testican-2 are reflective of mechanisms underlying progressive renal function loss, thus suggesting its applicability as a biomarker of early renal dysfunction and disease progression in the general population. P.R. Matias-Garcia: None. R. Wilson: None. Q. Guo: None. S.B. Zaghlool: None. S. Sharma: None. P. Schlosser: None. A. Köttgen: None. A. Peters: None. D.O. Mook-Kanamori: None. J. Graumann: None. W. König: None. K. Hveem: None. C. Jonasson: None. A. Butterworth: None. K. Suhre: None. C. Gieger: None. A. Teumer: None. M. Waldenberger: None. P03.25.A Gain-of-function variant of the calcium-sensing receptor gene is associated with longer survival on renal replacement therapy in Caucasian patients

H. M. Rothe 1,2 , S. Komolka 2 , C. Wanner 3 , G. Mayer 4

1 Centre for Nephrology and Metabolic Disorders, Weisswasser, Germany, 2 Coburg University for Applied Sciences, Coburg, Germany, 3 University of Würzburg, Division of Nephrology, University Hospital, Würzburg, Germany, 4 Medical University Innsbruck, Department of Internal Medicine IV, Nephrology and Hypertension, Innsbruck, Austria
Introduction: Patients with end-stage kidney disease require renal replacement therapy, i.e. dialysis or kidney transplantation, for survival. Here we report for the first time a genetic variant which is associated with longer survival on renal replacement therapy in Caucasian patients. Materials and Methods: We conducted a survival analysis of three European end-stage kidney disease patient populations in Austria and Bavaria. 198 patients (118 male, 80 female) were genotyped for three variants of the calcium-sensing receptor gene after signing the consent form. The study started in 2005 and survival analysis was performed in 2018. During these years all patients received care according to the standards of the Austrian and German societies of nephrology. Results: Survival on renal replacement therapy was significantly different (age and sex adjusted Cox regression p=0.04) between carriers of the glycine and arginine alleles at position 990 of the calcium-sensing receptor molecule (rs1042636.) The two rs1042636 cohorts AA (two arginine alleles) and GA/GG (at least one glycine allele) were in Hardy Weinberg equilibrium with a minor allele frequency of 7%. Conclusions: The glycine variant of rs1042636 calcium-sensing receptor molecules has been shown to result in a gain-of-function, as compared with the arginine variant, in cell culture studies with transfected human embryonic kidney cells. It was also associated with lower baseline parathyroid hormone levels in the Caucasian patient cohort of the Evaluation of Cinacalcet Hydrochloride Therapy to Lower Cardiovascular Events (EVOLVE) trial. Our study shows that it is associated with longer survival on renal replacement therapy in Caucasian patients. H.M. Rothe: None. S. Komolka: None. C. Wanner: None. G. Mayer: None. P03.26.B Genes regulating enteric nervous system development are impacted by Copy Number loss and modify penetrance in epistasis with RET

L. Kuil 1 , C. S. Tang 2 , J. D. Windster 1,3 , T. Le 4,5 , A. Karim 2,6 , B. M. de Graaf 1 , R. van der Helm 1 , Y. van Bever 1 , C. E. J. Sloots 3 , C. Meeussen 3 , D. Tibboel 3 , A. de Klein 1 , R. M. H. Wijnen 1 , J. Amiel 4,7,5 , S. Lyonnet 4,7,5 , M. Garcia-Barcelo 2 , P. K. H. Tam 2 , M. M. Alves 1 , A. S. Brooks 1 , R. M. W. Hofstra 1,8 , E. Brosens 1

1 Clinical Genetics department, Erasmus Medical Centre – Sophia Children’s Hospital, Rotterdam, Netherlands, 2 Department of Surgery, Li Ka Shing Faculty of Medicine, University of Hong Kong, Hong Kong, China, 3 Paediatric Surgery department, Erasmus Medical Centre – Sophia Children’s Hospital, Rotterdam, Netherlands, 4 Paris Descartes - Sorbonne Paris Cité University, Imagine Institute, Paris, France, 5 Laboratory of embryology and genetics of congenital malformations, INSERM UMR1163, Paris, France, 6 Chittagong Research Institute for Children Surgery (CRICS), Chittagong, Bangladesh, 7 Service de Génétique, Necker Enfants Malades University Hospital, APHP, Paris, France, 8 Stem Cells and Regenerative Medicine, UCL Great Ormond Street Institute of Child Health, London, United Kingdom
Hirschsprung disease (HSCR) is characterized by absence of enteric ganglia, primarily in the distal colon. Approximately 18% of patients have additional anatomical malformations or associated neurodevelopmental disorders (HSCR-complex). We hypothesized that genes affected by rare Copy Number Variants contribute to disease development in “HSCR-complex” patients. Indeed, we could confirm that Copy Number (CN) losses ( n = 11) are enriched in these patients ( n = 23), if no other causal variant was identified (p=3.64E −7 ). At least five patients had a large de novo CNV (3 losses, 2 gains). The identified rare CN losses are enriched for variant intolerant genes, overexpressed in the developing mouse enteric nervous system (p=1.760E-10): SLC8A1 , GNL1 , GABBR1 , MAPK8 , UFD1L , AKT3 , TBX2 , USP32 and TUBB . Additionally, a loss of function variant in TUBB was identified in a HSCR patient without a deleterious CNV, and AKT3 as well as UFD1L are impacted by CN losses in other patients. The contribution of the known HSCR predisposing risk haplotypes was comparable between patients with a pathogenic coding variant and controls (P=0.826). All other HSCR subgroups differed significantly with controls and between each subgroup (P=0.02 or lower), patients with isolated HSCR and without a RET coding variant had the highest burden, unaffected controls the lowest. Our zebrafish model reveals that disruption of six of these 9 genes - alone or via epistasis with ret- results in a HSCR phenotype. This confirms the epistatic effect of losing a copy of a gene regulating enteric nervous system development with the predisposed downregulation of RET expression. L. Kuil: None. C.S. Tang: None. J.D. Windster: None. T. Le: None. A. Karim: None. B.M. de Graaf: None. R. van der Helm: None. Y. van Bever: None. C.E.J. Sloots: None. C. Meeussen: None. D. Tibboel: None. A. de Klein: None. R.M.H. Wijnen: None. J. Amiel: None. S. Lyonnet: None. M. Garcia-Barcelo: None. P.K.H. Tam: None. M.M. Alves: None. A.S. Brooks: None. R.M.W. Hofstra: None. E. Brosens: None. P03.27.C Identification of disease-causing variants in children and adolescents with focal segmental glomerulosclerosis

S. Draut 1 , M. C. Braunisch 1,2 , K. M. Riedhammer 1,2 , P. M. Herr 1 , R. Günthner 1,2 , M. Wagner 1,3,4 , B. Alhaddad 1 , L. Renders 2 , U. Heemann 2 , C. Schmaderer 2 , J. Hoefele 1

1 Institute of Human Genetics, Klinikum rechts der Isar, Technical University of Munich, School of Medicine, Munich, Germany, 2 Department of Nephrology, Klinikum rechts der Isar, Technical University of Munich, School of Medicine, Munich, Germany, 3 Institute of Human Genetics, Helmholtz Zentrum München, Neuherberg, Germany, 4 Institute of Neurogenomics, Helmholtz Zentrum München, Neuherberg, Germany
Introduction: A monogenic cause can be identified in approximately 30% of young patients (< 25 years of age) with steroid-resistant nephrotic syndrome (SRNS). SRNS in children and adolescents is mostly inherited in an autosomal recessive pattern. The histological correlate of SRNS is often focal segmental glomerulosclerosis (FSGS). The aim of this study was to identify genetic causes in a cohort of children and adolescents with FSGS. Material and Methods: 35 unrelated individuals with disease onset <18 years of age and suspected hereditary FSGS were analyzed using exome sequencing. Suspected hereditary FSGS was defined if at least one of the following criteria was present: absence of a secondary cause, <18 years of age, kidney biopsy with suspicion of a hereditary cause, extrarenal manifestations, and/or positive familial history/consanguinity. Variants were rated according to the ACMG classification. The clinical phenotype was assessed using a standardized questionnaire. Results: In seven patients, (likely) pathogenic variants according to ACMG were identified in known disease-causing genes (diagnostic yield: 20%). Three variants in two different FSGS genes ( INF2 , TRPC6 ) had to be classified as VUS (Table 1). Conclusions: The diagnostic yield of 20% highlights the importance of genetic testing in patients <18 years of age with nephrotic syndrome. Early genetic diagnosis could allow a personalized treatment approach with weaning of unnecessary immunosuppressive treatment, avoidance of renal biopsies and provision of well-informed genetic counselling. Table 1 . Results of exome sequencing m, maternal; n.d., not determined; p, paternal; VUS, variant of unknown significance S. Draut: None. M.C. Braunisch: None. K.M. Riedhammer: None. P.M. Herr: None. R. Günthner: None. M. Wagner: None. B. Alhaddad: None. L. Renders: None. U. Heemann: None. C. Schmaderer: None. J. Hoefele: None. P03.29.B Genetic linkage analysis identifies a genomic region in 2q24.3 as a candidate modulator of reduced penetrance in heritable pulmonary arterial hypertension .

I. Madrigal 1,2 , I. Blanco 3,4 , J. Barberà 3,4 , C. Badenas 1,2 , D. Geiger 5 , P. Puigdevall 6 , L. Piccari 3 , R. Castelo 6,7

1 Biochemistry and Molecular Genetics, IDIBAPS, Hospital Clinic, Barcelona, Spain, 2 Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Barcelona, Spain, 3 Hospital Clínic de Barcelona, Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain, 4 Centro de Investigación Biomédica en Red de Enfermedades Respiratorias (CIBERES), Madrid, Spain, 5 Faculty of Computer Science, Technion Israel Institute of Technology, Haifa, Israel, 6 Department of Experimental and Health Sciences, Universitat Pompeu Fabra, Barcelona, Spain, 7 nstitut Hospital del Mar d’Investigacions Mèdiques (IMIM), Barcelona, Spain
Hereditary pulmonary arterial hypertension (hPAH) is an autosomal dominant disease caused, in most cases, by mutations in the BMPR2 gene. However, not all BMPR2 mutation carriers develop the disease, highlighting the presence of reduced penetrance. In order to identify genomic regions that could be contributing to incomplete penetrance, high-throughput genotyping arrays and next generation sequencing were conducted in an hPAH family. The family comprises 65 Spanish individuals spanning five different generations. One BMPR2 mutation (c.1472G> A; p.Arg491Gln) was identified in 22 members, but only 9 of them developed the disease. The SNP arrays analysis was performed in 33 family members and we identified a genomic region in 2q24.3, located at 38 Mb from the BMPR2 gene, which could contribute to increase the susceptibility of pulmonary hypertension in mutation carriers (Puigdevall et al., J Med Genet, 2019). Several genetic association studies have identified variants in this region associated with phenotypes involved in cardiovascular pathology, such as hypertension or ischemic cardiomyopathy, which could be related to some of the symptoms of pulmonary hypertension. In order to identify the specific variant/s that may act as genetic modifiers of hPAH within the candidate region, we have performed whole-genome sequencing in 16 BMPR2 mutation carriers: 6 affected individuals and 7 asymptomatic carriers. The identification of a possible genetic modifier in this family will allow us to advance in our understanding of reduced penetrance in hPAH. Acknowledgments: ISCIIII (PI15/00483), ‘fondos FEDER’, Agència de Gestió d’Ajuts Universitaris i de Recerca (2017SGR1134), “CERCA Programme / Generalitat de Catalunya” I. Madrigal: None. I. Blanco: None. J. Barberà: None. C. Badenas: None. D. Geiger: None. P. Puigdevall: None. L. Piccari: None. R. Castelo: None. P03.32.B HNF1B-related syndrome: genotype-phenotype correlation

C. P. Cristalli 1,2 , A. Mattiaccio 2 , C. Graziano 3 , I. Capelli 1 , O. Baraldi 1 , G. Comai 1 , M. Pariali 2,3 , L. Gargano 2,3 , M. Seri 3 , G. La Manna 1 , V. Mantovani 2,3

1 Nephrology Dialysis and Transplantation Unit, DIMES, Bologna University, Bologna, Italy, 2 Center for Applied Biomedical Reasearch (CRBA), Bologna University, Bologna, Italy, 3 Medical Genetics Unit, S.Orsola-Malpighi University Hospital, Bologna, Italy
Molecular defects of HNF1B are associated with RCAD syndrome, a clinically heterogeneous disorder characterized by diabetes (MODY5), kidney abnormalities (cysts), cholestasis and uro-genital malformations. We performed targeted NGS of 15 genes and MLPA on 118 patients recruited by nephrology or diabetology units. 19 index cases showed molecular defects in HNF1B gene (10 pediatric and 9 adult). Six patients carried a likely pathogenic novel variant (p.G83D p.R295C, c.-67C>T, c.1207-1G>A, p.S379*, p.Q484Sfs*22) and 4 a VUS (p.G76C, p.E105K, p.A453T, p.M532V). A functional analysis for c.-67C>T indicated reduced activity in Caco2 and Mcf7 cells. Nine cases showed the whole HNF1B gene deletion (de novo in five patients). The median age of diagnosis was 19 (SNVs) and 6 (deletion) years old, respectively. Clinical presentation included earlier onset in deleted patients (median 6 vs 19 years) and different spatial cysts localization (cortical for SNVs and medullary in at least 3 deleted cases). Diabetes was present in six patients only, possibly due to the young age of many cases involved. Two patients with urogenital malformation and 3 with cholestasis were identified. A 21-year-old woman had mild developmental delay: additional array-CGH was performed and a large (1.3 Mb) deletion was found. Contrary, a 29-years-old man homozygous for p.G83D did not show intellectual disability. In conclusion, the patients with SNVs developed a milder phenotype than those deleted, and the molecular defects were frequently of de novo origin. We confirm the wide phenotypic heterogeneity of HNF1B-related disorders and we point out that liver disease can be a major complication. C.P. Cristalli: None. A. Mattiaccio: None. C. Graziano: None. I. Capelli: None. O. Baraldi: None. G. Comai: None. M. Pariali: None. L. Gargano: None. M. Seri: None. G. La Manna: None. V. Mantovani: None. P03.33.C Impact of common genetic risk variants on familial aggregation of inflammatory bowel disease

H. Lee 1,2 , L. Hannes 1 , M. Vancamelbeke 1 , V. Ballet 3 , M. Ferrante 3,4 , S. Vermeire 3,4 , I. Cleynen 1

1 KU Leuven, Leuven, Belgium, 2 University of Ulsan College of Medicine, Seoul, Korea, Republic of, 3 University Hospitals Leuven, Leuven, Belgium, 4 KU leuven, Leuven, Belgium
Family history is the strongest risk factor for inflammatory bowel disease (IBD). The contribution of common genetic variants to familial aggregation in multiple-affected IBD families, however, remains unclear. We included 54 multiple-affected families (≥ 3 first-degree relatives affected), including 189 affected (IBD), and 133 unaffected first-degree relatives; as well as 1768 sporadic cases and 868 unrelated non-IBD controls. For all individuals, Immunochip genotypes were available. Weighted polygenic risk scores (PRS) were calculated using PRSice-2.0 across different p -value thresholds (pTs), and compared between groups. The association between PRS and familial IBD was further evaluated in age- and sex-adjusted generalized mixed regression models including family id as random effect. Using pT=0.05, the affected relatives had a higher PRS than unaffected relatives (P=1.0x10 −02 ), sporadic cases (P=4.6x10 −02 ), and non-IBD controls (P < 2.2x10 −16 ). The risk of IBD in families increased by 1.23-fold for every incremental standard deviation in PRS. However, the proportion of the explained variance between affected and unaffected family members was smaller than that between sporadic IBD and non-IBD controls (5% versus 17%, respectively). Of note, in familial IBD, variance explained by the PRS dropped dramatically for pT>0.01. A plausible hypothesis is that while familial cases differ from sporadic cases in low-risk (high p -value) variants, familial cases differ from their unaffected relatives mostly in high-risk (low p -value) variants. Higher PRS increases the risk for familial IBD as it does for sporadic IBD. The proportion of the explained variance in families however is limited, because of shared genetic background between family members. BIRD Grant H. Lee: None. L. Hannes: None. M. Vancamelbeke: None. V. Ballet: None. M. Ferrante: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Modest; Amgen, Biogen, Janssen, Pfizer, Takeda. D. Speakers Bureau/Honoraria (speakers bureau, symposia, and expert witness); Modest; Abbvie, Amgen, Biogen, Boehringer-Ingelheim, Falk, Ferring, Janssen, Lamepro, MSD, Mylan, Pfizer, Takeda. F. Consultant/Advisory Board; Modest; Abbvie, Boehringer-Ingelheim, Janssen, MSD, Pfizer, Sandoz, Takeda. S. Vermeire: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Modest; MSD, Abbvie, Takeda, Janssen, Pfizer. D. Speakers Bureau/Honoraria (speakers bureau, symposia, and expert witness); Modest; AbbVie, MSD, Takeda, Ferring, Hospira, Pfizer, Janssen, and Tillots. F. Consultant/Advisory Board; Modest; AbbVie, MSD, Takeda, Ferring, Genentech/Roche, Shire, Pfizer Inc, Galapagos, Mundipharma, Hospira, Celgene, Second Genome, Progenity, Lilly, Arena, Gilead and Janssen. I. Cleynen: None. P03.34.A Combined exome/genome sequencing with in-house variant data repository mining confirm USP53 as a causal gene for intrahepatic cholestasis

S. Alawbathani 1 , M. Rocha 1 , H. Arshad Cheema 2 , R. Boustany 3 , S. Nampoothiri 4 , A. Alswaid 5 , N. El Koofy 6 , V. Karageorgou 1 , I. Hövel 1 , S. Khan 1 , C. Beetz 1 , A. Rolfs 1,7 , P. Bauer 1 , A. M. Bertoli-Avella 1

1 Centogene AG, Rostock, Germany, 2 Children’s Hospital and Institute of Child Health, Lahore, Pakistan, 3 AUBMC American University of Beirut Medical Center, Beirut, Lebanon, 4 Amrita Institute of Medical Sciences, Kerala, India, 5 King Abdulaziz Medical City, Riyadh, Saudi Arabia, 6 Abu El Reesh Children’s Hospital, Cairo, Egypt, 7 University of Rostock, Rostock, Germany
Cholestatic liver disease is a significant cause of morbidity and mortality in the paediatric population, starting in the early postnatal life. Severity is variable, from benign idiopathic neonatal hepatitis to intrahepatic cholestasis leading to liver failure and transplant. There are several genes described for different genetic diseases that lead to cholestasis. USP53 has been recently suggested as a candidate gene for paediatric intrahepatic cholestasis based on one family with two affected siblings. Interestingly, USP53 interacts with tight junction protein 2 ( TJP2 ) for which pathogenic variants are known to cause progressive familial intrahepatic cholestasis. To date, the USP53 gene has not been linked to an OMIM phenotype. By combining exome/genome sequencing and mining our in-house variant database we identified five patients from unrelated families. All affected individuals display a similar phenotype, mainly including early onset intrahepatic cholestasis. Moreover, one family showed hearing loss as an additional phenotype. All patients were homozygotes for different loss-of-function variants in the USP53 gene. USP53 (ubiquitin specific peptidase 53) is a tight junction-associated protein that is known to involve in the survival of auditory hair cells and hearing. Our results confirm USP53 as a novel gene for autosomal recessive intrahepatic cholestasis. Additionally, we demonstrate that combined exome/genome sequencing and variant database mining is an effective approach to discover and confirm novel genes related to human diseases. This accelerate the process of gene discovery and ultimately shortens the diagnostic odyssey in rare genetic disorders. S. Alawbathani: A. Employment (full or part-time); Significant; Centogene AG, Rostock, Germany. M. Rocha: A. Employment (full or part-time); Significant; Centogene AG, Rostock, Germany. H. Arshad Cheema: None. R. Boustany: None. S. Nampoothiri: None. A. Alswaid: None. N. El Koofy: None. V. Karageorgou: A. Employment (full or part-time); Significant; Centogene AG, Rostock, Germany. I. Hövel: A. Employment (full or part-time); Significant; Centogene AG, Rostock, Germany. S. Khan: A. Employment (full or part-time); Significant; Centogene AG, Rostock, Germany. C. Beetz: A. Employment (full or part-time); Significant; Centogene AG, Rostock, Germany. A. Rolfs: A. Employment (full or part-time); Significant; Centogene AG, Rostock, Germany. P. Bauer: A. Employment (full or part-time); Significant; Centogene AG, Rostock, Germany. A. M. Bertoli-Avella: A. Employment (full or part-time); Significant; Centogene AG, Rostock, Germany. P03.35.B A common haplotype of the alternative serotonin transporter gene SLC6A4 promoter P2 impacts gene expression and shows disease association in women with Irritable Bowel Syndrome

S. Mohr 1 , N. Hattensperger 1 , C. Hammer 2 , C. Martinez 3 , S. Berens 4 , S. Schmitteckert 1 , L. A. Houghton 5,6 , M. Goebel-Stengel 7 , I. Vulic 8 , M. D’Amato 9,10 , T. Zheng 9,10 , H. Mönnikes 11 , F. Engel 4 , A. Gauss 12 , J. Tesarz 4 , V. Andresen 13 , T. Frieling 14 , J. Keller 13 , C. Pehl 15 , G. Clarke 16 , R. Spiller 17 , T. G. Dinan 16 , E. M. Quigley 16 , C. Beltrán 18 , E. A. Mayer 19 , G. Sayuk 20 , M. Gazouli 21 , L. Kapur-Pojskic 8 , M. Bustamante 22 , K. Rabionet 22 , X. Estivil 22 , G. Boeckxstaens 23 , M. M. Wouters 23 , M. Simrén 24 , G. A. Rappold 1 , M. Vicario 25 , J. Santos 25 , R. Schäfert 4,26 , J. Lorenzo Bermejo 27 , B. Niesler 1

1 Department of Human Molecular Genetics, Heidelberg University, Heidelberg, Germany, 2 Department of Cancer Immunology, Genentech, South San Francisco, CA, United States, 3 Institut de Recerca Biomèdica de Lleida, Lleida, Spain, 4 Department of General Internal Medicine and Psychosomatics, University Hospital Heidelberg, Heidelberg, Germany, 5 Division of Gastroenterology & Surgical Sciences, Leeds, United Kingdom, 6 Mayo Clinic, Jacksonville, FL, United States, 7 Helios Klinikum Rottweil, Rottweil, Germany, 8 Institute for Genetic Engineering and Biotechnology, Sarajevo, Bosnia and Herzegovina, 9 School of BioSciences, Monash University, Melbourne, Australia, 10 Karolinska Institute, Solna, Sweden, 11 Martin-Luther-Krankenhaus, Berlin, Germany, 12 Department of Gastroenterology, University Hospital Heidelberg, Heidelberg, Germany, 13 Israelitisches Krankenhaus, Hamburg, Germany, 14 Helios Klinik Krefeld, Krefeld, Germany, 15 Krankenhaus Vilsbiburg, Vilsbiburg, Germany, 16 Department of Psychiatry and Neurobehavioral Science, University College Cork, Cork, Ireland, 17 Nottingham Digestive Diseases Centre, University of Nottingham, Nottingham, United Kingdom, 18 Laboratory of Immuno-Gastroenterology, Universidad de Chile, Santiago de Chile, Chile, 19 Oppenheimer Centre for Neurobiology of Stress, University of California, Los Angeles, CA, United States, 20 Washington University School of Medicine, St. Louis, MO, United States, 21 Biology National and Kapodistrian University of Athens, Medical School Athens, Athens, Greece, 22 Centre for Genomic Regulation, Barcelona, Spain, 23 TARGID, University Hospital Leuven, Leuven, Belgium, 24 Institute of Medicine, University of Gothenburg, Gothenburg, Sweden, 25 Institut de Recerca Vall d’Hebron, Hospital Vall d’Hebron, Barcelona, Spain, 26 Universitätsspital Basel, Basel, Switzerland, 27 Institute of Medical Biometry and Informatics, Heidelberg University, Heidelberg, Germany
Background: Irritable bowel syndrome (IBS) is a gut-brain disorder in which imbalances within the central and peripheral serotonergic systems contribute to disease pathophysiology and thereby shape the phenotype. The serotonin transporter gene SLC6A4 has earlier been implicated in IBS. Material and Methods: We sequenced the alternative promoter P2 that primarily drives SLC6A4 expression in the gut and analyzed single nucleotide polymorphisms (SNP) for association with IBS. We also assessed their functional relevance by gene reporter assays, in silico analysis and comparative expression studies. In addition, we correlated detected SNPs with expression data and IBS symptoms. Results: Detected SNPs were linked in different haplotypes and the tagging SNP rs2020938 associated with female IBS with constipation (IBS-C) in a discovery sample comprising 287 individuals. Subsequent validation and meta-analysis of rs2020938 in 2175 IBS patients and 6128 controls from eight countries confirmed the association with female IBS-C. Expression data obtained from different intestinal regions showed that the alternative promoter drives SERT expression primarily in the small intestine. Additionally, gene reporter assays pointed to a functional impact of the SNPs within this region. In silico analyses indicated differential expression regulation by transcription factors that have previously been implicated in GI disorders. Further follow up in tissue samples and clinical data from IBS patients and controls revealed that rs2020938 correlates with differential expression in the jejunum and stool consistency. Conclusions: Our results indicate differential expression regulation driven by a functionally relevant polymorphic promoter in female IBS-C and consolidates the importance of SLC6A4 in IBS pathogenesis. S. Mohr: None. N. Hattensperger: None. C. Hammer: None. C. Martinez: None. S. Berens: None. S. Schmitteckert: None. L.A. Houghton: None. M. Goebel-Stengel: None. I. Vulic: None. M. D’Amato: None. T. Zheng: None. H. Mönnikes: None. F. Engel: None. A. Gauss: None. J. Tesarz: None. V. Andresen: None. T. Frieling: None. J. Keller: None. C. Pehl: None. G. Clarke: None. R. Spiller: None. T.G. Dinan: None. E.M. Quigley: None. C. Beltrán: None. E.A. Mayer: None. G. Sayuk: None. M. Gazouli: None. L. Kapur-Pojskic: None. M. Bustamante: None. K. Rabionet: None. X. Estivil: None. G. Boeckxstaens: None. M.M. Wouters: None. M. Simrén: None. G.A. Rappold: None. M. Vicario: None. J. Santos: None. R. Schäfert: None. J. Lorenzo Bermejo: None. B. Niesler: None. P03.36.C Application of NGS sequencing for improved diagnosis in the pediatric nephrology setting

O. Beltcheva 1 , K. Kamenarova 1 , K. Mihova 1 , G. Zlatanova 2 , F. Shakola 1 , D. Roussinov 2 , M. Gaydarova 2 , V. Mitev 1 , R. Kaneva 1

1 Molecular Medicine Center, Dept. of Medical Chemistry and Biochemistry, Medical University-Sofia, Sofia, Bulgaria, 2 SBAL Pediatric Diseases, Nephrology and Hemodialysis Clinic, Department of Pediatrics, Medical University - Sofia, Sofia, Bulgaria
Introduction: Renal disease first diagnosed in childhood often progresses towards chronic or end-stage kidney failure in adults. Identifying the molecular cause of the condition in each case may allow a more personalized approach to therapy and consequently increase the likelihood of preserving the renal function of the patients in adulthood. Materials and Methods: TruSight One Sequencing Panel (Illumina) on MiSeq platform was used for mutation screening in 10 children with hematuria, proteinuria and/or compromised renal function, who did not respond well to standard treatment or presented with unusual course of the disease. Sanger sequencing was applied for confirmation and establishing the origin of the mutations. The pathogenicity of each novel variant was evaluated according to the established ACMG criteria. Results: The NGS yielded both known and novel mutations in the sample set. In addition to well-known genes, such as WT1, GATA3, SALL1, AGXT and COL4A4/5, the analysis of the data revealed that pathogenic variants in CD96, HNF1? and TREX1 may also contribute to the etiology of renal disease in children. Conclusions: The application of massive parallel sequencing of a large gene panel allowed us to identify the molecular cause of renal disease in a number of pediatric patients where diagnoses were hindered by atypical presentation. In several of the cases the findings allowed us to expand the clinical spectrum of the phenotypic presentation of the mutations in certain genes. Grant references: D-73/2019; D01-285/17.12.2019 O. Beltcheva: None. K. Kamenarova: None. K. Mihova: None. G. Zlatanova: None. F. Shakola: None. D. Roussinov: None. M. Gaydarova: None. V. Mitev: None. R. Kaneva: None. P03.37.A New LUTO-candidate genes identified by systematic analysis of copy number variations in 152 patients

L. M. Schierbaum 1,2 , J. Fabian 2 , S. Herms 3 , S. Sivalingam 2,3 , S. Schneider 2 , S. Weber 4 , W. Rösch 5 , W. M. Merz 6 , M. Feldkötter 7 , H. Reutter 8,2 , B. Hoppe 1 , G. Schalk 1 , M. Zaniew 9 , A. Hilger 1,2

1 Division of Pediatric Nephrology, Department of Pediatrics, University Hospital Bonn, Bonn, Germany, 2 Institute of Human Genetics, Children’s Hospital, University of Bonn, Bonn, Germany, 3 Department of Genomics, Life & Brain Centre, Bonn, Germany, 4 Department of Pediatrics, University Hospital Marburg, Marburg, Germany, 5 Deparment of Pediatric Urology, St. Hedwig Krankenhaus Barmherzige Brüder, Regensburg, Germany, 6 Department of Obstetrics and Prenatal Medicine, University of Bonn, Bonn, Germany, 7 Division of Pediatric Nephrology, Children’s Hospital, University of Zürich, Zürich, Switzerland, 8 Department of Neonatology and Pediatric Intensive Care, Children’s Hospital, University of Bonn, Bonn, Germany, 9 Department of Pediatrics, University of Zielona Góra, Zielona Góra, Poland
Introduction: Lower urinary tract obstruction (LUTO) is caused by anatomical blockage of the bladder outflow tract or by functional impairment of urinary voiding. For anatomical blockages so far only variants in BNC2 have been described as causal. Genomic copy number variations (CNVs) have been previously described as a cause of congenital anomalies of the kidneys and the urinary tract. Used Methods: Array-based molecular karyotyping was performed in 152 LUTO patients. Raw intensity data were collected for all persons and CNVs were called with CNVPartition, QuantiSNP and PennCNV. Using PLINK version 1.07 and Excel the CNVs, identified in the patients, were compared to CNVs of 4168 controls. Overlapping CNVs between both groups were discarded. Further filtering steps were used. Results: We identified 30 potential disease causing CNVs: 8 deletions and 22 duplications. Based on expression of the comprised genes in developing mouse urethra we prioritized 6 CNVs. One of those CNVs comprised the gene GPC5 (MIM 602446), located on chromosome 13q31.3. Interestingly, of 8 patients reported in the literature with 13q duplications that comprise GPC5 , four showed urogenital malformations. So far, GPC5 was not yet correlated with a specific clinical phenotype. Conclusions: Systematic analysis of copy number variations in patients with LUTO is suitable for the identification of new candidate genes. We prioritized GPC5 as candidate gene. For further verification of this candidate gene, re-sequencing of LUTO patients to identify further mutation carriers and functional characterization in zebrafish is warranted. Acknowledgment: A.C.H. is supported by BONFOR grant O-149.0123. L.M. Schierbaum: None. J. Fabian: None. S. Herms: None. S. Sivalingam: None. S. Schneider: None. S. Weber: None. W. Rösch: None. W.M. Merz: None. M. Feldkötter: None. H. Reutter: None. B. Hoppe: None. G. Schalk: None. M. Zaniew: None. A. Hilger: None. P03.38.B ZSCAN12 , a candidate gene for congenital lower urinary tract obstruction (LUTO)

L. Waffenschmidt 1 , S. Schneider 1 , J. Altmüller 2,3 , H. Thiele 4 , S. Weber 5 , W. Rösch 6 , W. M. Merz 7 , M. Feldkötter 8 , B. Hoppe 9 , G. Schalk 9 , M. Zaniew 10 , H. Reutter 1,11 , M. F. Schreuder 12,13 , G. M. A. Beckers 14 , J. A. E. van Wijk 12 , A. C. Hilger 1,9

1 Institute of Human Genetics, Children’s Hospital, University of Bonn, Bonn, Germany, 2 Center for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne, Germany, 3 Cologne Center for Genomics, University of Cologne, Cologne, Germany, 4 University of Leipzig-Heart Center, Department of Internal Medicine/ Cardiology, Leipzig, Germany, 5 Department of Pediatrics, University Hospital Marburg, Marburg, Germany, 6 Department of Pediatric Urology, St. Hedwig Krankenhaus Barmherzige Brüder, Regensburg, Germany, 7 Department of Obstetrics and Prenatal Medicine, University of Bonn, Bonn, Germany, 8 Division of Pediatric Nephrology, Children’s Hospital, University of Zürich, Zürich, Switzerland, 9 Division of Pediatric Nephrology, Department of Pediatrics, University Hospital Bonn, Bonn, Germany, 10 Department of Pediatrics, University of Zielona Góra, Zielona Góra, Poland, 11 Department of Neonatology and Pediatric Intensive Care, Children’s Hospital, University of Bonn, Bonn, Germany, 12 Department of Pediatric Nephrology, VU University Medical Center, Pediatric Nephro-urology Center, Amsterdam, Netherlands, 13 Department of Pediatric Nephrology, Erasmus MC – Sophia Children’s Hospital, University Medical Center, Rotterdam, Netherlands, 14 Department of Pediatric Urology, VU University Medical Center, Amsterdam, Netherlands
Congenital lower urinary tract obstruction (LUTO) is usually characterised by urinary bladder outflow obstruction, it can be functional, based on a congenital dysfunction in voiding or on anatomical blockage of the urethra. LUTO has a birth prevalence of about three per 10.000 pregnancies. Whole-Exome-Sequencing (WES) in eight families revealed two rare variants in the ZSCAN12 gene. The first variant (c.1695-2A>C, minor allel frequency (MAF): 0,00005814) was identified in three affected brothers, the second variant (c.62T>C; p.Ile21Thr, MAF: 0,00002595) was identified in an affected father and his affected son. Re-sequencing of ZSCAN12 in 263 LUTO patients identified three further patients who all carried the same variant c.637T>C, p.Phe213Leu (MAF: 0.0002046). It has not been reported homozygous. Currently no genetic material of the parents is available for testing. ZSCAN12 is expressed in the developing mouse urethra at embryonic day 15.5 (GUDMAP: 5824; GUDMAP Consortium) and has been found to be differentially methylated in prostate carcinoma. Based on these preliminary data we suggest ZSCAN12 as a novel LUTO candidate gene. Further functional studies are warranted. A.C.H. is supported by BONFOR grants O-149.0123. L. Waffenschmidt: None. S. Schneider: None. J. Altmüller: None. H. Thiele: None. S. Weber: None. W. Rösch: None. W.M. Merz: None. M. Feldkötter: None. B. Hoppe: None. G. Schalk: None. M. Zaniew: None. H. Reutter: None. M.F. Schreuder: None. G.M.A. Beckers: None. J.A.E. van Wijk: None. A.C. Hilger: None. P03.39.C Biallelic mutations in the LSR gene cause a novel type of infantile intrahepatic cholestasis

T. Uehara 1 , M. Yamada 1 , S. Umetsu 2 , H. Nittono 3 , H. Suzuki 1 , T. Fujisawa 2 , T. Takenouchi 4 , A. Inui 2 , K. Kosaki 1

1 Center for Medical Genetics, Keio University Hospital, Tokyo, Japan, 2 Department of Pediatric Hepatology and Gastroenterology, Saiseikai Yokohama-shi Tobu Hospital, Kanagawa, Japan, 3 Junshin Clinic BA Institute, Tokyo, Japan, 4 Department of Pediatrics, Keio University Hospital, Tokyo, Japan
LSR is expressed in the liver and other organs, and represents a component protein of tricellular tight junctions. The roles of LSR in human health and disease have not been established. Only one patient with intrahepatic cholestasis with a rare variant of LSR has been reported. Herein, we report a patient who fulfilled this criterion establishing LSR deficiency as a novel cause of human liver disease. The proband was a 5-year-old female patient. At 5 months of age, the infant developed severe and uncontrollable itching. The results of blood examination at 9 months of age showed elevated serum AST, ALT and TBA. Her verbal milestones were slightly delayed. A needle biopsy of the liver at 5 years of age showed paucity of interlobular bile duct. At 6 years of age, whole-exome analysis of the samples from the patient and her parents was performed. The de novo non-synonymous variant (c.602C>T) and the maternally derived heterozygous frameshift variant (c.1396dup) were detected in the LSR gene. Immunohistochemical staining using anti-LSR antibodies showed absent expression of LSR at the tricellular tight junctions in the patient. Documentation of biallelic pathogenic variants of the LSR gene and lack of LSR protein expression in the liver in our patient with infantile intrahepatic cholestasis presented in this report establishes LSR deficiency as a novel cause of infantile intrahepatic cholestasis. T. Uehara: None. M. Yamada: None. S. Umetsu: None. H. Nittono: None. H. Suzuki: None. T. Fujisawa: None. T. Takenouchi: None. A. Inui: None. K. Kosaki: None. P03.40.A Neo-TAD formation on 7q36.3 causes lung agenesis in three affected fetuses

U. Melo 1 , J. Piard 2 , M. Klever 3 , M. Mensa 4 , R. Schöpflin 3 , M. Holtgrewe 3 , F. Arbez-Gindre 2 , V. Guigue 2 , D. Gaillard 2 , V. Kremer 2 , R. Ramanah 2 , C. Cabrol 2 , D. Beule 3 , F. Harms 3 , K. Kutsche 3 , M. Spielmann 3 , B. Fischer-Zirnsak 3 , L. Van Maldergem 3 , S. Mundlos 3

1 MPI, Berlin, Germany, 2 3Centre de Génétique Humaine, Université de Franche-Comté, Besançon, France, 3 1Max Planck Institute for Molecular Genetics, RG Development & Disease, Berlin, Germany, Berlin, Germany, 4 1Max Planck Institute for Molecular Genetics, RG Development & Disease, Berlin, Germany, Berlin, Dominican Republic
Lung development is a tightly regulated process under the control of a large number of signaling molecules. The sonic hedgehog ( SHH ) signaling pathway is one of the main regulators of lung development during embryogenesis and mutations in SHH -related genes can lead to lung malformation. Here we report on three fetuses from one family with severe malformation of the respiratory system. By ultrasound screening and autopsy after termination, a complete bilateral lung agenesis and near-complete agenesis of the tracheae was observed in all fetuses. Using a combination of array-CGH, Illumina whole-genome sequencing and Hi-C we identified a complex rearrangement at 7q36.3, ~580 kb downstream the SHH gene. The rearrangement consisted of a 449 kb triplication, a 232 kb duplication and a 61 kb deletion, involving parts of the SHH and NOM1/MNX1/UBE3C topologically associated domains (TADs). Due to incorporation of a boundary between two TADs in the triplicated CNV, the rearrangements lead to the formation of two neo-TADs each containing the known SHH enhancer MACS1 which is now placed next to MNX1 , a transcription factor involved in caudal development. This situation can be expected to result in an ectopic activation of MNX1 in the lung at a time and place where SHH is normally expressed; a misexpression that likely explains the lung aplasia. The rearrangement was present in mosaic state in the clinically unaffected father, explaining the recurrence of the phenotype in his offspring’s. Thus, whole-genome sequencing in combination with Hi-C is a powerful tool to resolve and functionally interpret complex rearrangements. U. Melo: None. J. Piard: None. M. Klever: None. M. Mensa: None. R. Schöpflin: None. M. Holtgrewe: None. F. Arbez-Gindre: None. V. Guigue: None. D. Gaillard: None. V. Kremer: None. R. Ramanah: None. C. Cabrol: None. D. Beule: None. F. Harms: None. K. Kutsche: None. M. Spielmann: None. B. Fischer-Zirnsak: None. L. Van Maldergem: None. S. Mundlos: None. P03.41.B Recessive and dominant patterns in Noonan Syndrome associated with LZTR1 variants

A. Cambra 1 , M. López-Blázquez 2 , J. Sánchez del Pozo 3 , J. Cruz-Rojo 3 , M. Güemes 4 , A. Carcavilla 5 , V. Seidel 6 , C. Medrano 2 , B. Ezquieta 1

1 Molecular Diagnostic Laboratory, Department of Laboratory Medicine, Hospital General Universitario Gregorio Marañón, Instituto de Investigación Sanitaria Gregorio Marañón (IiSGM), Madrid, Spain, 2 Pediatric Cardiology, Department of Pediatrics, Hospital General Universitario Gregorio Marañón, Madrid, Spain, 3 Pediatric Endocrinology, Department of Pediatrics, Hospital Universitario 12 de Octubre, Madrid, Spain, 4 Pediatric Endocrinology, Department of Pediatrics, Hospital Universitario Niño Jesús, Madrid, Spain, 5 Pediatric Endocrinology, Department of Pediatrics, Hospital Universitario La Paz, Madrid, Spain, 6 Clinical Genetics, Department of Pediatrics, Hospital General Universitario Gregorio Marañón, Madrid, Spain
Noonan syndrome (NS) is an autosomal dominant disease caused by germline mutations in genes involved in the Ras/MAPK pathway. Nevertheless, a recessive form related to biallelic variants in LZTR1 gene was described in 2018. Following publications discussed phenotype and inheritance patterns associated with this gene. Here we report seven unrelated patients with clinically suspected NS and LZTR1 variants. Clinical features are provided in table1. Three novel variants and four previously reported variants were identified. Two patients, NS1564 and NS960, presented a recessive form of NS. In the first patient, c.2074T>C;p.(F692L) and c.2070-2A>G were identified and the second one was homozygous for c.2074T>C;p.(F692L), both affect the BTB/POZ domain. In the rest of patients only one affected allele was identified: NS1414 had a stop-gain variant, c.1084C>T;p.(R362*), located on kelch-5 domain; NS895 presented c.742G>A;p.(G248R), which lies in kelch-4 domain; NS883 presented c.851G>A;p.(R284H), located on kelch-5 domain; NS1199 presented a splicing variant, c.791+1G>T; and the patient 99 was heterozygous for c.905C>T;p.(Ala302Glu), located on kelch-5 domain. Overall, these cases provide more evidence on the clinical phenotype of NS associated with LZTR1 variants. These results are in accordance with the model that specifies that the location throughout the gene determines the inheritance form of NS. Our experience indicates that LZTR1 is a not infrequent cause of NS, so this gene should be included in next-sequencing panels for molecular diagnosis of NS. A. Cambra: None. M. López-Blázquez: None. J. Sánchez del Pozo: None. J. Cruz-Rojo: None. M. Güemes: None. A. Carcavilla: None. V. Seidel: None. C. Medrano: None. B. Ezquieta: None. P03.42.C Profiling of microRNA isoforms in ulcerative colitis

D. Nikitina 1 , S. Juzėnas 1,2 , V. Šaltenienė 1 , A. Franke 2 , G. Kiudelis 3 , J. Kupčinskas 1,3 , L. Kupčinskas 1,3 , J. Skiecevičienė 1

1 Lithuanian University of Health Sciences, Medical Academy, Institute for Digestive Research, Laboratory of Clinical and Molecular Gastroenterology, Kaunas, Lithuania, 2 Kiel University, Institute of Clinical Molecular Biology, Kiel, Germany, 3 Lithuanian University of Health Sciences, Medical Academy, Department of Gastroenterology, Kaunas, Lithuania
Introduction: Recent research has shown that so far widely studied reference miRNA sequences in miRNA databases have a large number of isoforms that can be involved in other physiological pathways compared to the reference miRNA. Ulcerative colitis (UC) is a chronic disease of the digestive system of the autoimmune origin and is attributed to the group of inflammatory bowel diseases. The aim of this study was to perform isomiRNA analysis in ulcerative colitis tissue. Materials and Methods: In total, 76 individuals were included in this study (32 controls, 24 patients with active UC and 20 patients with UC in remission phase). Small RNA sequencing was performed using the TruSeq protocol and the HiSeq2500 platform (Illumina, USA). Bioinformatic microRNA analysis was performed using the following software tools: MiRDeep2, mirAligner, Cutadapt, FASTX, RNAfold, Bowtie, BioPerl, BLASTn and R. Results: The analysis revealed that the reference microRNA sequences make up 2.1% of all microRNA sequences and 97.9% are microRNA isoforms. Altered microRNAs and their isoforms (padj <0.01, log2FC> | 0.9 |) were detected in three groups: (i) active UC group compared to the HC (healthy control group) - 874; (ii) UC in the remission group compared to the HC - 93; (iii) active UC group compared to UC remission group - 632. The expression of 9 isomiRNA was strongly correlated with the intensity of the inflammation (rho> 0.7). Conclusions: An altered profile of microRNAs and their isoforms was found in UC colon tissue, which also correlated with inflammation intensity. D. Nikitina: None. S. Juzėnas: None. V. Šaltenienė: None. A. Franke: None. G. Kiudelis: None. J. Kupčinskas: None. L. Kupčinskas: None. J. Skiecevičienė: None. P03.43.A Screening for genetic polymorphisms in Bulgarian children with non-alcoholic fatty liver disease

D. Kofinova 1 , I. Yordanova 2 , P. Hadzhiyski 1 , R. Shentova-Eneva 1 , M. Baycheva 1 , P. Yaneva 1 , M. Panayotova 3 , A. Todorova 2 , D. Avdjieva-Tzavella 1

1 University Children’s Hospital "Prof. Ivan Mitev", Sofia, Bulgaria, 2 Genetic Medico-Diagnostic Laboratory Genica, Sofia, Bulgaria, 3 Trakia Hospital, Stara Zagora, Bulgaria
Introduction: Non-alcoholic fatty liver disease (NAFLD) is a highly prevalent liver disease in children. NAFLD is associated with insulin resistance, obesity, and dyslipidemia. NAFLD is a multifactorial disease. Growing evidence suggest the leading role of certain gene variants, associated with rapid progression of the liver disease and dyslipidemia. The aim of this study was to investigate whether various single-nucleotide polymorphisms play any role in the development of NAFLD in Bulgarian children. Materials and Methods: We prospectively included 22 patients with NAFLD and 10 healthy controls. Ten of the children with NAFLD underwent liver biopsy. PCR amplification with specific primers pairs for variants in the genes PNPLA3 (c.444C>G, p.I148 M), GCKR (c.1337C>T, p.P446L) and TM6SF2 ( c.499C>T, p.E167K) was performed followed by direct sequencing using BigDye Terminator v.3.1 Cycle Sequencing kit. Results: The GCKR P446L gene polymorphism was found in 75.0% (24/32) of the study participants (homo- or heterozygous carriers). It was significantly higher in homozygous patients with NAFLD than in healthy controls (54.5% vs. 10.0%, p=0.025). PNPLA3 I148M was detected in 9 of the patients and 7 of the controls. TM6SF2 E167K was the rarest genetic variant, only 3 children (2 patients and 1 healthy child) were carriers. Two of the patients, who underwent liver biopsy had significant fibrosis F2. Patient N1 was homozygous for PNPLA3 I148M and heterozygous for GCKR P446L. Patient N2 was GCKR P446L heterozygous carrier. Conclusions: GCKR P446L polymorphism is associated with development of NAFLD in Bulgarian children. Medical University Sofia, Grant "Young Investigator" Contract № D-142/02.05.2017 D. Kofinova: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Modest; Medical University Sofia, Grant "Young Investigator" Contract № D-142/02.05.2017. I. Yordanova: None. P. Hadzhiyski: None. R. Shentova-Eneva: None. M. Baycheva: None. P. Yaneva: None. M. Panayotova: None. A. Todorova: None. D. Avdjieva-Tzavella: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Modest; Medical University Sofia, Grant "Young Investigator" Contract № D-142/02.05.2017. P03.44.B Molecular characterization of familial intrahepatic cholestasis by multi-gene panel analysis

A. Mattiaccio 1,2 , G. Vitale 3 , C. P. Cristalli 1 , M. Pariali 1,4 , L. Gargano 1,4 , M. Seri 4 , P. Andreone 5,2 , V. Mantovani 1,4

1 Center for Applied Biomedical Research (CRBA), Bologna University, Bologna, Italy, 2 DIMEC, Bologna University, Bologna, Italy, 3 Internal Medicine for the Treatment of Severe Organ Failure, S. Orsola-Malpighi Hospital, Bologna, Italy, 4 Medical Genetics Unit, S.Orsola-Malpighi University Hospital, Bologna, Italy, 5 Department of Medical and Surgical Sciences, Maternal, Child and Adult Sciences, University Hospital, Modena, Italy
Introduction: Progressive Familial Intrahepatic Cholestasis (PFIC) is a group of autosomal recessive diseases that affects especially newborns and children, with progression to liver failure in the first decades of life. PFIC is classified into five types (PFIC1-5) based on the genetic defect involved in bile transport. Other benign late-onset phenotypes and non-progressive forms (BRIC, LPAC, DIC and ICP) are caused by heterozygous mutations in the same gene pattern. Materials and Methods: DNA analysis of 15 candidate genes involved in PFIC and other related benign phenotypes was carried out. Amplicon-based NGS on 80 patients was performed. Bioinformatic and statistic pipelines were applied. Results: A total of 184 different variants has been identified in our cohort: 18 pathogenic, 46 VUS, 44 likely benign and 76 benign. P/LP mutations were found in 12% of patients: 2 in ATP8B1 , 3 in ABCB11 , ABCB4 and TJP2 each, one in ABCC2 , JAG1 , NOTCH2 . Many patients had multiple variants in several genes. Patients had from 7 to 35 variants each and some SNPs were significantly associated with biochemical parameters and phenotypic features that could better explain clinics and accelerate the progression to liver failure. Conclusions: Our detection rate is according to other studies proposing multi-gene panels. Our analysis may be useful for the molecular diagnostics of PFIC and a better characterization and understanding of the linking between molecular defects and different subtypes of the disease. The high SNPs prevalence let us to hypothesize a synergistic haplotype effect in determining different multifactorial cholestasis phenotypes and overlapping features. A. Mattiaccio: None. G. Vitale: None. C.P. Cristalli: None. M. Pariali: None. L. Gargano: None. M. Seri: None. P. Andreone: None. V. Mantovani: None. P03.45.C Telomere length (TL) and oxidative stress in C57BL/6J mice

E. Kidd 1 , J. Pender 1 , M. J. Gatt 1 , J. Williams 2 , A. A. I. F. Blakemore 3 , E. Meimaridou 1 , A. J. Walley 4 , U. L. Fairbrother 1

1 London Metropolitan University, London, United Kingdom, 2 Queen Mary, London, United Kingdom, 3 Brunel University, London, United Kingdom, 4 St George’s University of London, London, United Kingdom
High levels of oxidative stress may lead to an increased rate of telomere shortening and contribute to loss of telomere integrity. Most in vivo studies have looked for correlations between biomarkers of oxidative stress and TL, and have used leucocytes. We have used a murine model to investigate the potential impact of oxidative stress on TL in a tissue-specific manner and to assess telomere oxidation directly. Our model is a C57BL/6J mouse strain with a naturally occurring nicotinamide nucleotide transhydrogenase (NNT) deficiency. The absence of Nnt results in high levels of reactive oxygen species (ROS) in cells. DNA was extracted from healthy control mice, Nnt-deficient mice and Nnt-rescued mice (Nnt reinserted at the blastocyst stage) and their telomeres analysed. Two qPCR methods were used: mmQPCR to assess relative telomere length (RTL), and a formamidopyrimidine DNA glycosylase (FPG) enzyme-based qPCR method to directly determine the extent of telomere oxidation. Analysis of RTL from kidney revealed no significant difference (p>0.05) between the three groups. This is predictable since rapidly dividing leucocytes probably best represent early life replicative responses and tissues such as kidney, a slowly dividing tissue, represents the heritable load of TL. QPCR analysis of the DNA extracts, before and after FPG digestion showed there was no significant difference in the ΔCT values between the three groups (p>0.05), implying there was no discernible difference in telomere oxidation levels. Further analysis will investigate tissues with variable metabolic and cellular turnover rates such as the brain, spleen, liver, heart and adipose tissue. E. Kidd: None. J. Pender: None. M.J. Gatt: None. J. Williams: None. A.A.I.F. Blakemore: None. E. Meimaridou: None. A.J. Walley: None. U.L. Fairbrother: None. P03.46.A Molecular markers of severity of nonalcoholic fatty liver disease

D. Sidorenko , V. Nazarov, S. Lapin, V. Emanuel

Pavlov First Saint Petersburg State Medical University, Saint-Petersburg, Russian Federation
Introduction: Nonalcoholic fatty liver disease (NAFLD) is one of the most common parenchymal liver disorders. There are no reliable markers for assessment of progression of NAFLD from steatosis to steatohepatitis that leads to liver fibrosis and cirrhosis. Polymorphism I148M in PNPLA3 gene is associated with inhibition of lipid hydrolysis. Pathological variants of SERPINA1 gene promote accumulation of mutant alpha-1-antitrypsin in endoplasmic reticulum. Polymorphisms in HFE gene leads to intracellular iron accumulation and fatty degeneration of liver. Materials and Methods: Group of 59 patients with NAFLD was selected. The assessment of structural changes in liver was carried out by transient elastometry with controlled attenuation parameter. Polymorphisms in PNPLA3, SERPINA1 and HFE genes were determined by real-time PCR. Results: Association between homozygous carriage of I148M polymorphism and increase of ALT was found (p=0.011). A connection was established between the presence of the polymorphism and more severe steatosis (p=0.045). AST activity was higher in PiZ carriers than in patients without changes in this gene (p=0.049). H63D and C282Y polymorphisms of the HFE gene are associated with higher ALT (p=0.020 and p=0.030). A correlation between changes in mentioned genes and more severe fibrosis was not demonstrated. Conclusions: Polymorphisms in the PNPLA3, SERPINA1, and HFE genes are associated with an increase in ALT and AST in patients with NAFLD. The homozygous variant of the I148M polymorphism in PNPLA3 gene and the heterozygous C282Y in HFE gene are associated with more severe steatosis. However, conflicting data regarding liver fibrosis rates need further study. D. Sidorenko: None. V. Nazarov: None. S. Lapin: None. V. Emanuel: None. P03.47.B Gene expression differences in non-obese, obese and previously obese subjects .

M. Tessarech 1 , E. Mileti 2 , K. Kwok 2 , P. Arner 3 , D. Bonneau 1 , E. Colin 1 , M. Rydén 3 , C. Daub 2

1 CHU Angers, Angers, France, 2 Karolinska Institutet, Stockholm, Sweden, 3 Karolinska University Hospital Huddinge, Stockholm, Sweden
Obesity is a worldwide public health issue associated with many comorbidities. Understanding its underlying causes and mechanisms is one of the keys to prevent and treat this condition. Besides obvious environmental causes, as physical inactivity or poor nutrition, genetics is one of the main component of obesity. Here, we perform a transcriptomic study by 5′Cap-mRNA sequencing of subcutaneous white adipose tissue of non-obese (NO), obese (OB) and previously obese (POB) subjects, at fasting and hyperinsulinemia states. Differential gene expression analysis showed few difference in the analysis of NO compared to POB subjects, while the comparison of OB subjects versus NO and versus POB displayed many transcriptomic changes, with most of the genes in common in both comparison. Moreover, many genes were specifically involved in expression changes between OB and POB subjects. Gene ontology analysis revealed that those genes were involved in metabolic and cellular process pathways and in cardiovascular diseases. Fasting and hyperinsulinemia metabolic states had little influence on the results. This study demonstrates that a major gene expression difference is observed between obese and lean subjects, whether they were never obese or got rid of their fat mass after a weight loss surgery, and that after this kind of surgery, many changes in gene expression appear. Further studies are needed to identify precisely which genes are involved and if they can be a target for personalized medicine in the treatment of obesity by weight loss surgery. M. Tessarech: None. E. Mileti: None. K. Kwok: None. P. Arner: None. D. Bonneau: None. E. Colin: None. M. Rydén: None. C. Daub: None. P03.49.A Autosomal Dominant Polycystic Kidney Disease in Israel: Genetic diagnosis reveals recurrent pathogenic variants

T. Paperna 1 , A. Mory 1 , A. Ofir 1 , N. Damouny 1 , D. Magen 2,3 , I. Maya 4,5 , K. Weiss 1 , H. Baris Feldman 6,5

1 The Genetics Institute, Rambam Health Care Campus, Haifa, Israel, 2 Pediatric Nephrology Institute, Rambam Health Care Campus, Haifa, Israel, 3 Faculty of Medicine, Technion, Haifa, Israel, 4 The Raphael Recanati Genetic Institute, Rabin Medical Center, Beilinson Hospital, Petah Tikva, Israel, 5 Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel, 6 The Genetics Institute, Tel Aviv Sourasky Medical Center, Haifa, Israel
Introduction: The genetic diagnosis of Autosomal Dominant Polycystic Kidney disease (ADPKD) is complicated by the genomic structure of the main causative gene PKD1 , due to the presence of overlapping pseudogenes. Classification of the missense variants is challenging as allelic heterogeneity is rife, and few recurrent variants are known. Data on ADPKD in Israeli populations is yet unavailable. Materials and methods: Patient samples ( n = 112) From Israel were referred to Rambam Genetics Institute for ADPKD gene panel testing ( PKD1, PKD1, HNF1B) . Next generation sequencing (NGS) was performed using either Trusight One (Illumina, n = 77), or exome sequencing (ES, n = 35); analysis employed the Genoox platform. NGS was supplemented by Sanger sequencing to achieve full coverage of panel. GANAB , DNAJB11 were included in ES testing. Results: A clinically significant variant (CSV) was identified in 82% of the patients ( PKD1 -88%, PKD2- 7%, HNF1B -5%). Three PKD1 CSVs were identified only following Sanger sequencing. Truncating CSVs comprised ~55% of pathogenic variants. Most CSVs (72%) were unique; however, eight PKD1 CSVs recurred in two or more patients, notably, in Jews of Persian and Ethiopian descent. HNF1B -related kidney disease was diagnosed in four individuals. Positive family history was linked with higher CSV detection rates, 81%, compared with 60% when negative. Conclusions: This first report on ADPKD genetics in Israel highlights recurrent CSVs in Israeli sub-ethnicities. HNF1B CSVs can account for an ADPKD-like phenotype. Family history was a strong predictor for detection of a CSV. Our experience underscores the importance of utilizing ES as well as Sanger sequencing for variant detection. T. Paperna: None. A. Mory: None. A. Ofir: None. N. Damouny: None. D. Magen: None. I. Maya: None. K. Weiss: None. H. Baris Feldman: None. P03.50.B Genome-wide association study identifies CDH12 as candidate gene for renal injury in patients with posterior urethral valves

L. F. M. van der Zanden 1 , I. A. L. M. van Rooij 1 , J. S. L. T. Quaedackers 2 , R. Nijman 2 , M. Steffens 3 , L. L. L. de Wall 1 , E. M. H. F. Bongers 1 , F. Schaefer 4 , M. Kirchner 5 , R. Behnisch 5 , A. K. Bayazit 6 , S. Caliskan 7 , L. Obrycki 8 , G. Montini 9,10 , A. Duzova 11 , M. Wuttke 12 , R. Jennings 13,14 , N. A. Hanley 13,14 , N. J. Milmoe 15 , P. J. D. Winyard 15 , K. Y. Renkema 16 , M. F. Schreuder 1 , N. Roeleveld 1 , W. F. J. Feitz 1

1 Radboud university medical center, Nijmegen, Netherlands, 2 University Medical Center Groningen, Groningen, Netherlands, 3 Isala, Zwolle, Netherlands, 4 University Hospital Heidelberg, Heidelberg, Germany, 5 University of Heidelberg, Heidelberg, Germany, 6 Cukurova University, Adana, Turkey, 7 Istanbul University-Cerrahpasa, Istanbul, Turkey, 8 Children`s Memorial Health Institute, Warsaw, Poland, 9 Fondazione IRCCS Ca’ Granda – Ospedale Maggiore Policlinico di Milano, Milan, Italy, 10 University of Milan, Milan, Italy, 11 Hacettepe University Faculty of Medicine, Ankara, Turkey, 12 University of Freiburg, Freiburg, Germany, 13 University of Manchester, Manchester, United Kingdom, 14 Manchester University NHS Foundation Trust, Manchester, United Kingdom, 15 UCL Great Ormond Street Institute of Child Health, London, United Kingdom, 16 University Medical Center Utrecht, Utrecht, Netherlands
Introduction: Posterior urethral valves (PUV) and ureteropelvic junction obstruction (UPJO) are congenital obstructive uropathies that may impair renal development. We performed the first GWAS for renal injury in obstructive uropathy patients. Methods: We included patients with pyeloplasty or PUV resection before age 18. Signs of renal injury were defined as dialysis, nephrectomy, kidney transplantation, estimated glomerular filtration rate<60 ml/min/1.73m 2 , high blood pressure, antihypertensive medication use, proteinuria, and/or one kidney functioning<45%. In the GWAS, we compared 141 and 216 obstructive uropathy patients with and without signs of renal injury 5 years post-surgery, respectively. For the associated SNPs, we performed stratified analyses for PUV and UPJO, and Kaplan-Meier and Cox regression analyses in the discovery and two replication samples of 102 PUV patients each. For the associated gene, we performed RNA and protein expression analyses in fetal tissues. Results: We observed suggestive associations with six SNPs in three loci, of which rs6874819 in CDH12 was the strongest ( P = 7.5×10 −7 ). This SNP also seemed to be associated with time to renal injury in the PUV discovery and replication samples. RNA expression analyses showed clear expression in fetal kidneys, which was confirmed by protein immunolocalization. Conclusions: This study identified CDH12 as candidate gene for renal injury in PUV. CDH12 is a target of the proinflammatory MCP-1-MCPIP pathway. We hypothesize that in PUV, MCP1 pathway upregulation due to increased urinary tract pressure is augmented in carriers of the CDH12 variant, exacerbating renal injury. Grant support: Dutch Kidney Foundation (13OKJ36), Netherlands Organisation for Scientific Research (91618036). L.F.M. van der Zanden: None. I.A.L.M. van Rooij: None. J.S.L.T. Quaedackers: None. R. Nijman: D. Speakers Bureau/Honoraria (speakers bureau, symposia, and expert witness); Modest; Astra Zeneca. M. Steffens: None. L.L.L. de Wall: None. E.M.H.F. Bongers: None. F. Schaefer: None. M. Kirchner: None. R. Behnisch: None. A.K. Bayazit: None. S. Caliskan: None. L. Obrycki: None. G. Montini: F. Consultant/Advisory Board; Modest; Merck. A. Duzova: None. M. Wuttke: None. R. Jennings: None. N.A. Hanley: F. Consultant/Advisory Board; Modest; APIS Assay Technologies. N.J. Milmoe: None. P.J.D. Winyard: None. K.Y. Renkema: None. M.F. Schreuder: None. N. Roeleveld: None. W.F.J. Feitz: None. P03.51.C RNA-seq approach to detect splicing alterations in primary ciliary dyskinesia and non-CF bronchiectasis

J. Legebeke 1 , G. Wheway 1 , H. Wai 1 , W. Walker 1,2 , J. S. Lucas 1,2 , D. Baralle 1,2

1 University of Southampton, Southampton, United Kingdom, 2 University Hospital Southampton, Southampton, United Kingdom
Mutations that cause PCD have been reported in >40 genes. Most cases are recessive, caused by biallelic changes in these genes. These account for around 70% of cases, suggesting that additional genes, and non-coding or synonymous changes in known genes, remain to be identified. Non-CF bronchiectasis is a heterogeneous group of disorders and adults may need to be investigated for PCD. Here we present an optimised pipeline for RNA analysis from nasal brushings taken from the inferior turbinate of patients with suspected PCD or non-CF bronchiectasis. We present analysis pipelines to investigate differences in gene expression, alternative splicing and the identification of deleterious splicing events. We developed gene-neutral and gene-specific analysis protocols, from both nasal brushings stored in RNA-later and cells from nasal brushings cultured at the air-liquid interface (ALI). We present this RNA-seq approach as a particularly useful diagnostic tool to investigate patients in whom a single pathogenic variant has been identified in a known PCD gene and where we suspect an alteration in splicing of the same gene due to a second intronic or synonymous / non-synonymous exonic variant. This represents a step towards using RNA analysis for increasing diagnostic yield in rare recessive respiratory disease. JL, DB and HT are supported by a NIHR Research Professorship to DB (RP- 2016-07-011). JL is further supported by Southampton NIHR BRC. GW is funded by a Wellcome Trust Seed Award in Science (Grant No. 204378/Z/16/Z) and a University of Southampton Faculty of Medicine Research Management Committee research grant. J. Legebeke: None. G. Wheway: None. H. Wai: None. W. Walker: None. J.S. Lucas: None. D. Baralle: None. P03.52.A Genetic testing for hereditary hyperparathyroidism in a large UK cohort

S. Mariathasan 1 , K. Andrews 2 , E. Thompson 3 , B. Challis 1,4 , S. Wilcox 2 , H. Pierce 2 , J. Hale 1 , S. Spiden 3 , G. Fuller 5 , H. Simpson 6 , B. Fish 7 , P. Jani 7 , I. Seetho 8 , R. Armstrong 2 , L. Izatt 9 , M. Joshi 10 , A. Velusami 10 , R. Casey 1 , S. Park 2

1 Department of Endocrinology, Cambridge University Hospitals NHS Foundation Trust, Cambridge, United Kingdom, 2 Department of Clinical Genetics, Cambridge University Hospitals NHS Foundation Trust, Cambridge, United Kingdom, 3 9.10. East Midlands and East of England NHS Genomic Laboratory Hub, Cambridge University Hospitals NHS Foundation Trust, Cambridge, United Kingdom, 4 Translational Science & Experimental Medicine, Research and Early Development, Cardiovascular, Renal and Metabolism, BioPharmaceuticals R&D, AstraZeneca,, Cambridge, United Kingdom, 5 9.10. East Midlands and East of England NHS Genomic Laboratory Hub, Cambridge University Hospitals NHS Foundation Trust, Cambridge, United Kingdom, 6 4. Department of Endocrinology, University College Hospital, London, United Kingdom, 7 Department of Head and Neck Surgery, Cambridge University Hospitals NHS Foundation Trust, Cambridge, United Kingdom, 8 Department of Endocrinology, Northwick Park Hospital, London North West University Hospital NHS Trust, London, United Kingdom, 9 Department of Clinical Genetics, Guy’s and St Thomas’ NHS Foundation Trust, London, United Kingdom, 10 Department of Endocrinology, Guy’s and St Thomas’ NHS Foundation Trust, London, United Kingdom
Primary hyperparathyroidism (PHPT) is common with a prevalence of 0.86%. 10% are familial. Syndromic PHPT includes multiple endocrine neoplasia (MEN)1, MEN4, MEN2A and CDC73-related disorders. Non-syndromic causes include familial hypocalciuric hypercalcaemia (FHH), familial isolated HPT and neonatal severe PHPT. Establishing the underlying genetic cause allows for targeted, cost effective management. The 2019 National Genomic Test Directory PHPT testing criteria are PHPT and calcium creatinine clearance ratio (CCCR) >0.02 presenting <35y or <45y with one of a) multi-glandular disease, or b) hyperplasia, or c) ossifying fibromas of the jaw, or d) a family history (FH) of PHPT. The testing criteria for FHH is CCCR <0.02. We reviewed 121 PHPT patients from the Cambridge and Guy’s Endocrine Genetics clinic over a 4y period referred with multi-glandular disease, relevant FH, CCCR <0.01, age <50y, or >1 risk factor. We offered NGS panel gene testing of all known PHPT genes. Of the 121 patients, (92F, mean age 41y), a pathogenic variant was identified in 16% (11 CASR , 6 MEN1 , 1 CDC73 , 1 AP2S1) and we detected 4 VUS’s. The mean CCCR was 0.0073 in those with a CASR pathogenic variant vs 0.016 in those without (p 0.003). A CCCR <0.01 had a sensitivity of 82% and specificity of 81% for FHH. A positive FH was a strong predictor of a pathogenic variant whereas multi-glandular disease was not. In summary, genetic testing is recommended in PHPT at any age with i) syndromic PHPT, ii) relevant FH iii) a CCCR<0.01 and iv) multiple risk factors. S. Mariathasan: None. K. Andrews: None. E. Thompson: None. B. Challis: None. S. Wilcox: None. H. Pierce: None. J. Hale: None. S. Spiden: None. G. Fuller: None. H. Simpson: None. B. Fish: None. P. Jani: None. I. Seetho: None. R. Armstrong: None. L. Izatt: None. M. Joshi: None. A. Velusami: None. R. Casey: None. S. Park: None. P03.55.A Biallelic loss of function GFRA1 variants cause bilateral renal agenesis

S. Khan 1 , I. Verma 2 , V. Arora 2 , R. Puri 2 , A. El-Hattab 3 , M. Rocha 1 , R. Merdzanic 1 , O. Paknia 1 , C. Beetz 1 , A. Rolfs 1,4 , A. Bertoli-Avella 1 , P. Bauer 1

1 Centogene AG,, Rostock, Germany, 2 Sir Ganga Ram Hospital, Institute of Genetics and Genomics, Rajinder Nagar, New Delhi, India, 3 Department of Clinical Sciences, College of Medicine, University of Sharjah, Sharjah, United Arab Emirates, 4 University of Rostock, Rostock, Germany
Congenital anomalies of the kidney and urinary tract comprise a broad spectrum of renal and urinary tract malformations with renal agenesis being at the most severe end of the spectrum. It usually results in death in utero or in the perinatal period. Only three genes have been identified so far as causing renal agenesis. By exome/genome sequencing within a diagnostic setting, with combined database analyses, we identified two unrelated patients presenting with congenital bilateral renal agenesis and homozygous loss of function variants (LoF) in the GFRA1 gene. Both patients had parental consanguinity and similarly affected, deceased siblings, suggesting an autosomal recessive inheritance. Patient 1 was homozygote for the nonsense variant NM_001348097.1:c.676C>T, p.(Arg226*). The second patient was homozygote for a frameshift variant (NM_001348097.1:c.1294delA, p.(Thr432Profs*13)). Both variants are novel, identified for first time in our database, and presumed to lead to LoF. The GFRA1 gene is not reported to be associated to any human phenotype in OMIM, but GFRA1 missense variants have been reported as associated to Hirschsprung disease in several patients, based on an oligogenic model. Gfra1-deficient mice demonstrate absence of enteric neurons and agenesis of the kidney, characteristics that are reminiscent of both Gdnf-and Ret-deficient mice. The phenotype described in our patients is identical to the renal phenotype reported in Gfra1 null mice, however, no abnormalities of the enteric nervous system was reported in these two cases. In conclusion, these findings strongly support the causal role of GFRA1 inactivating variants for an autosomal recessive form of bilateral renal agenesis. S. Khan: A. Employment (full or part-time); Significant; Centogene AG, Rostock, Germany. I. Verma: None. V. Arora: None. R. Puri: None. A. El-Hattab: None. M. Rocha: A. Employment (full or part-time); Significant; Centogene AG, Rostock, Germany. R. Merdzanic: A. Employment (full or part-time); Significant; Centogene AG, Rostock, Germany. O. Paknia: A. Employment (full or part-time); Significant; Centogene AG, Rostock, Germany. C. Beetz: A. Employment (full or part-time); Significant; Centogene AG, Rostock, Germany. A. Rolfs: A. Employment (full or part-time); Significant; Centogene AG, Rostock, Germany. A. Bertoli-Avella: A. Employment (full or part-time); Significant; Centogene AG, Rostock, Germany. P. Bauer: A. Employment (full or part-time); Significant; Centogene AG, Rostock, Germany. P03.56.B WES expands the mutational spectrum in patients with Silver-Russell syndrome features

R. Meyer 1 , M. Begemann 1 , C. Hübner 1 , D. Dey 1 , A. Küchler 2 , M. Elgizouli 2 , P. Kroisel 3 , G. Fekete 4 , L. Ambrozaityte 5 , A. Matuleviciene 5 , M. Elbracht 1 , T. Eggermann 1

1 Institute of Human Genetics, Medical Faculty, RWTH Aachen University, Aachen, Germany, 2 Institute of Human Genetics, University Hospital Essen, Essen, Germany, 3 Institute of Human Genetics, Medical University of Graz, Graz, Austria, 4 II. Department of Pediatrics, Semmelweis University, Budapest, Hungary, 5 Department of Human and Medical Genetics, Institute of Biomedical Sciences, Faculty of Medicine, Vilnius University, Vilnius, Lithuania
Silver-Russell syndrome (SRS) is one of the most prominent growth retardation disorders. However, both, the phenotype and underlying molecular alterations of SRS show a marked heterogeneity. SRS itself is an imprinting disorder, mainly associated with specific molecular changes at specific imprinted chromosomal regions. Disease causing point mutations are less common and vary widely in means of the affected gene. The suitability of panel-based NGS assays to identify monogenetic causes of SRS-like phenotypes has already been proven. The reasonableness of whole exome sequencing to identify point mutations in patients with Silver-Russel syndrome features however, is still the subject of ongoing discussions. We examined samples of 75 patients referred for molecular testing for SRS using different NGS-based approaches. The typical SRS-associated molecular alterations had been excluded before. The results of multi gene panel, whole exome sequencing, and trio-based whole exome sequencing approaches were compared. Taking together the results of all different approaches, in 19/75 patients a disease-causing variant could be identified. Most of these variants are associated with monogenetic differential diagnoses of Silver-Russell syndrome. The diagnostic yield raised from panel to whole exome sequencing to trio-based whole exome sequencing. Broadening the molecular sequencing approach increases the mutation detection rate in patients referred for molecular testing of SRS without one of the typical molecular alterations. Some of the monogenetic disorders detected in our cohort have an important impact on the clinical management and genetic counseling of affected families mainly in terms of recurrence risks, tumor development, and mental development. Funding: Deutsche Forschungsgemeinschaft (EG110/15-1). R. Meyer: None. M. Begemann: None. C. Hübner: None. D. Dey: None. A. Küchler: None. M. Elgizouli: None. P. Kroisel: None. G. Fekete: None. L. Ambrozaityte: None. A. Matuleviciene: None. M. Elbracht: None. T. Eggermann: None. P03.57.C ‘The pathogenicity analysis of mutations identified in AR gene in patients with AIS from Ukraine’

L. A. Livshits 1 , D. A. Sirokha 1 , O. V. Gorodna 1 , D. M. Lozhko 1 , G. B. Livshyts 1 , N. B. Zelinska 2

1 IMBG NANU, Kyiv, Ukraine, 2 Ukrainian Scientific and Practical Center for Endocrine Surgery, Transplantation of Endocrine Organs and Tissues of the Ministry of Health of Ukraine, Kyiv, Ukraine
Androgen Insensitivity Syndrome (AIS) is a Disorder of Sex Development (DSD) that occurs at a frequency of 1 in 20,000 live births, is the most common DSD in people with karyotype 46, XY, caused by mutations in the Androgen Receptor (AR) gene. More than 1000 AR gene mutations have been described in patients with AIS and prostate cancer. Almost 400 of them are located in ligand binding domain (LBD). The pathogenicity of mutations detected in AIS patients from Ukraine was analyzed. Directional sequencing of the PCR products of LBD-coding exons 7 and 8 was performed on DNA samples of AIS patients after informed consent. The substitution X:67722905 T>C (rs9332970), exon 7, missense mutation Ile843Thr was identified in PAIS-patient. In another PAIS-patient the substitution X: 67723745 C>T (rs137852594) samesence variant was identified. It was predicted the creation of a new enhancer motif for SRp55 splisosome protein, and as a consequence a new donor splice site at the end of the last exon 8. The substitution X:67722943 C> T (rs886041132), exon 7 - missense mutation Arg856Cys, was identified in CAIS-patient. Mutation X: 67722884 T> G, exon 7, a missense mutation Ile836Ser, was identified in CAIS-patient was not annotated in Varsome, gnomAD and ARDB and identified as novel. The effect of listed missense mutations was predicted according to SIFT and PolyPhen values and classified as damaging. Analysis the substitution Ile836Ser on 3D model showed that the mutant protein does not get the correct conformation and binds dihydrotestosterone ~ 460 times worse than wild-type AR. L.A. Livshits: None. D.A. Sirokha: None. O.V. Gorodna: None. D.M. Lozhko: None. G.B. Livshyts: None. N.B. Zelinska: None. P03.59.B Cell free DNA as potential marker of inflammatory diseases

D. Smolak 1,2 , G. Roman 3 , J. Budis 2,4,5 , M. Maronek 3 , B. Gromova 3 , R. Liptak 3 , M. Harsanyova 2,1 , J. Radvanszky 6,2 , T. Szemes 2,1,4

1 Department of Molecular Biology, Faculty of Natural Sciences, Comenius University, Bratislava, Slovakia, 2 Geneton Ltd., Bratislava, Slovakia, 3 Institute of Molecular Biomedicine, Faculty of Medicine, Comenius University, Bratislava, Slovakia, 4 Comenius University Science Park, Bratislava, Slovakia, 5 Slovak Center of Scientific and Technical Information, Bratislava, Slovakia, 6 Institute for Clinical and Translational Research, Biomedical Research Center, Slovak Academy of Sciences, Bratislava, Slovakia
Introduction: Ulcerative colitis is an inflammatory bowel disease (IBD) characterised by chronic inflammation of the human digestive tract. Despite efforts, the availability of effective non-invasive diagnostics assays is still limited. Cell free DNA (cfDNA) represents extracellular genetic information that already showed promises for various fields of genetic testing, including, non-invasive prenatal testing (NIPT) and cancer testing. Materials and methods: Using a murine dextran sulfate sodium (DSS) induced colitis model we collected blood from 46 laboratory mice, grouped by stage of ulcerative colitis modeled by the duration of DSS administration. cfDNA was isolated and sequenced using the MiSeq Illumina System. Preprocessed sequenced data were mapped against mouse genome (GRCm38.p6). Statistical analyses were performed by in-house Python and R scripts. Results: Besides the observed different fragment length distributions between control and colitis groups, we observed also a significantly increasing amount of mouse genomic material with the advancing stage of the disease, measured by the proportion of sequenced reads mappable to the mouse genome. In addition, to further investigate the pathophysiological processes behind the observed changes, we performed a sequence based differentiation of reads according to their mappability to the mouse nuclear and mitochondrial genomes, as well as of the detected bacterial communities. Conclusions: We observed significant differences between the control group and various disease stages in sequenced data. Our results suggest the utility of cfDNA as a potential screening marker for IBD and even its specific stages and pathophysiological processes behind them. Funded by: Slovak Research and Development Agency, grant APVV-17-0526. D. Smolak: A. Employment (full or part-time); Significant; Geneton Ltd.. G. Roman: None. J. Budis: A. Employment (full or part-time); Significant; Geneton Ltd.. M. Maronek: None. B. Gromova: None. R. Liptak: None. M. Harsanyova: A. Employment (full or part-time); Significant; Geneton Ltd. J. Radvanszky: A. Employment (full or part-time); Modest; Geneton Ltd. T. Szemes: A. Employment (full or part-time); Significant; Geneton Ltd.. P03.60.C Functional characterization of variants of unknown significance in the Wilson disease-associated ATP7B gene

A. Stalke , F. Hennig, E. Pfister, U. Baumann, M. Eilers, V. Schäffer, B. Vajen, N. Huge, T. Reinkens, B. Schlegelberger, T. Illig, B. Skawran

Hannover Medical School, Hannover, Germany
Introduction: Wilson disease (WD) is an autosomal recessive disorder of copper excess due to pathogenic ATP7B variants. ATP7B codes for a copper-transporting ATPase. Sequencing of ATP7B is an important tool to diagnose the disease. However, variants of unknown significance (VUS) are detected frequently, impeding a clear diagnosis. Functional analyses can help to unambiguously classify VUS. We aimed to functionally characterize four ATP7B VUS, detected in patients with clinically distinct or suspected WD. Methods: The ATP7B open reading frame was cloned into an expression vector and the VUS were generated by site-directed mutagenesis. After HEK293T cell transfection, ATP7B expression of the mutants was quantified at mRNA and protein level by qRT-PCR and western blot. To assess the mutant’s ability to export copper a copper-sensitive luciferase-based assay was used. Results: For all VUS ATP7B mRNA expression was significantly reduced compared to wild-type and a benign polymorphism. Three VUS also led to decreased protein expression and copper transport capacity. These VUS had been detected in clinically distinct WD patients. One VUS led to protein expression and copper transport capacity at wild-type level. It had been detected in a patient with suspected WD. Conclusions: Our results strongly suggest a pathogenic effect for three of the VUS. For one VUS our results hint at a benign effect. On-going immunofluorescence assays will further address a possible protein mislocalization. Besides clarifying the clinical relevance of variants, functional characterization will help to understand the variant’s pathomechanism contributing to personalized treatment with fewer side effects. Supported by: DFG A. Stalke: None. F. Hennig: None. E. Pfister: None. U. Baumann: None. M. Eilers: None. V. Schäffer: None. B. Vajen: None. N. Huge: None. T. Reinkens: None. B. Schlegelberger: None. T. Illig: None. B. Skawran: None.

P04 Skeletal, Connective Tissue, Ectodermal and Skin Disorders
P04.03.C Geleophysic and acromicric dysplasias: clinical and molecular review of 38 patients

P. Marzin 1,2 , B. Thierry 3,4 , A. Dancasius 1,2 , C. Michot 1,2 , S. Rondeau 1,2 , G. Baujat 1,2 , G. Phan 5 , M. Le Bourgeois 6 , D. Khraiche 7 , C. Delacourt 6 , V. Cormier-Daire 1,2

1 Fédération de Génétique et Institut Imagine, AP-HP, Hôpital Necker-Enfants Malades,, F-75015, France, 2 Université de Paris, UMR1163, INSERM, F-75 015, Paris, France, 3 Service d’ORL pédiatrique, AP-HP, Hôpital Necker-Enfants Malades, F-75015, France, 4 Université de Paris, UMR976, INSERM, F-75 010,, Paris, France, 5 Laboratoire de Cristallographie et RMN Biologiques, UMR 8015, CNRS, Université Paris Descartes, Faculté de Pharmacie, 4 Avenue de l'Observatoire, 75006, paris, France, 6 Service de Pneumologie et Allergologie Pédiatriques, AP-HP, Hôpital Necker-Enfants Malades, F-75 015, Paris, France, 7 Unité Médico-Chirurgicale de Cardiologie Congénital et Pédiatrique, AP-HP, Hôpital Necker-Enfants Malades, F-75 015,, Paris, France
Introduction: Geleophysic dysplasia (GD) and acromicric dysplasia (AD) are skeletal dysplasia characterized by short stature, brachydactyly and progressive joint limitation. Cardiorespiratory involvement may be responsible for a severe prognosis in GD. Dominant mutations in the FBN1 and LTBP3 genes are associated with AD and GD, whereas recessive mutations in the ADAMTSL2 gene only with GD. These three genes encode for proteins of microfibrillar network. The aim of this study is to improve understanding of natural history and genotype-phenotype correlations. Materials and methods: This retrospective study included patients with AD and GD followed in our center, with identified mutation(s). Data from the medical records were abstracted, recorded in an Excel spreadsheet and analyzed using simple frequency analysis. Results: We described clinical and molecular data from 38 patients (20GD, 18 AD) with a mean age of 17 years. Mutations in the FBN1 , ADAMTSL2 and LTBP3 genes were identified in 23, 12 and 3 patients, respectively. Early death occurred in nine cases. 45% of patients presented with valvulopathy, progressive in half of cases. Respiratory disorders were asthma (50%), non-cardiogenic restrictive syndrome (30%), laryngo-tracheo-bronchial involvement (18%) and acute non-cardiogenic pulmonary hypertension (four cases). Patients with a variant involving a cysteine in the FBN1 gene or mutations in the ADAMTSL2 gene presented a higher proportion of severe cardiorespiratory complications compared to a more benign course in patients with other mutations. Conclusions: Genetic findings could have importance not only in the diagnosis, but also in risk stratification and clinical management of patients with suspected acromelic dysplasia. P. Marzin: None. B. Thierry: None. A. Dancasius: None. C. Michot: None. S. Rondeau: None. G. Baujat: None. G. Phan: None. M. Le Bourgeois: None. D. Khraiche: None. C. Delacourt: None. V. Cormier-Daire: None. P04.04.A Acromesomelic dysplasia Maroteaux type, nine patients with seven novel NPR2 mutations

E. Kilic 1 , B. Cavdarli 2 , G. Buyukyilmaz 3 , M. Kilic 4

1 Division of Pediatric Genetics, Department of Pediatrics, University of Health Sciences, Ankara City Hospital, Ankara, Turkey, 2 Department of Medical Genetics, Ankara City Hospital, Ankara, Turkey, 3 Division of Pediatric Endocrinology, Department of Pediatrics, Ankara City Hospital, Ankara, Turkey, 4 Division of Metabolism, Department of Pediatrics, Sami Ulus Children Hospital, Ankara, Turkey
Background: Acromesomelic Dysplasia, type Maroteaux (AMDM), is an autosomal recessive skeletal dysplasia caused by loss of function mutations of NPR2 gene which encodes C-type natriuretic peptid reseptor B. This rare type of dwarfizm characterized with shortening of the middle and distal segments of the limbs. Herein we reported nine patients from eight families with seven novel NPR2 mutations. Methods: Nine patients were diagnosed AMDM in a single centre from 2015 to 2020 were enrolled in this study. Their clinical, radiological and molecular genetic findings examined retrospectively. The mutations in NPR2 gene were confirmed with direct sequencing. Results: All nine patiens in this group were admitted to hospital with the complaint of short stature. All had short limb type dwarfism with marked shortening middle and acral segment of the limbs. All had typical dysmorphic signs, brachydactyly, significant greater toes and normal intelligence. We identified one previously reported (c.2729 C>T), five novel missense (c.3029 A>G, c.1738-1739 del ins TT, c.1163 G>A, c.3058 C>T, c.329 G>A) and two novel nonsense (c.1213 delT fs*, c.14 C>A fs*) mutations. All new mutations identified in this group are likely pathogenic and meaningfull with the patients typical clinical presentations. Conclusions: Reporting new patients with novel mutations would contribute to better clinical dealination of this rare dysplasia and would help to established pathogenic mutations. References: 1. Maroteaux P, Martinelli B, Campailla E.[Acromesomelic dwarfism]. Presse Med. 1971; 79:1839-42.2. Khan S, Basit S, Khan MA, et al. Genetics of human isolated acromesomelic dysplasia. Eur J Med Genet. 2016;59:198-203. E. Kilic: None. B. Cavdarli: None. G. Buyukyilmaz: None. M. Kilic: None. P04.06.C Severe congenital cutis laxa with generalized muscle hypotonia due to ATP6V1A mutations

G. Vogt 1,2 , N. El Choubassi 1,2 , M. Holtgrewe 3 , S. Krause 4 , A. Lekaj 1 , R. Horvath 5 , M. Schülke 6 , T. Gardeitchik 7 , S. Mundlos 1,2 , Á. Herczegfalvi 8 , H. Lochmüller 9,10 , V. Karcagi 11 , U. Kornak 1,2,12 , B. Fischer-Zirnsak 1,2

1 Institute of Medical Genetics and Human Genetics, Charité - Universitätsmedizin, Berlin, Germany, 2 RG Development & Disease, Max Planck Institute for Molecular Genetics, Berlin, Germany, 3 Core Unit Bioinformatics-CUBI, Berlin Institute of Health, Berlin, Germany, 4 Department of Neurology, Friedrich-Baur-Institute, Munich, Germany, 5 Department of Clinical Neurosciences, University of Cambridge School of Clinical Medicine, Cambridge, United Kingdom, 6 Department of Neuropediatrics, Charité Universitätsmedizin, Berlin, Germany, 7 Department of Human Genetics, Radboud University Medical Centre, Nijmegen, Netherlands, 8 II. Department of Paediatric Neurology, Semmelweis Medical University, Budapest, Hungary, 9 Division of Neurology, Children’s Hospital of Eastern Ontario Research Institute, Ottawa, IL, Canada, 10 Brain and Mind Research Institute, Ottawa, IL, Canada, 11 Department of Molecular Genetics and Diagnostics, National Institute of Environmental Health, Budapest, Hungary, 12 Institute of Human Genetics, University Medical Center, Göttingen, Germany
Introduction: Autosomal recessive cutis laxa type 2D is a severe multisystem disease due to pathogenic variants affecting ATP6V1A , encoding a component of the V-Type H + ATPase. Affected individuals show a strong cutis laxa phenotype with large skin folds at birth, hypotonia and cardiac abnormalities. Material and Methods: We performed single whole exome sequencing (WES) to identify the causative molecular defect in our index patient. Furthermore, Sanger sequencing, qRT-PCR and Immunoblot analyses were performed to further investigate causality of the variants identified. Results: Here we report on two affected individuals from a non-consanguineous family from Hungary suffering from a multisystem condition with severe, generalized muscle hypotonia and and strong juvenile cutis laxa. One child died in infancy, while the other significantly improved. Using WES and co-segregation analyses we found our proband to be compound heterozygous for a novel missense and the first so far described nonsense pathogenic variant affecting ATP6V1A . Expression studies revealed a strong degradation of the allele carrying the nonsense alteration. Additionally, patients’ dermal fibroblasts showed strong alteration of Cathepsin D processing indicating alterations of lysosomal function. Conclusions: This is the second report on pathogenic variants in ATP6V1A as the cause for a severe condition with cutis laxa. Our data expand the clinical spectrum of ATP6V1A -related phenotypes and show that beside missense, also nonsense pathogenic variants are a possible cause of ATP6V1A -ARCL. Funding: The study was supported by the Deutsche Forschungsgemeinschaft (FI 2240/1-1) to BF-Z and by EURO-CDG-2 to UK. G. Vogt: None. N. El Choubassi: None. M. Holtgrewe: None. S. Krause: None. A. Lekaj: None. R. Horvath: None. M. Schülke: None. T. Gardeitchik: None. S. Mundlos: None. Á. Herczegfalvi: None. H. Lochmüller: None. V. Karcagi: None. U. Kornak: None. B. Fischer-Zirnsak: None. P04.07.A New duplication downstream of the BMP2 gene in a French family with Brachydactyly type A2 (BDA2)


1 Department of Biochemistry and Genetics, Angers University Hospital, ANGERS, France, 2 Department of Gynecology and Obstetrics, Angers University Hospital, ANGERS, France, 3 Pole of Biology Genetic Pathology, Lille University Hospital - Center of Biology Genetic Pathology, LILLE, France
Brachydactyly type A2 (BDA2, MIM 112600) is an autosomal dominant pathology characterized by deviation and shortening of the middle phalanx of the index and / or second toe. It may be due to mutations in the BMPR1B gene (MIM 603248) or the GDF5 gene (MIM 601146) but may also depend on structural changes in 20p12.3 involving the BMP2 gene. Indeed, microduplications downstream of the BMP2 gene have been described in 5 families (2 German and 3 Chinese). Using an SNP-array technique, we identified a new duplication of 45kb downstream of the BMP2 gene in a French family. We describe the phenotypic and genotypic characteristics of this family and review the literature. E. Colin: None. A. Guichet: None. F. Boussion: None. M. Tessarech: None. A. Ziegler: None. M. Gorce: None. C. Houdayer: None. S. Manouvrier: None. D. Bonneau: None. P04.11.B Loss-of-function variants in C3ORF52 result in localized autosomal recessive hypotrichosis

N. Cesarato 1 , L. Malki 2,3 , O. Sarig 2,3 , J. Mohamad 2,3 , T. Canter 4 , S. Assaf 2,3 , M. Pavlovsky 2 , D. Vodo 2,3 , Y. Anis 5 , O. Bihari 2 , K. Malovitski 2,3 , A. Gat 2 , H. Thiele 6 , B. E. Perez White 4 , L. Samuelov 2 , A. Nanda 7 , A. S. Paller 4 , R. C. Betz 1 , E. Sprecher 2,3

1 Institute of Human Genetics, University of Bonn, Bonn, Germany, 2 Division of Dermatology, Tel Aviv Sourasky Medical Center, Tel Aviv, Israel, 3 Department of Human Molecular Genetics & Biochemistry, Sackler Faculty of Medicine, Tel Aviv University, Ramat Aviv, Israel, 4 Department of Dermatology, Northwestern University Feinberg School of Medicine, Chicago, IL, United States, 5 Institute of Endocrinology, Tel Aviv Sourasky Medical Center, Tel Aviv, Israel, 6 Cologne Center for Genomics, University of Cologne, Cologne, Germany, 7 As’ad Al-Hamad Dermatology Center, Kuwait, Kuwait
Introduction: Localized autosomal recessive hypotrichosis has been associated with pathogenic variants in DSG4 , encoding a desmosomal protein as well as in LIPH and LPAR6 , encoding respectively lipase H, which catalyzes the formation of 2-acyl lysophosphatidic acid (LPA), and lysophosphatidic acid receptor 6, a receptor for LPA. LPA promotes hair growth and differentiation. In this study, we aimed at delineating the genetic basis of LAH in patients without pathogenic variants in these three genes. Materials and Methods: Variant analysis was conducted using exome and Sanger sequencing. We then performed RT-qPCR, immunofluorescence staining, immunoblotting, enzymatic and co-immunoprecipitation assays in cell cultures and hair follicles to evaluate the consequences of the identified variants. Results: We identified homozygous variants in C3ORF52 in four individuals from two families with LAH. C3ORF52 was found to be co-expressed with lipase H in the inner root sheath of the hair follicle and the two proteins were found to directly interact. The LAH-causing variants were associated with decreased C3ORF52 expression and resulted in markedly reduced lipase H-mediated LPA biosynthesis. Conclusions: We could connect C3ORF52 to the LPA-mediated signaling pathway which, together with LPAR6 and LIPH, plays a crucial role in hair growth. LAH can be caused by abnormal function of any of these three proteins. N. Cesarato: None. L. Malki: None. O. Sarig: None. J. Mohamad: None. T. Canter: None. S. Assaf: None. M. Pavlovsky: None. D. Vodo: None. Y. Anis: None. O. Bihari: None. K. Malovitski: None. A. Gat: None. H. Thiele: None. B.E. Perez White: None. L. Samuelov: None. A. Nanda: None. A.S. Paller: None. R.C. Betz: None. E. Sprecher: None. P04.12.C Evidence for a role of fibrillin-2 in de pathogenesis of carpal tunnel syndrome

S. Peeters 1 , A. Decramer 2 , S. Cain 3 , P. Houpt 4 , F. Verstreken 5 , J. Noyez 2 , C. Hermans 6 , W. Jacobs 7 , M. Lammens 8 , E. Fransen 1 , A. Kumar 9 , G. Vandeweyer 1 , B. Loeys 1 , W. Van Hul 1 , C. Baldock 3 , E. Boudin 1 , G. Mortier 1

1 Department of Medical Genetics, University of Antwerp and Antwerp University Hospital, Antwerp, Belgium, 2 Department of Orthopaedics and Traumatology, AZ Delta, Roeselare, Belgium, 3 Division of Cell-Matrix Biology and Regenerative Medicine, Wellcome Centre for Cell-Matrix Research, the University of Manchester, Manchester, United Kingdom, 4 Department of Plastic Surgery, Isala clinic Zwolle, Zwolle, Netherlands, 5 Department of Orthopaedic surgery, AZ Monica, Deurne, Belgium, 6 Center for Oncological Research Antwerp (CORE), University of Antwerp, Edegem (Belgium), Antwerp, Belgium, 7 Department of Forensic Medicine and Pathology, Antwerp University Hospital and University of Antwerp, Antwerp, Belgium, 8 Department of Pathological Anatomy, Antwerp University Hospital, Antwerp, Belgium, 9 Department of Paediatrics, Wellcome - MRC Cambridge Stem Cell Institute Cambridge, Cambridge, United Kingdom
Introduction: Carpal tunnel syndrome (CTS) is the most common form of peripheral entrapment neuropathy. Although CTS is quite common, its pathogenesis remains largely unknown. An estimated heritability index of 0.46 and a familial occurrence in 27-39% of the cases indicate that genetic factors must play a role in the pathogenesis. We report on a peculiar family in which CTS occurred in subsequent generations at an usually young age. Additional clinical features included brachydactyly and short Achilles tendons resulting in toe walking in childhood. Material and methods: We performed exome sequencing and genome-wide linkage analysis to identify the pathogenic variant in this family. Several functional assays were performed to further study the causality of the variant. In addition, we sequenced a cohort of 216 patients to assess the role of this new candidate gene in the pathogenesis of CTS in a larger population. Results: We identified a heterozygous variant (c.5009T>G; p.Phe1670Cys) in the fibrillin-2 ( FBN2 ) gene that co-segregated with the phenotype in the family. Functional assays showed that the FBN2 variant impaired integrin-mediated cell adhesion and migration. Moreover, we observed an increased TGF-β signaling and fibrosis in the carpal tissues of affected individuals. A variant burden test in a large cohort of CTS patients revealed a significantly increased frequency of rare FBN2 variants in patient alleles compared to controls. Conclusions: Our data strongly suggest a role of FBN2 in the pathogenesis of CTS. S. Peeters: None. A. Decramer: None. S. Cain: None. P. Houpt: None. F. Verstreken: None. J. Noyez: None. C. Hermans: None. W. Jacobs: None. M. Lammens: None. E. Fransen: None. A. Kumar: None. G. Vandeweyer: None. B. Loeys: None. W. Van Hul: None. C. Baldock: None. E. Boudin: None. G. Mortier: None. P04.14.B New pathways and variants in genes causing collagenopathies identified in patients with isolated cervical insufficiency

L. Volozonoka , D. Rots, I. Kempa, A. Kornete, D. Rezeberga, L. Gailīte, A. Miskova

Riga Stradins University, Riga, Latvia
Introduction: Cervical insufficiency is phenotypically distinguishable condition causing pregnancy loss or preterm delivery. A strong genetic component and few syndromic forms of cervical insufficiency are described. Knowledge about the genetics of human cervix remodelling during pregnancy is scarce - only 12 maternal genes and their variants are implicated in cervical insufficiency so far as identified by our systematic gene analysis before. Material and methods: We sequenced DNA from 21 patients with clinical presentation of isolated cervical insufficiency using Illumina’s TruSight One NGS kit. Genes having rare deleterious variants identified in our patients ( n = 694) were annotated for the pathway enrichments using ConsensusPathDB. Variants in genes from enriched phenotype-relevant pathways were manually curated based on variant’s pathogenicity, gene’s expression pattern, mode of inheritance and disease mechanism, to assign likelihood for contribution to the patients’ phenotype. Results: Pathway-enrichment analysis revealed increased variant burden in 15 pathways (max p -value 2.47E-03) playing roles in tissue mechanical and biomechanical properties, i.e. collagen biosynthesis, cell-extracellular matrix communications and previously unnoticed pathways of hemidesmosome assembly, laminins, integrins and focal adhesion. Fourteen variants of unknown significance and (likely) pathogenic variants in 11 genes - TGM2 , ITGA6 , COMP , COL11A1 , including COL1A1 , COL1A2 , COL6A3 , P3H1 , PLOD1 , PPIB and TNXB causing collagenopathies - in 11 patients were classified as potential to increase susceptibility to the development of non-syndromic cervical insufficiency. Conclusions: Cervical insufficiency being an expression point in a phenotypic continuum of collagenopathies should be investigated further using multiple approaches. This might open up new opportunities to improve patient evaluation and management in the future. L. Volozonoka: None. D. Rots: None. I. Kempa: None. A. Kornete: None. D. Rezeberga: None. L. Gailīte: None. A. Miskova: None. P04.15.C The phenotypic spectrum of Chitayat syndrome: Six additional affected individuals

A. Suter 1 , A. Abad Perez 1 , F. Santos-Simarro 2 , P. Toerring 3 , R. Ramos-Mejia 4 , K. Heath 2 , V. Huckstadt 5 , M. Parrón-Pajares 6 , M. Mensah 1,7 , W. Hülsemann 8 , M. Holtgrewe 1,9 , S. Mundlos 1,10 , U. Kornak 1,11 , O. Bartsch 12 , N. Ehmke 1,10

1 Institute of Medical and Human Genetics, Charité - Universitätsmedizin Berlin, Berlin, Germany, 2 Institute of Medical and Molecular Genetics and Skeletal dysplasia multidisciplinary Unit (UMDE), Hospital Universitario La Paz and CIBERER, ISCIII, Madrid, Spain, 3 Department of Clinical Genetics, Odense University Hospital, Odense, Denmark, 4 Growth and Development Department, Garrahan Hospital, Buenos Aires, Argentina, 5 Genetics Department, Garrahan Hospital, Buenos Aires, Argentina, 6 Department of Radiology and Skeletal dysplasia multidisciplinary Unit (UMDE), Hospital Universitario la Paz, Madrid, Spain, 7 Berlin Institute of Health (BIH), Anna-Louisa-Karsch-Str. 2, 10178, Berlin, Germany, 8 Handchirurgie Kinderkrankenhaus Wilhelmstift, Hamburg, Germany, 9 Core Unit Bioinformatics – CUBI, Berlin Institute of Health, Chariteplatz 1, Berlin, Germany, 10 RG Development & Disease, Max Planck Institute for Molecular Genetics, Berlin, Germany, 11 Institut für Humangenetik, Universität Göttingen, Göttingen, Germany, 12 Institute of Human Genetics, University Medical Centre of the Johannes Gutenberg University, Mainz, Germany
Introduction: Chitayat Syndrome is characterized by hand hyperphalangism with shortened index fingers and ulnar deviation, hallux valgus, severe respiratory distress requiring assisted ventilation and mild facial dysmorphism. It is caused by the recurrent heterozygous missense variant NM_006494.2:c.266A>G; p.Tyr89Cys in ERF, which encodes the ETS2 repressor factor (ERF) protein. The ETS family of transcription factors are involved in cellular proliferation and differentiation, embryological development, hematopoiesis, lymphocyte function and apoptosis. The exact pathomechanism of Chitayat syndrome remains unknown. So far, only seven individuals with molecularly confirmed Chitayat syndrome have been documented in the literature. Methods: We performed a systematic phenotype analysis of six unpublished individuals with Chitayat syndrome. Results: We found unreported phenotypic features and a great variability of pulmonary manifestations. Only one individual presented with a history of assisted ventilation, and one of the remaining presented with an atypical phenotype lacking any obvious pulmonary compromise. Conclusions: Our findings extend the phenotype spectrum associated with the recurrent ERF variant c.266A>G and contribute to improved diagnostic strategies in individuals with hand hyperphalangism. A. Suter: None. A. Abad Perez: None. F. Santos-Simarro: None. P. Toerring: None. R. Ramos-Mejia: None. K. Heath: None. V. Huckstadt: None. M. Parrón-Pajares: None. M. Mensah: None. W. Hülsemann: None. M. Holtgrewe: None. S. Mundlos: None. U. Kornak: None. O. Bartsch: None. N. Ehmke: None. P04.16.A First family from Germany with early-onset chondrocalcinosis and osteoarthritis (CCAL1, OMIM %600668) caused by TNFRSF11B gene mutation

A. C. Blumberg 1 , M. Wingendorf 2 , I. Spier 3 , V. Riehmer 4 , O. Bartsch 1

1 Institute of Human Genetics, University Medical Centre of the Johannes Gutenberg University Mainz, Mainz, Germany, 2 Practice for Orthopaedics and Trauma Surgery, Kirchen/Sieg, Germany, 3 Institute of Human Genetics, University Medical Centre Bonn, Bonn, Germany, 4 Institute of Human Genetics, University Medical Centre Cologne, Cologne, Germany
Chondrocalcinosis (CCAL) is a common condition caused by calcium pyrophosphate accumulation and resulting in destruction of articular cartilage, affecting 4-7% of adults. CCAL is usually seen in polygenic disorders (hypercalcemia, gout, arthritis), but in rare cases runs in families as a dominant trait (usually CCAL2, MIM #118600, ANKH gene). CCAL1 is extremely rare with only four families known to date: an Ashkenazy-Jewish kindred (PMIDs 2383078 and 29578045), a family from Maine (PMID 7887424), a family from the Netherlands and France (PMID 24743232), and a Long Island NY family (PMID 29578045). These families all demonstrated a specific heterozygous TNFRSF11B variant, c.1205A>T, modifying the stop codon (AAT>ATT) and predicting an elongated protein (p.Ter402Leuext*19). We report another 4-generation CCAL1 kindred from Rhineland-Palatinate (comprising 8 females and 3 males) also demonstrating the TNFRSF11B c.1205A>T variant. All patients (11/11, 100%) had early-onset osteoarthritis and chondrocalcinosis with an onset of symptoms between 16 and 30 years, and all had swelling of joints, motion restriction, and intense pain. Three probands (3/10, 30%; no data in a deceased family member) also had digital polyarthritis. First, knee, elbow, ankle and wrist joints were affected, followed by spinal destruction. Remarkably, ball-and-socket joints (e.g. hip joints) were only minimally affected (only an affected 30-year-old male). The early onset and familial clustering led to the molecular diagnosis. This is the first CCAL1 family from Germany, providing further understanding of the clinical signs and treatment (including physical therapy, orthoses, antirheumatic drugs, experimental treatment with interleukin inhibitors, and multiple joint surgery). A.C. Blumberg: None. M. Wingendorf: None. I. Spier: None. V. Riehmer: None. O. Bartsch: None. P04.17.B Three families with suspected Ehlers-Danlos syndrome and glycine substitution in COL1A1 and COL1A2 genes

S. Döhnert 1 , H. Reichenbach 2 , S. Demuth 3 , S. Weidensee 3 , K. Wilhelm 4 , I. Schreyer 4 , B. Eichhorn 1

1 MVZ Mitteldeutscher Praxisverbund Humangenetik MVZ, Dresden, Germany, 2 MVZ Mitteldeutscher Praxisverbund Humangenetik MVZ, Leipzig, Germany, 3 MVZ Mitteldeutscher Praxisverbund Humangenetik MVZ, Erfurt, Germany, 4 Zentrum für ambulante Medizin, Uniklinikum Jena gGmbH, Jena, Germany
Introduction: Glycine substitutions in the Gly-X-Y-triplet disrupt the type I collagen triple helix structure and folding, and typically result in osteogenesis imperfecta (OI) and have not yet been associated with Ehlers-Danlos syndrome (EDS). OI is characterized by bone fragility, whereas EDS affects the connective tissue resulting in joint hypermobility, skin hyperextensibility, and tissue fragility. Although, an OI/EDS overlap” syndrome characterized by clinical hallmarks of EDS and mild or absent OI-phenotype is also described. The underlying mutation spectrum is wide, but includes glycine substitutions. Material and Methods: Here, we report three patients mainly showing joint hypermobility and unusual skin. Panel diagnostic via sequencing by synthesis was conducted including candidate genes contributing to connective tissue disorders. Sanger sequencing was used for analyses of segregation patterns in parents. Results: All index patients carried each an undescribed, heterozygous glycine substitution. In two cases the variant (c.1270G>A/p.(Gly424Ser) in COL1A1 gene; c.434G>A/p.(Gly145Arg) in COL1A2 gene) was maternal inherited and segregated with the symptoms in both families. In the other case the substitution occurred de novo (c.179G>A/p.(Gly60Asp) in COL1A2 gene). Comparable to our patients, glycine substitutions in the more N-terminal region of both genes are already described with “OI/EDS overlap” syndrome. In detail, one of our families showed an EDS-phenotype with additional minimal OI-symptoms (short stature, blue sclera), whereas the others were clinical classified as EDS-phenotype. Conclusions: Despite the detection of glycine substitution, the patients could manifest a more typical EDS phenotype rather than OI. Certainly, subclinical symptoms for OI can additionally occur and should be considered. S. Döhnert: None. H. Reichenbach: None. S. Demuth: None. S. Weidensee: None. K. Wilhelm: None. I. Schreyer: None. B. Eichhorn: None. P04.20.B Morphological and molecular characterization of ectopic cartilage in Crouzon Syndrome

E. Candelo

Fundacion Valle del Lili, Cali, Colombia
Background: Craniosynostosis is the premature closure of the skull sutures. Patients with these condition undergo several surgical procedures, which are not able to fully restore appearance and functionality due to insufficient bone growth and suture refusion. Most mutations disrupt genes that encode for members of the fibroblast growth factor receptor (FGFR) family. Materials and Methods: A knock out mouse with Fgfr2c342Y/C342Y was generated for phenotypic analysis by histological staining of sections of WT, Fgfrc342y/+ heterozygous (HET) and Fgfr2c342Y/C342Y homozygous (HOM) mouse embryos from embryonic day (E) 14.5 and E 16.5 stages. Morphological characterisation of the ectopic cartilages in the mutant specimens was done against the HET and WT by calculating the surface area, perimeter, width on the parietal cartilage in each genotype and cellular characterisation by obtaining the cellular density and shape descriptors. Molecular characterisation was determined by mmunohistochemistry of Collagen I and II. Results: We identified a significant parietal cartilage expansion in HOM embryos since E14.5 with higher perimeter, surface area, and high cellular density. Additionally, characteristic cell morphology comprised; high cellular area and perimeter and circular or rounded shape. These findings were associated with abnormal intensity and localization pattern of Collagen type I and II. Conclusions: A possible switch off on the intramembranous ossification of the parietal and frontal primordial bone and switch on of endochondral ossification process supported by the cellular, morphological and molecular findings of the abnormal cartilage expansion lead to a novel hypothesis of coronal suture fusion that can future drug target on this condition. E. Candelo: None. P04.22.A The first case of Dyssegmental Dysplasia Rolland-Desbuquois type with a variant in HSPG2

T. Kalayci 1 , N. Balanda 2 , C. R. Ferreira 3 , U. Altunoglu 1,4

1 Istanbul University, Istanbu Faculty of Medicine, Medical Genetics Department, Istanbul, Turkey, 2 Undiagnosed Diseases Program, Common Fund, National Institutes of Health, Bethesda, MD, United States, 3 Medical Genomics and Metabolic Genetics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD, United States, 4 Koc University, School of Medicine, Medical Genetics Department, Istanbul, Turkey
Introduction: Dyssegmental dysplasia (DD) is a rare, autosomal recessive disorder characterized by micromelia and anisospondyly. Two distinct forms are described; Silverman-Handmaker (DDSH) characterized by severe shortening of limbs and perinatal lethality, and Rolland-Desbuquois (DDRD) with survival beyond infancy and milder skeletal findings. DDSH is caused by biallelic variants in HSPG2 but the aetiology of DDRD has not been explained. Here, we present the first DDRD patient reported to have an HSPG2 variant. Materials and Methods: Detailed phenotyping and next-generation sequencing were performed. Results: The patient is a 15-month-old female with short bones, flared metaphyses, camptodactyly, anisospondyly, pes equinovarus, flat face, micrognathia, cleft palate and the history of respiratory distress in infancy. She was diagnosed as DDRD given milder skeletal findings and a prolonged survival in comparison to DDSH. Whole exome analysis revealed a homozygous NM_005529.7:c.9970G>A(p.Gly3324Arg) variant on HSPG2 which was predicted to be pathogenic by in silico analyses. Conclusions: The phenotypic heterogeneity and clinical overlap between DDSH and DDRD raise the question of whether HSPG2 variants lead to distinct DD forms or represent a single disorder with variable phenotypic spectrum. In a review of the literature, we recognised three patients reported as DDSH whereas they had milder skeletal findings and survival age between 13 months and 7 years, compatible with DDRD. Strikingly, they harboured novel HSPG2 variants, one of which was detected in our case. In the light of all these findings, we conclude that DDSH and DDRD are allelic and represent a single disorder with a spectrum of severity. T. Kalayci: None. N. Balanda: None. C.R. Ferreira: None. U. Altunoglu: None. P04.23.B FROM CEREBRAL HEMORRAGE AND SUSPICTION OF CHILD ABUSE TO DIAGNOSIS OF FAMILIAR EHLERS DANLOS SYNDROME DUE TO DELECION IN COL5A1 GENE

P. Marin Reina 1 , A. Zuñiga Cabrera 1 , A. Parra Llorca 1 , P. Molina Aguilar 2 , J. Blasco 2 , A. Monzo Blasco 2 , M. Evole Buselli 1

1 Hospital UyP La Fe, Valencia, Spain, 2 Forensic Department, Valencia, Spain
Introduction: The misdiagnosis of Ehler Danlos syndrome (EDS) and child abuse is well known, usually in relationship to skin lesions or bone fractures. At our case, the first manifestation was an aborted lethal episode in an infant due to a cerebral hemorrhage. Clinical Case: A 4-month-old boy was founded unconscious after sleep. He was admitted to the pediatric intensive care unit with a subdural and subarachnoid hemorrhage and severe cerebral edema. Child abuse was suspected. Familiar antecedents: Mother, 34 years, first gravida. She was followed up during infancy due to small size. She has joint hypermobility. Her brother died at sixth month due to supposed sudden infant death. One 50 years old aunt presented a cerebral hemorrhage. Healthy father, 30 years old. No consanguinity. The child presented hypermovility and skin hyperextensibility. EDS vascular type was suspected. NGS analysis was performed using Illumina (NextSeq 500) technology with a ClearSeq Inherited Disease panel (Agilent Thecnologies) and consisted in the analysis of the coding and intronic regions of collagen deficiency related gene. The study did not show any pathogenic variant and was completed by analysis of gene dosage by MLPA (P331; P332), resulting in patient HETEROZYGOUS for deletion of exons 5-7 of the COL5A1 gene. Presence of deletion was confirmed in affected familial members. Conclusions: Vascular complications in nonvascular subtypes of the EDS could be life-threatening. - Misdiagnosis of child abuse should be considered in EDS - The relevance of study microdeletions in cases not diagnosed by gene sequencing. P. Marin Reina: None. A. Zuñiga Cabrera: None. A. Parra Llorca: None. P. Molina Aguilar: None. J. Blasco: None. A. Monzo Blasco: None. M. Evole Buselli: None. P04.25.A Follow-up findings and genotype-phenotype correlation in 18 Turkish patients with biallelic mutation in the FKBP10 gene

A. Yüksel Ülker 1 , D. Uludag Alkaya 1 , G. Yeşil 2 , G. Toksoy 3 , Z. Uyguner 3 , B. Tüysüz 1

1 Istanbul University, Cerrahpasa Medical Faculty, Istanbul, Turkey, 2 Bezmialem Vakif University, Medical Faculty, Department of Medical Genetics, Istanbul, Turkey, 3 Istanbul University, Medical Faculty, Department of Medical Genetics, Istanbul, Turkey
Introduction: Osteogenesis Imperfecta (OI)-typeXI and Bruck syndrome type-I are two very rare disorders caused by biallelic FKBP10 gene mutations. While both disorders are characterized by early onset bone fractures, progressive bone deformity and scoliosis, Bruck syndrome type 1 also has congenital contractures and pterygium. FKBP10/KRT14 mutations have been reported to be present in patients with OI-typeXI and Epidermolysis bullosa simplex (OIEBS). The aim of this study is to investigate the clinical findings and genotype-phenotype correlation in the Turkish cohort with FKBP10 mutation. Materials and Methods: Eighteen patients from fifteen families were enrolled. FKBP10 mutations studied via sanger sequencing or next generation sequencing. The parents of all patients had consanguineous marriages. 16 patients were followed up for 6.2 years (7 months-15.5 years). Results: We identified p.Gly300Ter in eleven patients and p.Gly278Argfs*95 in one patient who live same region. Ten of these patients were compatible with OI-XI, two patients had Bruck1 phenotype. One patient with Bruck1 who lives different region had a novel (c.1256+1G>A) mutation. We also found same mutation (c.321_353del) in five patient with OIEBS who lives same city. Pamidronate treatment was given to 16 follow-up patients. Conclusions: The clinical course of the patients was progressive; bone fractures were started in the infantile period (15/18) and continued as more than 1 fracture (9/16) per year. During the follow-up period, severe limb deformity (7/16) and scoliosis (11/16) developed and no patient could walk (16/16). Although most families are not related, they had the same mutation which is attributed to the founder effect. A. Yüksel Ülker: None. D. Uludag Alkaya: None. G. Yeşil: None. G. Toksoy: None. Z. Uyguner: None. B. Tüysüz: None. P04.26.B Wide sutures and fontanels, delayed speech development and a hoarse voice useful signs in diagnosing KBG syndrome, clinical, molecular and cytogenetic description of 12 patients .

A. Kutkowska-Kazmierczak 1 , M. Gos 1 , J. Castaneda 1 , M. Boczar 1 , A. Barczyk 1 , A. Pietrzyk 1 , O. Kordowska 1 , T. Gambin 1 , P. Własienko 1 , B. Wiśniowiecka-Kowalnik 1 , K. Kowalczyk 1 , M. Bartnik-Głaska 1 , M. Smyk 1 , M. Paczkowska 1 , I. Plaskota 1 , K. Niepokój 1 , J. Kelly 2 , B. Nowakowska 1 , E. Obersztyn 1 , J. Bal 1

1 Institute of the Mother and Child, Warsaw, Poland, 2 Quest Diagnostics Nichols Institute, Chantilly, VA, United States
Introduction: KBG syndrome is a neurodevelopmental disorder characterized by short stature, macrodontia, developmental delay, behavioral problems as hyperactivity, velopharyngeal insufficiency causing feeding problems and speech delay and by delayed closing of sutures and fontanels. Most of the patients with KBG syndrome have a mutation of ANKRD11 gene or micro-rearrangement encompassing this gene. Patients and methods: We present clinical evaluations of 12 patients with the clinical features of KBG syndrome at the age from 4 months to 17 years. The mutation analysis in these patients was performed using panel next generation sequencing (6pts), whole exome sequencing (WES) (2pts) and array CGH (4pts). Results: Besides typical dysmorphic features for KBG syndrome found in all patients, the majority of patients had wide sutures and fontanels (7/12), delayed speech (9/11), feeding problems (9/12). Hoarse voice was noticed in 4 patients and short stature, below 2 SD in two patients, between -1-2 SD in 7 patients. Abnormal behavior was noted in 4 patients, macrodontia in 3 patients. Conclusions: The fact that the delayed closing of sutures and fontanels was observed in more than half of our patients with KBG syndrome caused by mutations or rearrangements of ANKRD11 gene confirms the role of this gene in skull and suture formation. Speech delay is found in majority of patients with this syndrome. Rarely described sign - hoarse voice could reinforce clinical knowledge in diagnosing KBG syndrome. The studies on craniosynostoses were supported from Institute of Mother and Child in Warsaw intramural grant no. OPK-510-18-63 A. Kutkowska-Kazmierczak: None. M. Gos: None. J. Castaneda: None. M. Boczar: None. A. Barczyk: None. A. Pietrzyk: None. O. Kordowska: None. T. Gambin: None. P. Własienko: None. B. Wiśniowiecka-Kowalnik: None. K. Kowalczyk: None. M. Bartnik-Głaska: None. M. Smyk: None. M. Paczkowska: None. I. Plaskota: None. K. Niepokój: None. J. Kelly: None. B. Nowakowska: None. E. Obersztyn: None. J. Bal: None. P04.27.C Patients with suspected Erythrodermia variabilis of a large family carry variants in GJB3 and KRT10

A. Hotz 1 , S. Redler 2 , J. Kopp 1 , A. Zimmer 1 , J. Fischer 1

1 Institute of Human Genetics, University Medical Center Freiburg, Faculty of Medicine, Freiburg, Germany, 2 Institute of Human Genetics, Heinrich-Heine-University, Medical Faculty, Düsseldorf, Germany
Introduction: Erythrokeratoderma variabilis (EKV) is a rare skin disease characterized by two features: red skin areas with sharp borderlines, which tend to shift positions; and hyperkeratosis, which can either be generalized or localized as thickened plaques. EKV is mostly inherited in an autosomal dominant manner and is mainly caused by pathogenic variants in GJB3 , GJB4 or GJA1 . Results: We describe a large family with twelve patients of five generations affected by a skin disorder. The index patient shows typical features of EKV. Mutation analysis in seven affected members from three generations using Next Generation and Sanger sequencing methods revealed that all of them carry both the heterozygous unclear variant c.783G>C, p.(Gln261His) in GJB3 and the novel heterozygous pathogenic variant c.1346A>C, p.(Tyr449Ser) in KRT10 . Pathogenic variants in KRT10 lead to Epidermolytic ichthyosis, which is characterized by diffuse erythroderma and blistering at birth and progressive hyperkeratosis later in life and is usually inherited in an autosomal dominant manner. Furthermore, two unaffected members were analyzed, one of them carries none of the variants, the other only the GJB3 variant. Conclusions: It is remarkable, that all seven tested affected family members from three generations carry both variants in GJB3 and KRT10 . Although the KRT10 variant is pathogenic, it cannot be excluded that the GJB3 variant has an impact on the phenotype, since the initial suspected diagnosis was EKV. However, it cannot be excluded that the GJB3 variant is neutral. In this case, analysis of patients with suspected EKV should include pathogenic variants in KRT10 . A. Hotz: None. S. Redler: None. J. Kopp: None. A. Zimmer: None. J. Fischer: None. P04.30.C When textbooks fall short - earlier detection of Hypochondroplasia - a large UK cohort experience and systematic review .

A. Sabir 1 , E. Morley 2 , J. Sheikh 2 , A. Singh 2 , A. Cocca 1 , M. Cheung 1 , M. Irving 1

1 Guy’s and St Thomas’ Hospital, London, United Kingdom, 2 University of Birmingham, Birmingham, United Kingdom
Introduction: Hypochondroplasia (HCH) is a rare autosomal dominant skeletal dysplasia caused by FGFR3 mutations leading to disproportionate short stature. Classically HCH presents in toddlers or school-age children, as limb-to-trunk disproportion is often mild and easily overlooked during infancy. We report experiences from our UK HCH cohort of ~30 patients and review recent literature to explore the antenatal detection of HCH. Methods: We reviewed the rate of antenatal HCH detection in our 29 patient cohort and describe relevant case-date. We then conducted a systemic, retrospective literature review using PUBMED and MEDLINE, identifying antenatally suspected / detected HCH cases. Paper inclusion criteria: Antenatally suspected HCH. Molecular confirmation of denovo HCH. Exclusion criteria: unclear antenatal scanning history. Results: Antenatally suspected (and concurrent or deferred molecularly confirmation) HCH has been reported 10 times in the literature (between 2004-2019) and in 6 of our 29 patient cohort. Key sonographic markers included reduced; femur length, biparietal diameter and humeral length. Conclusions: HCH is increasingly detected both antenatally and in infancy, contrary to previous descriptions. This is likely due to greater HCH awareness, improved imaging and easier molecular testing. Thus one should consider HCH outside the classical presenting period. Studying the natural history of earlier cases is important with the advent of several targeted FGFR3 therapies currently in trials for Achondroplasia (Biomarin’s Vosorotide, Ascendis’ Transchon, QED’s Infagratinib and Pfizer’s TA-46) as such therapies may soon be trialled in HCH and are likely most efficacious during earliest skeletal maturation. A. Sabir: None. E. Morley: None. J. Sheikh: None. A. Singh: None. A. Cocca: None. M. Cheung: None. M. Irving: None. P04.31.A Mono-allelic deleterious variants in autosomal recessive skeletal dysplasia genes are strongly associated with idiopathic short stature

C. T. Thiel 1 , N. Hauer 1 , C. Vogl 1 , S. Uebe 1 , H. Sticht 2 , A. B. Ekici 1 , C. Kraus 1 , H. Dörr 3 , A. Reis 1

1 Institute of Human Genetics, Friedrich-Alexander-Universität Erlangen-Nürnberg FAU, Erlangen, Germany, 2 Institute of Biochemistry, Friedrich-Alexander-Universität Erlangen-Nürnberg FAU, Erlangen, Germany, 3 Department of Pediatrics and Adolescent Medicine, Friedrich-Alexander-Universität Erlangen-Nürnberg FAU, Erlangen, Germany
Short stature is a common concern in childhood with an incidence of 3%. After exome analysis about 60% remain without diagnosis (idiopathic short stature, ISS). Recent identification of heterozygous mutations in ACAN in 1.4% of ISS individuals confirmed that single, large-effect variants strongly affect height (appr. 15cm final adult height). We therefore hypothesized that heterozygous variants in other autosomal recessive skeletal dysplasia genes also contribute to ISS. We selected exome data from 368 ISS families where previous analysis showed no mutations in a known growth-associated gene and selected 114 genes known to be involved in autosomal recessive inherited skeletal dysplasia, extracted from the “Nosology and Classification of Genetic Skeletal Disorders” (Bonafe et al. 2015). From a total of 391 heterozygous variants we selected 33 variants in 26 genes which were classified as ACMG class 4/5 and compatible with segregation in the families and were thus considered strong candidates for ISS. This accounted for 31 patients (8.4%). No class 4/5 variant in any of the 26 genes was detected in 343 controls (p<1e-85). Frequently affected genes were ACAN (6 individuals) and NPR2 (3 individuals) representing 2.5% of the study group. In addition to missense variants in 17 genes, a nonsense variant was observed in ADAMTS10 , CHST3 , CYP27B1 , TCIRG1 , TRIP11 , WDR35 and CUL7 . Our findings suggest a strong association between rare mono-allelic deleterious variants in genes for recessive skeletal dysplasia and idiopathic short stature and provide further evidence for the contribution of rare, large-effect variants to the etiology of this common trait. C.T. Thiel: None. N. Hauer: None. C. Vogl: None. S. Uebe: None. H. Sticht: None. A.B. Ekici: None. C. Kraus: None. H. Dörr: None. A. Reis: None. P04.32.B IRAK1 gene SNPs rs1059703 and rs3027898 polymorphisms is associated with psoriasis

L. Kucinskas 1 , A. Bruzaite 1 , J. Cepaite 1 , V. Kucinskiene 2 , R. Ugenskiene 1 , S. Valiukeviciene 2

1 Institute of Biological System and Genetic Research LUHS, Kaunas, Lithuania, 2 Department of Skin and Venereal Diseases, Lithuanian University of Health Sciences (LUHS), Hospital of LUHS Kauno Klinikos, Kaunas, Lithuania
Psoriasis vulgaris and psoriatic arthritis are multifactorial diseases with an important genetic component. The clinical significance of IRAK1 genes SNPs rs1059703 ir rs3027898 are unknown for psoriasis pathogenesis. The aim of present study was to determinate IRAK1 gene SNPs rs1059703 and rs3027898 clinical importance for psoriatic arthritis and psoriasis development. Methods . 106 patients with psoriasis from Skin and Veneral disease department of Lithuanian University of Helath Sciences hospital and 52 healthy control group subjects were genotyped. The IRAK1 gene SNPs rs1059703 and rs3027898 genotyping was performed using Mva1269I (BSMI) and Eco57I (AcuI) restriction enzymes. Data and statistical analysis was performed using, Microsoft Office Excel“ and, IBM SPSS Statistics 20.0“ programs. Results . The number of patients of IRAK1 gene rs3027898 with C/A, C/C and C genotypes outnumbered control group (32 persons (30 %) vs 5 persons (10 %). The number of patients of IRAK1 rs1059703 with C/T, C/C and C genotype was higher compare with control group (30 persons (30 %) vs 6 persons (11 %). The statistical analysis of patients genotypes and psoriatic arthritis corelation showed no significant data of association (p < 0,05). Odds of ratio calculation (OR) showed IRAK1 rs3027898 C/A, C/C, C and IRAK1 rs1059703 C/T, C/C and C genotypes association with psoriasis (OR 4,06) and (OR 3,39) respectively. Conlusions . Statistically significant association of IRAK1 rs3027898 and IRAK1 rs3027898 SNPs with psoriatic arthritis was not determinated. IRAK1 rs3027898 C/A, C/C, C genotypes and IRAK1 rs1059703 C/T, C/C and C genotypes are associated with psoriasis development. L. Kucinskas: None. A. Bruzaite: None. J. Cepaite: None. V. Kucinskiene: None. R. Ugenskiene: None. S. Valiukeviciene: None. P04.33.C Skin cancer screening is indicated in IWC patients due to the identification of heterozygous KRT10 variants in patients with MAUIE syndrome

B. Burger 1 , A. Ghosh 1,2 , C. K. Y. Ng 3 , S. Piscuoglio 1 , I. Spoerri 1 , P. H. Itin 1 , K. Greer 4 , D. Elbaum 5

1 University of Basel and University Hospital Basel, Basel, Switzerland, 2 University of Zurich and ETH Zurich, Zurich, Switzerland, 3 University of Bern, Bern, Switzerland, 4 University of Virginia, Charlottesville, VA, United States, 5 Mercy Hospitals of Bakersfield, Bakersfield, CA, United States
Ichthyosis with confetti (IWC) is an autosomal dominant skin disorder characterized by a congenital erythema and the development of numerous pale spots. About 20% of the patients are reported to develop skin cancer at young adulthood. All IWC patients are carriers of a heterozygous keratin (KRT)10 or KRT1 variant leading to a shifted reading frame and the translation of an aberrant carboxyl (C)-terminus. This results in the nuclear localization of the KRT10/KRT1 protein instead of cytoplasmic. About 20 years ago, two patients were described as MAUIE patients. They showed the typical IWC-phenotype including the development of pale spots. Both patients developed aggressive and fatal cutaneous squamous cell carcinoma (cSCC). A genetic analysis is not yet done. Our study aimed to identify an IWC-correlated frameshift variant in both patients and to clarify whether they suffered from IWC. We performed whole exome sequencing on genomic DNA from FFPE cSCC tissue of both deceased patients and screened for variants similar to IWC-associated variants. A heterozygous KRT10 variant, encoding for the aberrantly shifted C-terminus, was identified in each patient and the nuclear localization of KRT10 was confirmed by immunofluorescence staining. Our study reclassified both MAUIE patients as patients with a specific form of IWC. The fatal course of the disease is not typical for the other IWC forms, but it emphasizes the importance of skin cancer screening in all IWC-patients. We recommend the regular intensive examination of their skin for cancers and a good education of the patients with reference to their disease. B. Burger: None. A. Ghosh: None. C.K.Y. Ng: None. S. Piscuoglio: None. I. Spoerri: None. P.H. Itin: None. K. Greer: None. D. Elbaum: None. P04.34.A Kinesin family member 13A , KIF13A , is a novel gene for hypomelanosis of Ito


The University of Hong Kong, Hong Kong, China
Introduction: Hypomelanosis of Ito is a neurocutaneous syndrome, characterized by distinctive skin manifestations, with or without multisystem involvements. While the exact cause of hypomelanosis of Ito is unknown, chromosomal mosaicism as a result of sporadic gene mutation has been suggested in literature. Materials and Methods: In our Clinics for Rare diseases Referral (CRareDr), we encountered a 3-year-old girl presenting with characteristic skin pigmentation and developmental delay, suggesting hypomelanosis of Ito This case was enrolled in our undiagnosed diseases program (UDP) for ending diagnostic odyssey. We initiated genetic analysis for neurodevelopmental impairment using whole-genome (WGS) and whole exome sequencing (WGS). Results: A heterozygous de novo variant NM_022113.6: c.2357dupA; p.Asn786Lysfs*18 in the KIF13A gene was identified in the patient. The variant is a single nucleotide duplication causing a shift in the translational reading frame and premature termination of the protein. The variant is absent in control population in gnomAD exomes and genomes. Conclusions: KIF13A is a relatively new gene with a limited number of studies on its function and has not been reported to cause any hereditary conditions. To date, KIF13A is known to interact with adaptor complex AP-1, forming a complex that is responsible for the transportation of a number of proteins and receptors to plasma membrane and endosomal organelles. The role of KIF13A in melanosome biogenesis is well-demonstrated in in-vitro studies and may explain the underlying pathogenesis of skin hypopigmentation in hypomelanosis of Ito. C. Lam: None. P04.37.A Functional evidence supporting the hypothesis that Neanderthals and Denisovans had a genetically specified high bone mass negatively selected in anatomically modern humans

N. Roca-Ayats 1 , I. Maceda 2 , C. Bruque 3 , M. Cozar 1 , N. Garcia-Giralt 4 , L. Mellibovsky 4 , W. van Hul 5 , O. Lao 2 , D. Grinberg 1 , S. Balcells 1

1 University of Barcelona, CIBERER, IBUB, IRSJD, Barcelona, Spain, 2 Centre Nacional d’Anàlisi Genòmica CNAG-CRG, Barcelona, Spain, 3 Centro Nacional de Genética Médica, ANLIS, Instituto de Biología y Medicina Experimental, CONICET, Buenos Aires, Argentina, 4 Musculoskeletal Research Group, IMIM (Hospital del Mar Medical Research Institute), Centro de Investigación Biomédica en Red de Fragilidad y Envejecimiento Saludable (CIBERFES), ISCIII, Barcelona, Spain, 5 Department of Medical Genetics, University of Antwerp, Antwerp, Belgium
Introduction: Bone density underwent gracilization through hominin evolution; while Neanderthals and Denisovans had robust and dense bones, anatomically modern humans (AMH) show a more gracile skeleton. In this context, mutations in the first β-propeller of LRP5 result in high bone mass (HBM) in AMH, suggesting this gene as a suitable candidate for studying the hominin evolution of bone mass. Material and Methods: Variants were looked for in the publically available Neanderthal and Denisovan genomes and functionally tested by a luciferase assay in Saos-2 cells. A molecular homology model of LRP5 was generated using MODELLER (v.9.22). Introgression was inferred in the Simons Genome Diversity Project using the CRF method. Evidences of selective pressures at LRP5 in AMH were sought using PopHuman browser. Results: We identified five Neanderthal or Denisovan missense mutations in LRP5 . Four displayed significantly greater Wnt pathway stimulation, and two also showed reduced inhibition by DKK1. Modelling suggested that these mutations either affected the 3D structure of the first β-propeller, or impinged on the interaction of LRP5 with DKK1. We observed that the LRP5 genomic region in AMH is depleted of introgression from Neanderthals and/or Denisovans and that the functional Neanderthal and Denisovan mutations are at a very low frequency in current human populations. Conclusions: The variants identified in Neanderthal and Denisovan genomes, together with the evolutionary analyses in the LRP5 region suggest that this gene may have been involved in HBM determination in archaic hominins. Funding: Spanish MINECO (SAF2016-75948-R; PGC2018-098574-B-I00); CIBERER (U720); AGAUR (2017 SGR 937). N. Roca-Ayats: None. I. Maceda: None. C. Bruque: None. M. Cozar: None. N. Garcia-Giralt: None. L. Mellibovsky: None. W. van Hul: None. O. Lao: None. D. Grinberg: None. S. Balcells: None. P04.39.C Looking for genetic modifiers in Marfan syndrome

E. Ramos-Luis 1,2 , G. Teixidó 3 , J. Limieres 3 , R. Cruz 4,2 , C. Granato 3 , B. Sobrino 5,2 , J. Amigo 5,2 , A. Blanco-Verea 1,2 , A. Carracedo 5,2,4 , A. Evangelista 3 , M. Brion 1,2

1 Xenética Cardiovascular, Instituto de Investigación Sanitaria de Santiago, Santiago de Compostela, Spain, 2 Grupo de Medicina Xenómica, Universidade de Santiago de Compostela, Santiago de Compostela, Spain, 3 Hospital Vall d’Hebron, Servicio de Cardiología, Barcelona, Spain, 4 CIBER enfermedades Raras, Santiago de Compostela, Spain, 5 Fundación Pública Galega de Medicina Xenómica, Santiago de Compostela, Spain
Introduction: Marfan syndrome is an autosomal dominant disorder of connective tissue manifested in the ocular, skeletal and cardiovascular systems. It is most frequently caused by mutations in FBN1 with high clinical penetrance, but showing a high inter and intra-familial variability, that cannot be explained by the current knowledge of genotype-phenotype correlation. In order to improve knowledge of the whole genetic background of this disease, this study aims to identify genetic modifiers of the phenotypic expression of Marfan syndrome, looking for the intra-familial variability with known causal mutation in FBN1. Material and Methods: A total of 22 Marfan syndrome patients with different severity from 8 families with known FBN1 mutation, were exome sequenced. For exome wide gene-set analyses F-SKAT (familial sequence kernell association test) was performed. At the intra-family level, non-common rare protein changing variants among first-degree relatives with severe and mild phenotypes were selected for discriminant analysis of principal components (DAPC). Results: F-SKAT test showed no gene with significant differences after Bonferroni test between severe and mild phenotype group of patients. Nevertheless, DAPC showed two genes of retinal homeostasis, NXNL1 gene and RP1L1 gene, as one of the principal contributors to the discrimination analysis between mild and severe phenotype. Conclusions: The genetic architecture of Marfan syndrome is very complex, however, as it was previously described in the literature, this study point to the genes related to retinal homeostasis as possible genetic modifiers of the disease. Founding: Instituto de Salud Carlos III and Fondo Europeo de Desarrollo Regional (PI14/01062, PI16/00903) E. Ramos-Luis: None. G. Teixidó: None. J. Limieres: None. R. Cruz: None. C. Granato: None. B. Sobrino: None. J. Amigo: None. A. Blanco-Verea: None. A. Carracedo: None. A. Evangelista: None. M. Brion: None. P04.40.A Reciprocal translocation as a possible cause of familial connective tissue disorder

B. Herr, K. Komlosi, A. Tzschach, J. Fischer, B. Gläser

Department of Human Genetics, Freiburg, Germany
Introduction: Monogenic disorders due to apparently balanced reciprocal translocations are rarely reported. There are few reports of interstitial deletions involving FBN1 -gene and only one case of a patient with Marfan syndrome and a complex chromosome rearrangement. However, no disruption of FBN1 due to a translocation has been reported so far. Clinical report: We report on a mother and daughter with clinical symptoms of connective tissue disorder. The 39-year-old mother presented with congenital strabismus, muscle weakness, muscle hypotrophy of forearms and calves, reduced exercise tolerance and muscle and joint pain. Skeletal manifestations included arachnodactyly, joint laxity and foot deformity. Her father and a paternal uncle had skeletal abnormalities and died unexpectedly in young adulthood. Her 18-year-old daughter fulfilled the Ghent criteria for Marfan syndrome. Methods and Results: Connective tissue panel diagnostics did not reveal pathogenic variants in the mother. R-banded karyotyping of lymphocyte cultures revealed the same reciprocal translocation in mother and daughter: 46,XX,t(2;15)(q22;q21.1). Chromosome microarray analysis revealed no imbalances. Subsequent FisH analysis with region-specific BAC probes for 2q and 15q narrowed down the translocation break point to a region of approximately 110 kb from g.43663778 proximal the 3`end of FBN1 to g.48772449 in intron 32-33 of FBN1 . Conclusions: In order to prove a possible disruption of FBN1 we plan to perform long range PCR, next generation mapping and RNA sequencing. We emphasize the importance of a combination of cytogenetic and molecular diagnostic techniques to reveal the cause of monogenic diseases typically caused by point mutations. B. Herr: None. K. Komlosi: None. A. Tzschach: None. J. Fischer: None. B. Gläser: None. P04.41.B Variant classification in Marfan syndrome: illustrating an underestimated issue in DNA-diagnostics

G. Pals 1,2,3 , S. A. Stolk 1 , A. Maugeri 1 , D. Micha 2 , M. A. M. M. Mannens 1

1 Amsterdam UMC, Genome Diagnostics laboratory, Amsterdam, Netherlands, 2 Centre for Connective tissue disorders, Amsterdam, Netherlands, 3 Prodia laboratories, Jakarta, Indonesia
Introduction: Correct classification is crucial for reliable diagnostic DNA-testing of patients. The classification of variants ranges from benign (class 1) to pathogenic (class 5). General recommendations for classification have been developed, like the American College of Medical Genetics (ACMG). Variant classification differs per method used. We compared three different guidelines, by reanalyzing patients’ data of the FBN1 gene. Materials and Methods: Variants in the FBN1 gene, found in our lab, were used to compare the outcome of three different variant classifications: the ACMG guidelines, the methods of our accredited laboratory (ISO15189-RvA-M174) and recommendations from the Revised Ghent nosology of Marfan syndrome (Loeys et al. 2010). Results: We classified, by using our guidelines, 869 variants as pathogenic. 77 of these are classified as benign according to Loeys et al. or VUS according to ACMG. A large number of variants that we classified as benign would be classified as likely benign or VUS according to the other guidelines. Conclusions: Comparison of various variant interpretations shows that pathogenic variants were classified as VUS or even benign by using the other guidelines. This was mainly caused by using an incorrect consensus sequence for the amino acid motif of the 47 EGF domains in fibrillin-1 (Loeys et al.) or not using specific properties of the protein. Different use of frequency data causes a large difference in interpretation of benign variants. These data emphasize that knowledge of the disease and the protein involved are indispensable in correct interpretation of variants. G. Pals: None. S.A. Stolk: None. A. Maugeri: None. D. Micha: None. M.A.M.M. Mannens: None. P04.42.C Identifying the molecular cause in Marfan and Marfan like phenotype in a cohort of patients from Romania - 5-year single center experience

M. Puiu 1 , A. Chirita-Emandi 1 , N. Andreescu 1 , G. Doros 2 , A. Popoiu 2 , A. Dumitrescu 3 , A. Lacatusu 4 , C. Zimbru 5

1 Genetics Discipline, Center of Genomic Medicine, “Victor Babes,” University of Medicine and Pharmacy, Timisoara, Romania, 2 Pediatric Department University of Medicine and Pharmacy “Victor Babes”, Timisoara, Romania, 3 Clinical Emergency Hospital for Children “Louis Turcanu”, Timisoara, Romania, 4 “Victor Babes,” University of Medicine and Pharmacy, Timisoara, Romania, 5 Department of Automation and Applied Informatics, Politehnica University, Timisoara, Romania
Background: Connective tissue disorder represent a group of life-threatening disorders. The prevalence of these diseases is relatively low, but Marfan, Ehlers-Danlos, and Loeys-Dietz syndromes are ubiquitous, affect both genders and involve several organs, particularly the cardiovascular system, eyes, and skeleton. Mortality generally results from cardiovascular complications, mainly aortic dissection. Identifying the molecular cause can lead to a different therapeutic management and the possibility for prevention of reoccurrence in the family. Aim: We aimed to retrospectively evaluate the diagnostic yield in these group o patients and present the variants identified. Methods: Using targeted Next Generation Sequencing (NGS) with a panel for 174 genes (TruSightCardio Illumina) related to cardiologic diseases we screened 52 patients (37 males/15 females) evaluated for a Marfan/Marfan like phenotype using Ghent nosology, between 2015-2020 at the Center for Genomic Medicine University of Medicine and Pharmacy Timisoara, Romania. Results: Diagnosis was identified in 55.7% (29/52) patients, most(25) pathogenic/likely pathogenic variants were found in FBN1 gene, 3 splicesite, 5 frameshift and 17 missense variants, including two variants associated with neonatal Marfan Syndrome (NM_000138.4:c.3143T>C and c.3217G>A). Of these, 10 are previously unreported variants. Nine out of 52 patients originated from 3 families. Diagnosis in 3 other patients involved COL3A1, TGFBR1 and SLC2A10 genes. Conclusions: This study broadens the spectrum of pathogenic variants in FBN1 gene, providing an update of the molecular basis of Marfan Syndrome in Romania. M. Puiu: None. A. Chirita-Emandi: None. N. Andreescu: None. G. Doros: None. A. Popoiu: None. A. Dumitrescu: None. A. Lacatusu: None. C. Zimbru: None. P04.44.B Characterization of the pathogenetic role of POLD1 gene in Mandibular hypoplasia, Deafness, Progeroid features and Lipodystrophy (MDPL) Syndrome

C. De Masi 1 , P. Spitalieri 1 , M. Murdocca 1 , V. Ferradini 1 , M. D’Adamo 2 , P. Sbraccia 2 , M. Sanchez 3 , I. Udroiu 4 , J. Marinaccio 4 , G. Novelli 1 , A. Sgura 4 , M. D’Apice 5 , F. Sangiuolo 1

1 Dep. of Biomedicine and Prevention, Tor Vergata University, Rome, Italy, 2 Dep. of Systems Medicine, Tor Vergata University, Rome, Italy, 3 Core Facilities Center - Section of Cytometry, Istituto Superiore di Sanità, Rome, Italy, 4 Dep. of Science. "Roma Tre" University, Rome, Italy, 5 Laboratory of Medical Genetics, Tor Vergata Hospital, Rome, Italy
MDPL defines a multisystem disorder identified in 22 patients worldwide and associated to mutations in POLD1 gene, encoding the DNA polymerase δ, involved in DNA replication and repair. We identified an in-frame deletion (p.Ser605del), segregating for the first time as an autosomal dominantmutation, in a MDPL patient from whom dermal fibroblasts (HDFs) have been derived. Cells displayed in vitro abnormalities of nuclear envelope morphology, presence of micronuclei, a large number of autophagosomesand a significant accumulation of prelamin A, strongly linked to altered cellular proliferation and genomicinstability. DNA damage-induced treatment in MDPL-HDFs revealed a poor capacity of DNA repair, an arrestin G0/G1 phase transition, and a lower number of cells in the S phase. Moreover, Western Blot analysesshowed a nuclear reduction of POLD1 in MDPL-HDFs compared to WT in basal condition but a statisticallysignificant and persistent increase after cisplatin/1-Gy X-ray damage when compared to WT. Analysis of telomere length exhibited an increased rate of telomere shortening, accompanied by anaccumulation of unrepaired telomeric DNA damage. Furthermore, the evaluation of H2AX and TelomereInduced Foci after 1-Gy X-ray treatment enabled us to figure out that POLD1 repairs less efficiently DNAdamage and its activity is mainly associated with the homologous recombination phase. Finally, we also generated human induced pluripotent stem cells from patient’s fibroblasts showing thepeculiar presence of micronuclei. Knowing the mechanistic basis for the association of DNA damage and failed DNA repair with aging wouldopen up new therapeutic possibilities for this rare disease. C. De Masi: None. P. Spitalieri: None. M. Murdocca: None. V. Ferradini: None. M. D’Adamo: None. P. Sbraccia: None. M. Sanchez: None. I. Udroiu: None. J. Marinaccio: None. G. Novelli: None. A. Sgura: None. M. D’Apice: None. F. Sangiuolo: None. P04.45.C Molecular analysis of Polish patients with Mendelian Disorders of Cornification

K. Wertheim-Tysarowska 1 , K. Osipowicz 2 , A. Kutkowska-Kaźmierczak 1 , J. Sawicka 1 , M. Jackiewicz 1 , O. Kordowska 1 , M. Gos 1 , A. M. Rygiel 1 , K. Niepokój 1 , K. Woźniak 2 , C. Kowalewski 2 , J. Bal 1

1 Medical Genetic Department, Institute of Mother and Child, Warsaw, Poland, 2 Department of Dermatology and Immunodermatology, Warsaw Medical University, Warsaw, Poland
Introduction: Mendelian Disorders of Cornification (MeDOC) comprise syndromic or non-syndromic heterogeneous disorders manifesting by skin hyperkeratosis and/or scaling. Mutations in approximately 40 different genes can cause MeDOCs, that can be inherited in autosomal recessive/dominant or X-linked patterns. Identification of molecular defect is essential for prognosis, proper medical interventions and genetic counseling. Nonetheless, genetic diversity, low incidence of majority of MeDOCs and overlapping phenotypes make diagnosis challenging. The aim of the study was to characterize molecularly Polish MeDOC patients. Materials and methods: A group of 161 patients with clinical symptoms of MeDOC was enrolled to the study. Genetic analysis was performed using self-designed customized NGS panel. Sanger sequencing or MLPA were used for mutations verification. Results: We identified full genotype in 125 (78%) patients. In 17 (11%) patients we didn’t detect any mutation, while in 19 (12%) we found mutation in one allele only, despite expected autosomal recessive inheritance. Overall, we detected mutations in 27 distinct genes. The 33 patients had mutations in more than one gene involved in skin barrier functioning: 13 of them had pathogenic mutations in two genes, while in remaining 20 cases variant of unknown significance was detected in at least one of the genes. Conclusions: Our diagnostic strategy proved to be an efficient and sensitive diagnostic tool for MeDOC patients. Our data provide further information regarding molecular epidemiology of skin barrier defects and also focus on the presence of secondary mutations, which have important impact on genetic counseling and, presumably, may affect therapy. Supported by NCN:2014/13/D/NZ5/03304 K. Wertheim-Tysarowska: None. K. Osipowicz: None. A. Kutkowska-Kaźmierczak: None. J. Sawicka: None. M. Jackiewicz: None. O. Kordowska: None. M. Gos: None. A.M. Rygiel: None. K. Niepokój: None. K. Woźniak: None. C. Kowalewski: None. J. Bal: None. P04.49.A CRISPR/Cas9-based analysis of a missense variant in Paxip1 suggests subtle effects on palatal process distance

N. Ishorst 1,2 , E. Z. Kvon 2 , Y. Zhu 2 , S. Tran 2 , I. Plajzer-Frick 2 , C. S. Pickle 2 , S. Hölzel 1 , A. Harrington 2 , J. Godoy 2 , J. A. Akiyama 2 , L. Selleri 3,4,5,6,7 , I. C. Welsh 3 , M. M. Nöthen 1 , K. U. Ludwig 1 , E. Mangold 1 , D. E. Dickel 2 , L. A. Pennacchio 2,8,9 , A. Visel 2,8,10

1 Institute of Human Genetics, University of Bonn, School of Medicine & University Hospital Bonn, Bonn, Germany, 2 Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, Berkeley, CA, United States, 3 Program in Craniofacial Biology, University of California at San Francisco, San Francisco, CA, United States, 4 Institute of Human Genetics, University of California at San Francisco, San Francisco, CA, United States, 5 Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California at San Francisco, San Francisco, CA, United States, 6 Department of Orofacial Sciences, University of California at San Francisco, San Francisco, CA, United States, 7 Department of Anatomy, University of California at San Francisco, San Francisco, CA, United States, 8 U.S. Department of Energy Joint Genome Institute, Berkeley, CA, United States, 9 Comparative Biochemistry Program, University of California Berkeley, Berkeley, CA, United States, 10 School of Natural Sciences, University of California Merced, Merced, CA, United States
Nonsyndromic cleft lip with/without cleft palate (nsCL/P) is among the most common congenital malformations and has a multifactorial etiology. Common risk loci explain parts of the trait heritability, but a substantial fraction needs to be elucidated. Parts of the missing heritability could be explained by rare, penetrant mutations that might occur de novo in genes potentially involved in craniofacial development. A previous study by our group identified three nsCL/P susceptibility genes with rare recurrent de novo mutations ( PAXIP1 , MDN1 , ANK1 ). The present study aims at assessing a contribution of the rare mutations found in these nsCL/P susceptibility genes to cleft development by using CRISPR/Cas9 generated mouse models. We created five knock-in mouse strains that carried human missense mutations in Paxip1 (1 mutation) , Ank1 (1 mutation), and Mdn1 (3 different mutations). Embryos at embryonic day E18.5 were dissected and assessed for craniofacial anomalies using skeletal preparations and microscopic assessment. No craniofacial anomalies were observed for E18.5 embryos in the F 0 generation in any of the five knock-in strains. In the F 1 generation, 2/31 E18.5 pups carrying the knocked-in Paxip1 missense mutation in a heterozygous state presented with abnormal body posture, a broader snout and a subtle increase in distance between the palatal processes, suggesting a role of the missense variant in Paxip1 in craniofacial development. Further follow-up studies in mice homozygous for the variants and more precise phenotyping methods are ongoing. N. Ishorst: None. E.Z. Kvon: None. Y. Zhu: None. S. Tran: None. I. Plajzer-Frick: None. C.S. Pickle: None. S. Hölzel: None. A. Harrington: None. J. Godoy: None. J.A. Akiyama: None. L. Selleri: None. I.C. Welsh: None. M.M. Nöthen: None. K.U. Ludwig: None. E. Mangold: None. D.E. Dickel: None. L.A. Pennacchio: None. A. Visel: None. P04.50.B High-throughput screen for rare variants in candidate genes for orofacial clefting

B. Zametica 1 , F. Thieme 1 , J. Fazaal 1 , N. Ishorst 1 , E. Ongkosuwito 2 , C. Carels 3 , I. A. L. M. van Rooij 4 , E. Mangold 5 , K. U. Ludwig 1

1 Institute of Human Genetics, University of Bonn, School of Medicine & University Hospital Bonn, Bonn, Germany, Bonn, Germany, 2 Department of Orthodontics and Craniofacial Biology, Radboud Institute for Molecular Life Sciences, Radboudumc, Nijmegen, Netherlands, 3 Department of Human Genetics and Department of Oral Health Sciences, KU Leuven and University Hospitals KU Leuven, Leuven, Belgium, 4 Department for Health Evidence, Radboud Institute for Health Sciences, Radboud university medical center (Radboudumc), Nijmegen, Netherlands, 5 Institute of Human Genetics, University Hospital of Bonn, Bonn, Germany
Nonsyndromic cleft lip with/without cleft palate (nsCL/P) is a common birth defect with multifactorial etiology. Genome-wide association studies (GWAS) have revealed >40 risk loci that contribute to nsCL/P risk, and it has been suggested that ~30% of phenotypic variance is explained by common genetic variation. In the absence of surgery, nsCL/P is a detrimental phenotype, which implies a considerable effect on reproductive fitness. Thus, rare variants are likely to also contribute to nsCL/P etiology. First studies have suggested nsCL/P candidate genes that harbor causative rare variants, or a burden thereof. These studies included the analysis of established “syndromic” genes ( n = 6 genes), genes with established role in craniofacial development ( n = 5), and the identification of mutations in genes of the epithelial cadherin-p120-catenin complex segregating within multiply affected families ( n = 5). However, independent replication is lacking. Here, we aimed at providing further genetic evidence for an involvement of these genes in nsCL/P etiology, through the identification of additional evidence for rare variants from a large Central-European cohort. Using single molecule molecular inversion probes (smMIPs), we obtained high-confident sequencing results for 14 of the 16 genes in 711 nsCL/P cases, 761 population-based controls, and 234 case-parent trios. At individual level, we identified four novel loss-of-function (LoF) variants (3 cases / 1 control). The variants observed in cases were LoF-mutations in CDH1 , CTNND1 and MTRR . Further statistical analyses, including analyses of missense variants, burden and pedigree analyses, are currently ongoing and will help to further understand the etiology of nsCL/P. B. Zametica: None. F. Thieme: None. J. Fazaal: None. N. Ishorst: None. E. Ongkosuwito: None. C. Carels: None. I.A.L.M. van Rooij: None. E. Mangold: None. K.U. Ludwig: None. P04.52.A Phenotypic spectrum of NPR2 mutations: study of 12 patients with acromesomelic dysplasia, Maroteaux type and 8 patients with short stature .

C. Michot 1 , S. Prévost 1 , C. Haudry 1 , A. Tourré 1 , A. Dieux-Coeslier 2 , M. Mathieu-Dramard 3 , M. Rossi 4 , A. Toutain 5 , G. Baujat 1 , S. Rondeau 1 , V. Cormier-Daire 1

1 APHP - Hôpital Necker-Enfants Malades, Paris, France, 2 CHU de Lille - Centre de Biologie Pathologie Génétique, Lille, France, 3 CHU Amiens-Picardie - Site Sud, Amiens, France, 4 CHU de Lyon HCL - GH Est-Hôpital Femme Mère Enfant, Bron, France, 5 CHRU de Tours - Hôpital Bretonneau, Tours, France
Introduction: Acromesomelic dysplasia, Maroteaux type (AMDM, MIM 602875) associates disproportionate short stature, short limbs and extremities with cone-shaped epiphyses. AMDM is caused by biallelic mutations of NPR2 (natriuretic peptide receptor B). Heterozygous NPR2 mutations are involved in idiopathic or dyschondrosteosis-like short stature. Materials and methods: Our study reviewed the clinical and molecular findings of AMDM and short stature patients with NPR2 mutations. 12 AMDM DNAs and more than 200 short stature DNAs were tested, after SHOX MLPA testing, in the molecular biology laboratory of Necker Hospital. Results: NPR2 mutations were identified in all AMDM cases and in 8 short stature patients. 23 different mutations were identified: nonsense, frameshift, splice and missense ones in AMDM and mostly missense variants in short stature. Heterozygous parents of AMDM patients were relatively small. All AMDM patients were of short birth and final heights (125-130 cm). Apart small extremities, 6 had large hallux and 7 had curved radius with limited pronation. Out of 12 cases, 8 had hyperlaxity, 7 scoliosis and 8 dental misalignment and arched palate. All heterozygous patients were of normal birth size and grew between -2,5 to -1,5 SD. They had normal envergure/height ratio, even if X-rays showed thick radius. 2/8 had IV and V brachymetacarpia. Conclusions: our study supports AMDM genetic homogeneity. We underline the importance of specific assessments, including laxity, scoliosis and orthodontics. We confirm that NPR2 heterozygous mutations should be considered in short stature with minor skeletal changes. NPR2 function in C-natriuretic peptide signaling might lead to therapeutics options. C. Michot: None. S. Prévost: None. C. Haudry: None. A. Tourré: None. A. Dieux-Coeslier: None. M. Mathieu-Dramard: None. M. Rossi: None. A. Toutain: None. G. Baujat: None. S. Rondeau: None. V. Cormier-Daire: None. P04.55.A Non-syndromic cleft lip with/without cleft palate: Genome-wide association study in patients from The Netherlands and Belgium identifies a suggestive risk locus at 16p12.1 and supports SH3PXD2A as a clefting susceptibility gene .

I. van Rooij 1 , K. U. Ludwig 2 , J. Welzenbach 2 , N. Ishorst 2 , M. Thonissen 3 , T. E. Galesloot 1 , E. Ongkosuwito 3 , S. J. Bergé 4 , K. Aldhorae 5 , A. Rojas-Martinez 6 , L. Kiemeney 7 , J. Vermeersch 8 , H. Brunner 9 , N. Roeleveld 1 , K. Devriendt 10 , T. Dormaar 11 , G. Hens 12 , M. Kanpp 13 , C. Carels 14,8 , E. Mangold 2

1 Radboud university medical center, Radboud Institute for Health Sciences, Department for Health Evidence, Nijmegen, Netherlands, 2 Institute of Human Genetics, University of Bonn, School of Medicine & University Hospital Bonn, Bonn, Bonn, Germany, 3 Radboud Institute for Health Sciences, Department of Dentistry, Section of Orthodontics and Craniofacial Biology, Radboud university medical center, Nijmegen, Netherlands, 4 Radboud university medical center, Department of Oral and Maxillofacial Surgery, Nijmegen, Netherlands, 5 Orthodontic Department, College of Dentistry, Thamar University, Thamar, Zambia, 6 Tecnologico de Monterrey, School of Medicine, and Universidad Autonoma de Nuevo Leon, Centro de Investigación y Desarrollo en Ciencias de la Salud, Monterrey, Mexico, 7 Radboud university medical center, Radboud Institute for Health Sciences, Department for Health Evidence and Department of Urology, Nijmegen, Netherlands, 8 Department of Human Genetics, KU Leuven, Leuven, Belgium, 9 Radboud university medical center, Department of Human Genetics, and Donders Institute for Brain, Cognition and Behaviour, Nijmegen, Netherlands, 10 Center for Human Genetics, University Hospitals Leuven, KU Leuven, Leuven, Belgium, 11 Oral and Maxillofacial Surgery, University Hospitals Leuven, Leuven, Belgium, 12 Department of Neurosciences, Experimental Otorhinolaryngology, KU Leuven, Leuven, Belgium, 13 Institute of Medical Biometry, Informatics and Epidemiology, University of Bonn, Bonn, Germany, 14 Orthodontics, University Hospitals Leuven, Leuven, Belgium
Nonsyndromic cleft lip with or without cleft palate (nsCL/P) ranks among the most common human congenital malformations. The etiology of nsCL/P is multifactorial, whereby genetic risk factors, environmental exposures, and potential gene-environment interactions all contribute to susceptibility. The present project is a genome-wide association study (GWAS) in a medium-sized nsCL/P cohort encompassing a total of 285 nsCL/P patients and 1,212 controls from The Netherlands and Belgium. Twenty of 40 previously reported nsCL/P susceptibility loci were replicated, and at the well-established nsCL/P susceptibility locus at chromosome 8q24.21, the analyses identified 62 genome-wide significant SNVs. These findings underline the validity of this sample. SNV-based analysis of the data identified a yet unreported suggestive locus at chromosome 16p12.1 ( p -value of the lead SNV: 4.17×10-07). This association was replicated in two of three patient/control replication series (Central European and Yemeni). The lead SNV rs56383345 maps to a 930 kb non-coding region at 16p12.1, and is not located in any currently known regulatory element. Gene-based analysis of the GWAS data prioritized SH3PXD2A at chromosome 10q24.33 as a candidate gene for nsCL/P. To date, support for this gene as a cleft gene has been restricted to data from zebrafish and a knockout mouse model. The present GWAS is the first to implicate SH3PXD2A in non-syndromic cleft formation in humans. In summary, although performed in a relatively small sample, the present GWAS generated novel insights into nsCL/P etiology, and demonstrates how medium-sized, clinically well characterized GWAS samples can improve knowledge of the genetic basis of nsCL/P. I. van Rooij: None. K.U. Ludwig: None. J. Welzenbach: None. N. Ishorst: None. M. Thonissen: None. T.E. Galesloot: None. E. Ongkosuwito: None. S.J. Bergé: None. K. Aldhorae: None. A. Rojas-Martinez: None. L. Kiemeney: None. J. Vermeersch: None. H. Brunner: None. N. Roeleveld: None. K. Devriendt: None. T. Dormaar: None. G. Hens: None. M. Kanpp: None. C. Carels: None. E. Mangold: None. P04.56.B GWAS candidate genes for cleft lip with or without cleft palate: Analysis of expression data during embryonic craniofacial development .

A. Siewert 1 , B. Reiz 2 , J. Welzenbach 1 , H. Dickten 2 , K. U. Ludwig 1

1 Institute of Human Genetics, University of Bonn, School of Medicine & University Hospital Bonn, Bonn, Germany, 2 FASTGenomics, Comma Soft AG, Bonn, Germany
Molecular defects during craniofacial embryonic development may result in nonsyndromic cleft lip with/without cleft palate (nsCL/P), one of the most common birth defects. Despite the possibility of surgical correction, follow-up treatment is long-term and requires an interdisciplinary approach. Also, epidemiological data indicate that individuals with nsCL/P show an increased risk for morbidities and suffer from a considerable psycho-social burden. The molecular pathways underlying these observations are currently unknown. We here used nsCL/P-GWAS data and array-based expression data from embryonic mice (gestational days E10.5-E12.5, Hooper et. al 2017), to identify pathways in which nsCL/P candidate genes are enriched. The 45 nsCL/P risk loci comprise ~400 genes within their topologically associated domains. Using gene-based analysis, we identified 86 genes with enrichment of common variants, suggesting their involvement in nsCL/P etiology. For each of these genes, we analyzed the expression pattern in ectodermal or mesodermal tissues of mandibular, maxillary and fronto-nasal prominences, and performed hierarchical clustering. One of the three clusters we identified comprised 33 genes, including established nsCL/P-genes like IRF6 and TFAP2a . A pathway analysis of genes within this cluster revealed a significant overrepresentation of cancer-related pathways and processes related to molecular signaling. Interestingly, affecteds with nsCL/P have been reported to be at increased risk of cancer. We will next extend our approach at single-cell level, using systematic data as recently provided (Cao et. al 2019) and the analytical ecosystem FASTGenomics (Scholz et. al 2018). This will generate novel insights into gene regulatory networks across development, and their contribution to nsCL/P. A. Siewert: None. B. Reiz: A. Employment (full or part-time); Significant; Comma Soft AG. J. Welzenbach: None. H. Dickten: A. Employment (full or part-time); Significant; Comma Soft AG. K.U. Ludwig: None. P04.59.B Severe Osteogenesis imperfecta with oligodontia: think of MESD

S. Moosa 1,2,3 , G. L. Yamamoto 4,5,3 , L. Garbes 6 , K. Keupp 6 , A. Beleza-Meireles 7,8 , C. A. Moreno 9 , E. R. Valadares 10 , S. B. de Sousa 7,11 , S. Maia 7,12 , J. Saraiva 7,13 , R. S. Honjo 14 , C. A. Kim 15 , H. Cabral de Menezes 16 , E. Lausch 17 , P. V. Lorini 18,19 , A. Lamounier Jr 10 , T. C. B. Carniero 10 , C. Giunta 20 , M. Rohrbach 20 , M. Janner 21 , O. Semler 22 , F. Beleggia 23 , Y. Li 24 , G. Yigit 24 , N. Reintjes 25 , J. Altmüller 26 , P. Nürnberg 26 , D. P. Cavalcanti 9 , B. Zabel 17,27 , M. L. Warman 28 , D. R. Bertola 14,5 , B. Wollnik 24,29 , C. Netzer 23,30

1 Stellenbosch University, Tygerberg, South Africa, 2 University Medical Center Göttingen, Göttingen, Germany, 3 Boston Children’s Hospital and Harvard Medical School, Boston, MA, United States, 4 Unidade de Genética, Instituto da Criança do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo, São Paulo, 05403-000, Brazil;, Brazil, 5 Instituto de Biociências da Universidade de São Paulo, São Paulo, Brazil, 6 Institute of Human Genetics, University Hospital of Cologne and Faculty of Medicine, University of Cologne, Cologne, Germany, 7 Medical Genetics Unit, Hospital Pediátrico de Coimbra, Centro Hospitalar e Universitário de Coimbra, Coimbra, Portugal, 8 Department of Clinical Genetics, St Michael’s Hospital, University Hospitals Bristol, Bristol, United Kingdom, 9 Skeletal Dysplasia Group, Department of Medical Genetics, Faculty of Medical Sciences, University of Campinas, Campinas, Brazil, 10 Hospital das Clínicas da Faculdade de Medicina da Universidade Federal de Minas Gerais, Belo Horizonte, Belo Horizonte, Brazil, 11 University Clinic of Genetics, Faculty of Medicine, University of Coimbra, Coimbra, Portugal, 12 University Clinic of Genetics, Faculty of Medicine, University of Coimbra,, Coimbra, Portugal, 13 University Clinic of Pediatrics, Faculty of Medicine, University of Coimbra, Coimbra, Portugal, 14 Unidade de Genética, Instituto da Criança do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo, São Paulo, Brazil, 15 Instituto de Biociências da Universidade de São Paulo, São Paulo, Portugal, 16 Unidade de Endocrinologia, Instituto da Criança do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo, São Paulo, Portugal, 17 Division of Genetics, Children’s Hospital, University of Freiburg, Freiburg, Germany, 18 Stellenbosch UniversityDivision of Genetics, Children’s Hospital, University of Freiburg, Freiburg, Germany, 19 Institute of Human Genetics, University Hospital Halle, Martin Luther University Halle-Wittenberg, Halle, Germany, 20 Connective Tissue Unit, Division of Metabolism, University Children’s Hospital Zurich, Zurich, Switzerland, 21 Division of Pediatric Endocrinology, Diabetology and Metabolism, Department of Pediatrics, Inselspital, Bern University Hospital, Bern, Switzerland, 22 Faculty of Medicine, University of Cologne, 50931 Cologne, Germany; Department of Pediatrics, University Hospital Cologne, Cologne, Germany, 23 Institute of Human Genetics, University Hospital of Cologne, Cologne, Germany, 24 Institute of Human Genetics, University Medical Center Göttingen, Göttingen, Germany, 25 Institute of Human Genetics, University Hospital of Cologne, Cologne, Ghana, 26 Cologne Center for Genomics, University of Cologne, Cologne, Germany, 27 Medical Faculty of the University of Magdeburg, Magdeburg, Germany, 28 Orthopaedic Research Laboratories, Department of Orthopaedic Surgery, Boston Children’s Hospital and Harvard Medical School, Boston, MA, United States, 29 Cluster of Excellence, Multiscale Bioimaging: from Molecular Machines to Networks of Excitable Cells (MBExC), University of Göttingen, Göttingen, Germany, 30 Faculty of Medicine, University of Cologne, Cologne, Germany
Osteogenesis imperfecta (OI) comprises a genetically heterogeneous group of skeletal fragility diseases. It is often clinically impossible to predict the underlying causative gene, thus necessitating broad multi-gene panel testing or exome sequencing. We report on five independent families with a progressively-deforming type of OI with the distinguishing feature of oligodontia. We identified four homozygous truncation or frameshift mutations in a novel gene for OI, namely MESD. MESD encodes an endoplasmic reticulum (ER) chaperone protein for the canonical Wingless-related integration site (WNT) signaling receptors LRP5 and LRP6. Because complete absence of MESD causes embryonic lethality in mice, we hypothesized that the OI-associated mutations are hypomorphic alleles since these mutations occur downstream of the chaperone activity domain but upstream of ER-retention domain. This would be consistent with the clinical phenotypes of skeletal fragility and oligodontia in persons deficient for LRP5 and LRP6, respectively. Further functional analysis showed that OI-associated MESD mutations produce hypomorphic alleles whose failure to remain within the ER significantly reduces but does not completely eliminate LRP5 and LRP6 trafficking. Since these individuals have no eye abnormalities (which occur in individuals completely lacking LRP5) and have neither limb nor brain patterning defects (both of which occur in mice completely lacking LRP6), we infer that bone mass accrual and dental patterning are more sensitive to reduced canonical WNT signaling than are other developmental processes. Multigene panels do not yet include MESD. Therefore, if an undiagnosed patient with severe OI and oligodontia presents, it may be prudent to think first of MESD. S. Moosa: None. G.L. Yamamoto: None. L. Garbes: None. K. Keupp: None. A. Beleza-Meireles: None. C.A. Moreno: None. E.R. Valadares: None. S.B. de Sousa: None. S. Maia: None. J. Saraiva: None. R.S. Honjo: None. C.A. Kim: None. H. Cabral de Menezes: None. E. Lausch: None. P.V. Lorini: None. A. Lamounier Jr: None. T.C.B. Carniero: None. C. Giunta: None. M. Rohrbach: None. M. Janner: None. O. Semler: None. F. Beleggia: None. Y. Li: None. G. Yigit: None. N. Reintjes: None. J. Altmüller: None. P. Nürnberg: None. D.P. Cavalcanti: None. B. Zabel: None. M.L. Warman: None. D.R. Bertola: None. B. Wollnik: None. C. Netzer: None. P04.61.A Adult osteopetrosis with osteonecrosis of the jaws and abnormal osteoclast resorption due to a LRRK1 splice site mutation

A. Howaldt 1 , A. Hennig 1 , T. Rolvien 2 , U. Roessler 1 , A. Knaus 3 , S. Boettger 4 , S. Geißler 1 , R. Oheim 2 , M. Amling 2 , H. Howaldt 4 , U. Kornak 5

1 Charité-Universitaetsmedizin Berlin, Berlin, Germany, 2 University Medical Center Hamburg-Eppendorf, Hamburg, Germany, 3 School of Medicine & University Hospital Bonn, Bonn, Germany, 4 Justus Liebig Universität Gießen, Gießen, Germany, 5 Universitaetsmedizin Göttingen, Göttingen, Germany
We report on a 34 year-old patient with sandwich vertebrae, platyspondyly, osteosclerosis of long bones, pathologic fractures, and anemia. In the third decade, he developed osteonecrosis of the jaws, which was progressive in spite of repeated surgical treatment. An iliac crest bone biopsy revealed an elevated trabecular density (BV/TV [%] 32.2 vs. 21.5 + 14.8) and the presence of hypermineralized cartilage remnants leading to an overall increase in mineralization (CaMean [wt%] 25.65 vs. 21.8). We also found large multinucleated osteoclasts with abnormal morphology and inadequate bone resorption. Although these findings were reminiscent of autosomal dominant osteopetrosis type 2, no mutations in CLCN7 were detected. Subsequent whole exome sequencing identified the novel homozygous splice-site mutation c.261G>A in the gene LRRK1 . cDNA sequencing showed nearly complete skipping of exon 3 leading to a frameshift (p.Ala34Profs*33), very likely leading to a loss of function. LRRK1 encodes a protein kinase and its dysfunction can cause osteosclerotic metaphyseal dysplasia (OSMD; OMIM # 615198). Osteoclasts differentiated in vitro from the patient’s peripheral blood monocytes formed quickly and became unusually large. In resorption assays these cells formed large areas with superficial pseudo-resorption but excavated only few resorption pits (resorption pit surface surface [%] 1.3 + 0.6 vs. 15 + 3.6; p < 0.01). Phosphorylation of L-plastin was strongly reduced in patient-derived osteoclasts, thus corroborating a LRRK1 loss of function (0.26 + 0.1 vs. 1 + 0.08; p < 0.01). Our analysis indicates a strong overlap of LRRK1 -related OSMD with other late-onset forms of osteopetrosis, but an exceptionally leaky osteoclast resorption. A. Howaldt: None. A. Hennig: None. T. Rolvien: None. U. Roessler: None. A. Knaus: None. S. Boettger: None. S. Geißler: None. R. Oheim: None. M. Amling: None. H. Howaldt: None. U. Kornak: None. P04.62.B The lrp5 knockout zebrafish as a model to study osteoporosis

P. J. Coucke , J. Bek, H. De Saffel, A. De Clercq, A. Boel, A. Willaert

Center for Medical Genetics, Gent, Belgium
Osteoporosis-pseudoglioma syndrome (OPPG) is an autosomal recessive disorder characterized by reduced bone mass and strength, caused by loss-of-function mutations in LRP5 (low-density lipoprotein receptor-related protein 5), a co-receptor in the WNT signaling pathway. Here we describe the first reliable genetically induced form of osteoporosis in the zebrafish in a lrp5 knockout (KO). Molecular analysis confirmed reduced lrp5 transcripts and absence of Lpr5 protein. Reduced survival was observed in lrp5 KO larvae after the onset of mineralization. The fraction of larvae that did survive, displayed delayed ossification as was shown via mineral staining. Subsequently, these fish were grown to adulthood and analyzed via µCT. This revealed decreased bone volume and decreased bone mineralization in the vertebral column, which are hallmark features of osteoporosis. Next, we performed CRISPR/Cas9 mediated mutagenesis targeted at lrp5 followed by F0 phenotype analysis (crispant screening). Miseq sequencing of the mosaic larvae revealed out-of-frame mutations in on average 90% of the reads. Consequently, we showed a similar skeletal phenotype in the F0 ‘crispants’ to that of the stable lrp5 -/- larvae, showing that crispant screening in zebrafish is a promising approach to functionally screen a large set of osteoporosis candidate genes. In summary, we present lrp5 −/− zebrafish as the first genetic osteoporosis zebrafish model that can be used as a screening platform for potential therapeutics. Additionally, we show that F0 crispant screening can be used for functional validation of candidate genes for osteoporosis as high rates of out-of-frame mutations in the lrp5 gene are sufficient to induce phenotypes. P.J. Coucke: None. J. Bek: None. H. De Saffel: None. A. De Clercq: None. A. Boel: None. A. Willaert: None. P04.64.A The p.R321C mutation in p62 protein, associated to Paget’s Disease of Bone, leads to a relocation of the protein in human osteoclasts

N. Gestoso-Uzal 1,2 , R. Usategui-Martín 1,2 , C. Gutiérrez-Cerrajero 2,1 , A. I. Turrión Nieves 1,3 , J. Del Pino-Montes 1,3 , R. González-Sarmiento 2,1,4

1 Institute of Biomedical Research of Salamanca (IBSAL), Salamanca, Spain, 2 Molecular Medicine Unit, Department of Medicine, University of Salamanca, Salamanca, Spain, 3 Rheumatology Service, University Hospital of Salamanca, Salamanca, Spain, 4 Institute of Molecular and Cellular Biology of Cancer (IBMCC), University of Salamanca-CSIC, Salamanca, Spain
Background: The most important known genetic factor predisposing to Paget´s disease of bone (PDB) is mutation in SQSTM1 gene, which encodes p62 protein. We detected the c.961C>T SQSTM1 gene mutation in three PDB patients, that causes the p.R321C mutation. It has been reported that p.R321C mutation causes a blockade of autophagy and an activation of NF-kB pathway. The aim of this study was to characterize the effect of the p.R321C mutation in human osteoclasts. Methods: Peripheral blood mononuclear cells (PBMCs) were isolated from blood samples for generating human osteoclasts. Cells were differentiated by adding M-CSF and RANKL for 3 weeks. Human osteoclasts were transiently transfected with total plasmid DNA (pCEFL-Flag-SQSTM1-321C and pCEFL-Flag-SQSTM1-321R constructs). Finally, we performed a co-immunofluorescence assay to detect Flag and p62. Results and discussion: Our results showed that, while 321R-p62 protein remained scattered though the cytoplasm, 321C-p62 was concentrated in the periphery of osteoclasts. This relocation of p62 protein caused by p.R321C mutation could be a consequence of autophagy blockade, which has been reported that could have a crucial role in the etiology of PDB. The p.R321C mutation causes a blockade of autophagy and an activation of NF-kB pathway, therefore the alteration of the cell metabolism could be the cause of the relocation of p62 protein in human osteoclasts. Conclusion: The p.R321C mutation in p62 protein leads to a relocation of the protein in human osteoclasts. This reinforces the hypothesis that an alteration of autophagy could be involved in PDB etiology. This work was supported by FIS-FEDER:PI16/01920. N. Gestoso-Uzal: None. R. Usategui-Martín: None. C. Gutiérrez-Cerrajero: None. A.I. Turrión Nieves: None. J. Del Pino-Montes: None. R. González-Sarmiento: None. P04.66.C Penttinen syndrome and infantile myofibromatosis in a patient carrying the tyrosine kinase-activating variant Asn666Ser in PDGFRB

D. Horn 1 , A. T. Abad-Perez 1 , V. Stephan 2

1 Institute of Medical Genetics and Human Genetics, Charité-Universitätsmedizin, Berlin, Germany, 2 Sana Children´s Hospital Lichtenberg, Berlin, Germany
Introduction: Penttinen syndrome belongs to the group of progeroid disorders and is characterized by progressive subcutaneous lipoatrophy, sclerotic skin lesions and acro-osteolysis. This very rare condition is caused by a few distinct missense mutations of PDGFRB , other distinct mutations of this gene have been shown to be associated with different conditions such as infantile myofibromatosis. Material and Methods: Here, we describe a 5-year-old male patient with infantile myofibromatosis diagnosed in his first month of life who in addition developed a prematurely aged appearance with lipoatrophy, scar-like skin nodules on the hands and joint contractures. Results: Sanger sequencing revealed the heterozygous de novo mutation c.1997A>G; p.Asn666Ser in the PDGFRB gene. Recently, this mutation has been described in two patients affected with a severe form of Penttinen syndrome presenting a more pronounced connective tissue destruction. Conclusions: This is the first report of an individual affected with both conditions, infantile myofibromatosis and Penttinen syndrome. This mutation lead to increased phosphorylation of downstream signalling proteins such as PTPN11. Imatinib is a strong inhibitor of phosphorylation of these targets representing a treatment option. D. Horn: None. A.T. Abad-Perez: None. V. Stephan: None. P04.67.A Blood secreted miRNA hsa-miR-374b-5p inhibits osteoblast-differentiating gene UHMK1 and is upregulated in periodontitis patients

R. Wiehe 1 , D. Bajric 2 , A. El Sharawy 3 , A. Keller 4 , H. Dommisch 1 , A. Schaefer 1

1 Charité-Universitymedicine Berlin, Institute for Dental and Craniofacial Sciences, Department of Periodontology and Synoptic Dentistry, Berlin, Germany, 2 University Medical Center Schleswig-Holstein, Campus Kiel, Department of Operative Dentistry and Periodontology, Kiel, Germany, 3 Institute for Clinical Molecular Biology, Christian-Albrechts-University Kiel, Kiel, Germany, 4 Clinical Bioinformatics, Saarland University, Saarbrücken, Saarbrücken, Germany
microRNAs interact with the 3’UTRs of target mRNAs to suppress gene expression. MicroRNAs act tissue specifically but some are secreted into blood. Recent studies reported upregulation of specific miRNAs in the inflamed oral mucosa of periodontitis patients. The regulatory targets of these miRNAs may reveal genes that are conducive to oral health. If secreted into blood, these miRNAs could serve as biomarkers to diagnose periodontitis before clinical manifestation. To identify such miRNAs, we quantified 863 miRNAs in the blood of 18 periodontitis patients and 70 periodontitis-free controls (Geniom Biochip array). miRNA hsa-miR-421 and hsa-miR-374b-5p were most significantly upregulated in blood of periodontitis cases (P adjusted < 5x10 −6 ). Two previous studies that profiled microRNAs in the inflamed and healthy oral mucosa independently reported >2fold upregulation of hsa-miR-374b-5p, but not hsa-miR-421. Using mirVana miRNA mimics (ThermoFisher Scientific), we overexpressed hsa-miR-374b-5p in primary gingival fibroblasts (3 biological and 3 technical replicates) and quantified the mRNA levels in 24h transfected and mock-transfected cells (Clariom D Array, ThermoFisher Scientific). The gene U2AF homology motif kinase 1 ( UHMK1 ) was significantly downregulated in the miRNA mimic transfected cells (Fold Change= -1.8, P=0.00025). Two binding sites of hsa-miR-374b-5p were predicted at the 3’UTR of UHMK1 . To validate the functional mechanism, we cloned the respective 3’UTR of UHMK1 into a reporter plasmid (pGL4.24, Promega). After co-transfection of the plasmid and hsa-miR-374b-5p mimic into HeLa cells for 24 hours, the reporter gene expression was significantly reduced (Fold Change= -1.5, p=0.02, 3 independent replicates). UHMK1 has a role in the coordination of osteoblast differentiation. R. Wiehe: None. D. Bajric: None. A. El Sharawy: None. A. Keller: None. H. Dommisch: None. A. Schaefer: None. P04.70.A POP1 -Skeletal Dysplasias : Description of two new families .


CHU Liège, Liège, Belgium
Background: POP1 mutations should be considered in anauxetic dysplasia (AD) patients without RMRP mutations but also in skeletal dysplasia (SD) of different severities. To date, only 5 patients were reported. Methods: Cartilage Hair Hypoplasia (CHH) was suspected in two siblings based on clinical and radiological evaluation. In family 1, a 4 years boy was investigated. His older sister is also affected by SD but currently refuses to pursue genetic testing. In family 2, two sisters presented a severe short stature : the older has 120 cm as final adult height. The second aged of 13 also suffered from an immune deficiency. Results: A skeletal dysplasia gene panel revealed in the POP1 gene:Family N°1 : a composite heterozygous variant c.1846A>C, class3 (paternally inherited) and c.1747G>T, class 4 (maternally inherited). Family N°2 : a homozygous variant c.632G>A, class 4. Discussion : POP1 patients are characterized by severe short stature of prenatal-onset and very short adult height (classically less than 1 meter, but our adult patients were 120 cm and near 160 cm), hypodontia, midface hypoplasia, mild intellectual disability and radiological specific findings including systematic bullet-shaped middle phalanges. Notably, none of the 5 published cases were reported with immune deficiency. Conclusions: We describe two new families with POP1 mutations encountered in patients clinically suspected for CHH and achondroplasia, enhancing that the phenotypical spectrum is larger than AD type 2. Larger cohorts of patients are needed to improve the clinical characterization, especially the risk of having associated symptoms such as immune deficiency. J. Harvengt: None. S. Alkan: None. B. Florkin: None. S. Bulk: None. V. Bours: None. P04.71.B Coding variants of the CARD14 NF-kB adaptor are associated with palmar plantar pustulosis

A. Niaouris 1 , S. Haddad 1 , A. PushpaRajah 2 , N. Benzian-Olsson 1 , P. Baum 3 , S. Visvanathan 4 , T. he APRICOT and PLUM study team 2 , J. Barker 2 , C. Smith 2 , F. Capon 1

1 King’s College London, London, United Kingdom, 2 St John’s Institute of Dermatology, Guy’s and St Thomas’ NHS Foundation Trust, London, United Kingdom, 3 Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach, Germany, 4 Boehringer Ingelheim Pharma GmbH & Co. KG, Ridgefield, CT, United States
Caspase recruitment domain containing protein 14 (CARD14) is a soluble NF-kB adaptor protein, which is mostly expressed in keratinocytes. In the presence of inflammatory stimuli, free CARD14 molecules oligomerise, recruit a BCL10/MALT1 signalosome and activate NF-kB signalling. Gain-of-function CARD14 variants have previously been associated with inflammatory skin diseases such as psoriasis vulgaris and generalised pustular psoriasis. However, the involvement of CARD14 in palmar plantar pustulosis (PPP), a localised form of pustular psoriasis, remains unclear. Here, we analysed CARD14 changes in 160 PPP cases (125 females, 35 males; mean age of onset: 41 years) ascertained across the UK. We identified 11 rare variants predicted to have a deleterious effect on protein function. A burden test (comparing PPP cases vs controls sequenced by the ExAC consortium) demonstrated a significant association between these changes and PPP (OR: 2.5; 95% CI: 1.39-4.65; p = 0.0023). We also detected a low-frequency allele with deleterious potential (E422K) that occurred more frequently in affected individuals compared to controls (OR: 1.7; 95% CI: 1.07-2.69; p= 0.025). To assess the effect of disease-associated variants, CARD14 FLAG-tagged plasmids were mutagenized and transfected into HEK293 cells. The depletion of free CARD14 from the cytoplasm (which is an indirect measurement of protein oligomerization) was then analysed by western blotting. This confirmed that most variants promote the formation of insoluble CARD14 oligomers. These findings implicate CARD14 in the pathogenesis of PPP. They also suggest that the proteins encoded by disease alleles are prone to constitutive oligomerization, which will cause abnormal NF-kB activation. A. Niaouris: None. S. Haddad: None. A. PushpaRajah: None. N. Benzian-Olsson: None. P. Baum: None. S. Visvanathan: None. T. he APRICOT and PLUM study team: None. J. Barker: None. C. Smith: None. F. Capon: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Significant; 3 Boehringer Ingelheim Pharma GmbH & Co. P04.74.B Further delineation of Roifman syndrome: report of four new Spanish patients

I. Valenzuela 1 , I. Cusco 1 , I. Paramonov 1 , F. López-Grondona 1 , S. Garcia-Miñaur 2 , M. Palomares-Bralo 2 , A. Martin 1 , J. Riviere 1 , M. Garcia-Prat 1 , P. Soler 1 , R. Colobran 1 , E. Garcia-Arumí 1 , E. Tizzano 1

1 Hospital Vall d’Hebron, Barcelona, Spain, 2 Hospital La Paz, Madrid, Spain
Roifman Syndrome (RS, OMIM 300258) is a rare disorder characterized by pre- and postnatal growth retardation, cognitive delay, spondyloepiphyseal dysplasia, antibody deficiency and facial dysmorphism. Biallelic mutations in RNU4ATAC have been reported as the molecular cause. Our series expand the phenotypic spectrum of Roifman patients. We report four patients from three unrelated families with RS. Due to the complexity of variant annotation in RNU4ATAC , molecular studies are not usually straightforward. Indeed, in families 1 and 2 exome sequencing was inconclusive. In patient 1 the diagnosis was finally uncovered by genome sequencing, in sisters 2 and 3 by Sanger sequencing upon clinical suspicion and in patient 4 by a customized gene panel. Main clinical characteristics include skeletal dysplasia (4/4), short stature (4/4), intellectual disability (4/4), antibody deficiency (3/4) and retinal dystrophy (2/4). Main dysmorphic features included markedly long philtrum and thin upper lip. Hepatomegaly was identified in 2/4 and patient 1 presented hepatic cavernoma (unreported so far in Roifamn patients). Molecular results showed variants n.8C>T and n.50 G>A (patient 1); variant n.13C>T and n.17 G>C (sisters 2 and 3) and two novel variants, n.37G>A and n.53C>T (patient 4). Segregation analysis indicated that each variant was inherited from a healthy parent in the three families. The inclusion of new patients with Roifman increases the knowledge of mutational spectrum and it’s essential to delineate the phenotype of this syndrome. The recognition of the syndrome is indispensable for the orientation of molecular diagnosis, prompt follow up and counseling. I. Valenzuela: None. I. Cusco: None. I. Paramonov: None. F. López-Grondona: None. S. Garcia-Miñaur: None. M. Palomares-Bralo: None. A. Martin: None. J. Riviere: None. M. Garcia-Prat: None. P. Soler: None. R. Colobran: None. E. Garcia-Arumí: None. E. Tizzano: None. P04.77.B Identification of the deregulated NEK1 protein network in skeletal ciliopathies

C. Vogl 1 , K. Kessler 1 , A. Giessl 2 , P. Kirchner 1 , C. Büttner 1 , A. B. Ekici 1 , A. Reis 1 , C. T. Thiel 1

1 Institute of Human Genetics, FAU Erlangen-Nürnberg, Erlangen, Germany, 2 Department of Ophthalmology, FAU Erlangen-Nürnberg, Erlangen, Germany
The primary cilium is an organelle, which is present on almost all vertebrate cells. Cilia consist of the axoneme and the basal body and present various ciliary membrane receptors, which receive extracellular signals. Those signals are transmitted into the cell, where they modulate different intracellular signaling pathways. Mutations in ciliary proteins are involved in formation, maintenance and function of the primary cilium and associated with developmental defects, like brain malformations, polydactyly, kidney cysts and skeletal abnormalities. This phenotypic spectrum is present among patients with short-rib polydactyly syndromes (SRPS). This group constitutes the most frequent lethal autosomal recessive osteochondrodysplasias. Diallelic mutations in NEK1, encoding for a basal body protein, are the underlying cause of SRPS II. To gain further insight into the pathogenic mechanism, we used two approaches: RNA sequencing of CRISPR/Cas9 mediated genomic edited HEK293T cells and scRNASeq in limb buds of E13 embryos of the Nek1 knock down mouse, and respectively compared the knock out and the wildtype in order to find differentially expressed genes. Analysis of significantly enriched pathways revealed an activation of genes in early growth response and cell cycle control and silencing of different collagens and ribosomal and histone proteins. One of the main findings was a severely reduced expression of Acan, coding for Aggrecan the most abundant cartilaginous protein, explaining the severe growth phenotype. These results underline that the NEK1 associated defect of the primary cilium causes the deficiency in cell cycle and gene expression, mainly aggrecan, in the growth plate. C. Vogl: None. K. Kessler: None. A. Giessl: None. P. Kirchner: None. C. Büttner: None. A.B. Ekici: None. A. Reis: None. C.T. Thiel: None. P04.79.A Novel variant in PLAG1 in a familiar case of Silver-Russell syndrome

A. Pereda 1 , Y. Vado 2 , I. Llano-Rivas 3 , N. Gorria-Redondo 4 , I. Díez 5 , G. Perez de Nanclares 2

1 Rare Diseases Research Group, Molecular (Epi)Genetics Laboratory, BioAraba National Health Research Institute, Vitoria-Gasteiz, Spain, 2 Rare Diseases Research Group, Molecular (Epi)Genetics Laboratory, BioAraba National Health Research Institute, Vitoria-Gasteiz, Araba, Spain, 3 Service of Genetics, BioCruces Health Research Institute, Hospital Universitario Cruces., Barakaldo, Bizkaia, Spain, 4 Service of Paediatric Neurology, Araba University Hospital, Vitoria-Gasteiz, Araba, Spain, 5 Department of Pediatric Endocrinology. BioAraba Health Research Institute, Araba University Hospital-Txagorritxu, Vitoria-Gasteiz, Araba, Spain
Introduction: Silver-Russell syndrome is a rare imprinting disorder characterized by prenatal (SGA) and postnatal growth retardation (PNGR), relative macrocephaly at birth, a prominent forehead, body asymmetry and feeding difficulties and/or low BMI. The most common underlying mechanisms are hypomethylation at H19/IGF2: IG-DMR and UPD(7)mat. Rarely, rearrangements involving 11p15, or inactivating point variants have been identified (maternal CDKN1C or paternal IGF2 variants). Recently PLAG1 and HMGA2 genes, which participate in the IGF2 pathway, have been associated with this syndrome. Patients and Methods: We present a 9 years-old girl who was initially referred to pediatric endocrinology because of short stature. A clinical geneticist suggested the clinical diagnosis of SRS (NHC score=4: SGA, PNGR, prominent forehead and problems for feeding manifested as poor food intake). She also showed dental crowding with delayed teeth eruption, triangular face, small and bulbous nose, prominent maxillary, high ojival palate, fine voice, fine hair and learning difficulties. Her mother and maternal grandmother presented the same phenotype. After discarding alterations in 11p15 and chromosome 7 (MS-MLPA), a custom NGS panel for genes associated with growth was run. Results: The study of the NGS panel revealed an heterozygous novel variant within exon 5 of PLAG1 [NM_001114634.2:c.551delA; p.(Lys184Serfs*45)], bioinformatically predicted as likely pathogenic (ACMG: PVS1; PM2; PP1). Familial studies by Sanger sequencing confirmed cosegregation. Conclusions: We report the second familial case of SRS caused by a novel PLAG1 frameshift alteration, and we expand the clinical spectrum to the previously described. Funding: Instituto de Salud Carlos III (PI16/00073); Basque Department of Health (GV2017/111040). A. Pereda: None. Y. Vado: None. I. Llano-Rivas: None. N. Gorria-Redondo: None. I. Díez: None. G. Perez de Nanclares: None. P04.80.B Increased yield of genetic diagnoses in skeletal ciliopathies using massively parallel sequencing, structural variant and RNA analyses

A. Hammarsjo 1 , M. Pettersson 1 , D. Chitayat 2,3 , A. Handa 4 , F. Taylan 5 , D. Batkovskyte 5 , B. Anderlid 1 , A. E. Lin 6 , K. Shimizu 7 , A. Beleza-Meireles 8,9 , B. Chung 10,11 , U. Voss 12 , A. Nordgren 1 , G. Nishimura 13 , A. Lindstrand 1 , G. Grigelioniene 1

1 Department of Molecular Medicine and Surgery, Center for Molecular Medicine, Karolinska Institutet, and Clinical Genetics, Karolinska University Laboratory, Karolinska University Hospital, Stockholm, Sweden, 2 Division of Clinical and Metabolic Genetics, The Hospital for Sick Children, University of Toronto, Toronto, ON, Canada, 3 The Prenatal Diagnosis and Medical Genetics Program, Department of Obstetrics and Gynecology, Mount Sinai Hospital, University of Toronto, Toronto, ON, Canada, 4 Department of Radiology, University of Iowa Hospitals and Clinics, Iowa City, IA, United States, 5 Department of Molecular Medicine and Surgery, Center for Molecular Medicine, Karolinska Institutet, Stockholm, Sweden, 6 Medical Genetics, MassGeneral Hospital for Children, Boston, MA, United States, 7 Division of Medical Genetics, Saitama Children’s Medical Center, Saitama, Japan, 8 Department of Clinical Genetics and Genomics, Guy’s Hospital, Guy’s and St Thomas’ NHS Foundation Trust, London, United Kingdom, 9 Centre for Craniofacial and Regenerative Biology, King’s College London, London, United Kingdom, 10 Department of Pediatrics and Adolescent Medicine, The University of Hong Kong and Shenzhen Hospital, Futian District, Shenzhen, China, 11 Department of Pediatrics and Adolescent Medicine, Queen Mary Hospital, The University of Hong Kong, Hong Kong, China, 12 Department of Pediatric Radiology, Karolinska University Hospital, Stockholm, Sweden, 13 Department of Pediatric Imaging, Tokyo Metropolitan Children’s Medical Center, Tokyo, Japan
Introduction: Cilia are important for cell migration and signaling using multiple pathways and proteins essential for development of many body organs and systems, including the skeleton. Skeletal ciliopathies are a heterogenous group of disorders with overlapping features, caused by pathogenic variants in at least 30 genes, coding for different structural cilia proteins. Materials and Methods: We examined 28 probands with radiographic findings of skeletal ciliopathies. For single nucleotide variant (SNV) analysis, we used whole exome and genome sequencing with in silico filtering for variants in known skeletal ciliopathy genes. Results: We identified biallelic variants in 23 patients in DYNC2H1 , KIAA0753, WDR19 , C2CD3 , TTC21B , EVC and EVC2 . Subsequent examination of four potential splice variants showed that cDNA analyses are important in facilitating molecular diagnoses in a significant proportion (20%) of the patients and that aberrant splicing in DYNC2H1 , EVC and KIAA0753 can be confirmed with RNA extracted from blood. Copy number variant (CNV) analyses was performed for the same cohort and we found one individual with an intragenic deletion of DYNC2H1 and another with 1q24q25-microdeletion syndrome. Conclusions: In conclusion, combined SNV, CNV and RNA analyses result in a high yield of genetic diagnoses (86%) in a cohort of patients with a clinical, autopsy and radiological findings consistent with skeletal ciliopathy. Grants: AnH - Sällskapet Barnavård and Karolinska Institutet. GG - Stiftelsen Samariten, Promobilia and Stiftelsen Frimurare. GG, AL and AN - Stockholm County Council. AN and AL - The Swedish Research Council. AN - The Swedish Childhood Cancer Foundation. A. Hammarsjo: None. M. Pettersson: None. D. Chitayat: None. A. Handa: None. F. Taylan: None. D. Batkovskyte: None. B. Anderlid: None. A.E. Lin: None. K. Shimizu: None. A. Beleza-Meireles: None. B. Chung: None. U. Voss: None. A. Nordgren: None. G. Nishimura: None. A. Lindstrand: None. G. Grigelioniene: None. P04.82.A Lack of bone mineralization in a TANGO1 deficient patient

B. Guillemyn 1 , S. Nampoothiri 2 , O. Foresti 3 , I. Raote 3 , V. Malhotra 3 , F. Malfait 1 , S. Symoens 1

1 Center for Medical Genetics Ghent, Ghent University Hospital, Department of Biomolecular Medicine, Ghent, Belgium, 2 Department of Pediatric Genetics, Amrita Institute of Medical Sciences & Research Center, Cochin, India, 3 Center for Genomic Regulation, The Barcelona Institute of Science and Technology, Barcelona, Spain
Introduction: TANGO1 (Transport and Golgi organization-1) encodes a transmembrane protein, located at Endoplasmic Reticulum (ER) exit sites. TANGO1 binds collagen in the lumen, COPII-coats in the cytoplasm and recruits membranes from the ERGIC (ER-Golgi intermediate compartment) to create an export route for bulky cargos like collagen. Recently aberrant expression of truncated TANGO1 was linked to a severe collagenopathy associated with primary obesity, diabetes mellitus and intellectual disability. We show that complete loss of TANGO1 results in human embryonic lethality with near-total bone loss. Material and Methods: Fetal DNA and parental blood/fibroblast samples were obtained. Whole exome sequencing (WES) was performed. RTqPCR, Western blot, immunocytochemical staining and collagen secretion assays were conducted. Results: Ultrasound examination of a 13-weeks-old fetus of healthy Indian consanguineous parents revealed tetramicromelia, increased nuchal translucency, absent nasal bone, pingpong appearance of the skull, shortened limb segments, and extreme skeletal hypomineralization. The pregnancy was terminated and the infantogram showed an almost boneless fetus. Fetal WES detected a homozygous 4-basepair deletion in TANGO1 , heterozygously present in both parents. Parental fibroblast studies demonstrated decreased TANGO1 expression and protein, and severe defects in collagen secretion. Conclusions: Our findings underscore the importance of TANGO1 in cellular protein secretion. While absence of TANGO1 results in human embryonic lethality, aberrant expression of truncated TANGO1 results in a severe collagenopathy. This further supports the physiological role of TANGO1 in collagen export, which is necessary for bone mineralization, skin and tissue biogenesis. Moreover, the data clearly reveal the severity of human pathologies in association with levels of functional TANGO1 expression. B. Guillemyn: None. S. Nampoothiri: None. O. Foresti: None. I. Raote: None. V. Malhotra: None. F. Malfait: None. S. Symoens: None. P04.83.B TAR syndrome: clinical and molecular characterization of a cohort of 26 patients and description of novel non-coding variants of RBM8A

S. Boussion 1,2 , F. Escande 2,3 , A. Jourdain 2,3 , T. Smol 2,4 , P. Brunelle 2,3 , C. Duhamel 2 , Y. Alembik 5 , T. Attié-Bitach 6 , G. Baujat 7 , A. Bazin 8 , M. Bonnière 6 , P. Carassou 9 , D. Carles 10 , L. Devisme 2,11 , C. Goizet 12 , A. Goldenberg 13 , S. Grotto 14 , A. Guichet 15 , P. Jouk 16 , L. Loeuillet 17 , C. Mechler 18 , C. Michot 7 , F. Pelluard 19 , A. Putoux 20,21 , S. Whalen 22 , J. Ghoumid 1,2 , S. Manouvrier 1,2 , F. Petit 1,2

1 CHU Lille, Clinique de Génétique, Centre de Référence Anomalies du Développement, Lille, France, 2 Université de Lille, EA7364, Lille, France, 3 CHU Lille, Service de Biochimie et Oncologie Moléculaire, Lille, France, 4 CHU Lille, Institut de Génétique Médicale, Lille, France, 5 Service de Génétique Médicale, CHU Strasbourg, Strasbourg, France, 6 Unité d’Embryofoetopathologie, Service Histologie Embryologie et Cytogénétique, Hôpital Necker-Enfants Malades, AP-HP, Paris, France, 7 Service de Génétique Clinique, Hôpital Necker – Enfants Malades, AP-HP, INSERM UMR 1163, Institut IMAGINE, Paris, France, 8 Service de Diagnostic Anténatal, Hôpital René Dubois, Pontoise, France, 9 Service d’Hématologie, CHR Metz-Thionville, Metz, France, 10 Service d’Anatomo-Pathologie, CHU Bordeaux, Bordeaux, France, 11 Institut d’Anatomo-Pathologie, CHU Lille, Lille, France, 12 Service de Génétique Médicale, CHU Bordeaux, et laboratoire MRGM, INSERM U1211, Univ. Bordeaux, Bordeaux, France, 13 Département de Génétique, et centre de référence anomalies du développement et syndromes malformatifs, CHU de Rouen, Centre Normand de Génomique et de Médecine Personnalisée, Rouen, France, 14 Département de Génétique, Hôpital Robert Debré, AP-HP, Paris, France, 15 Service de Génétique, CHU Angers, Angers, France, 16 Département de Génétique, CHU Grenoble-Alpes, Grenoble, France, 17 Service d’Anatomo-Cytopathologie, Hôpital Cochin, AP-HP, Paris, France, 18 Unité de Foetopathologie, Hôpital Robert Debré, AP-HP, Paris, France, 19 INSERM U1053-UMR BaRITOn et Unité de Foetopathologie, Service de pathologie, Hôpital Pellegrin, CHU Bordeaux, Bordeaux, France, 20 Service de Génétique, Hospices Civils de Lyon, Lyon, France, 21 GENDEV Team, CRNL, INSERM U1028, CNRS UMR 5292, UCBL1, Lyon, France, 22 UF de Génétique Clinique, Centre de Référence Maladies Rares des Anomalies du développement et syndromes malformatifs, Hôpital Armand Trousseau, AP-HP, Paris, France
Thrombocytopenia-Absent Radius (TAR) syndrome is characterized by radial defect and neonatal thrombocytopenia. It is caused by bi-allelic variants of RBM8A gene (1q21.1) with the association of a null allele and a hypomorphic non-coding variant. RBM8A encodes Y14, a core protein of the Exon Junction Complex involved in mRNA maturation. To date, only two hypomorphic variants have been identified. We report on a cohort of 26 patients affected with TAR syndrome. Half patients carried a 1q21.1 deletion and one of the two known hypomorphic variants. Four novel non-coding variants of RBM8A were identified in the remaining patients. We developed experimental models enabling their functional characterization in vitro. Two variants, located respectively in the 5’-UTR and 3’-UTR regions, are responsible for a diminished expression whereas two intronic variants alter splicing. Our results bring new insights into the molecular knowledge of TAR syndrome and enabled us to propose genetic counseling for patients’ families. S. Boussion: None. F. Escande: None. A. Jourdain: None. T. Smol: None. P. Brunelle: None. C. Duhamel: None. Y. Alembik: None. T. Attié-Bitach: None. G. Baujat: None. A. Bazin: None. M. Bonnière: None. P. Carassou: None. D. Carles: None. L. Devisme: None. C. Goizet: None. A. Goldenberg: None. S. Grotto: None. A. Guichet: None. P. Jouk: None. L. Loeuillet: None. C. Mechler: None. C. Michot: None. F. Pelluard: None. A. Putoux: None. S. Whalen: None. J. Ghoumid: None. S. Manouvrier: None. F. Petit: None.

P05 Cardiovascular Disorders
P05.01.A Improving family screening of abdominal aorta aneurysm patients

R. L. Lechner, A. S. IJpma, D. Heijsman, J. W. Roos-Hesselink, H. J. M. Verhagen, S. ten Raa, R. M. W. Hofstra, D. F. Majoor-Krakauer

Erasmus MC, Rotterdam, Netherlands
Relatives of abdominal aneurysm patients (AAA) have an increased risk of developing aneurysms. To improve early detection and reduce risk of rupture in relatives, guidelines recommend screening of first degree relatives. Current guidelines do not specify the method or extend of aorta screening in relatives. In practice, mostly male relatives are referred for abdominal echographic screening of the abdominal aorta, possibly missing thoracic aneurysms (TAA) in relatives. To determine in which part of the aorta affected relatives of AAA patients have an aneurysm or dilatation, we reviewed the results of family history taking and family screening of families of 471 AAA patients with familial aneurysms of which medical records and/or CT images relatives were available. In total 475 affected relatives (311 male, 164 female) in 237 families had an aorta dilatation or aneurysm: 258 (54,3%) had abdominal and 147 (31%) thoracic aneurysm. Male relatives were more likely to have AAA (40% vs 28%, p < 0.05), females to have TAA (32% vs 17%, p < 0.05). Co-occurrence of AAA and TAA was observed in 7% of relatives. In 17% an iliac and in 2% a popliteal dilation occurred. In conclusion: relatives of AAA patients have an increased risk for dilatation of the abdominal and the thoracic aorta. To prevent underdiagnosis of aortic aneurysm in relatives and improve early detection of aortic aneurysms, a CT-scan of the total aorta and the iliac arteries should be considered as the method of choice for family screening. Conflicts of interest: none. Funding: Lijf en Leven Foundation R.L. Lechner: None. A.S. IJpma: None. D. Heijsman: None. J.W. Roos-Hesselink: None. H.J.M. Verhagen: None. S. ten Raa: None. R.M.W. Hofstra: None. D.F. Majoor-Krakauer: None. P05.02.B Yield of whole exome sequencing of aneurysm genes in 858 patients with an abdominal aorta aneurysm

R. L. Lechner , A. S. IJpma, D. Heijsman, J. W. Roos-Hesselink, H. J. M. Verhagen, S. ten Raa, R. M. W. Hofstra, D. F. Majoor-Krakauer

Erasmus MC, Rotterdam, Netherlands
A growing number of genes are associated with risk for aortic aneurysms. Similar mechanisms of remodeling of the extracellular matrix influencing the integrity of the aortic wall may play a role in abdominal and thoracic aneurysms. This helps to explain co-occurrence of AAA and TAA (25%), relatives with AAA in familial TAA and relatives with TAA in familial AAA. To establish the role of aneurysm genes in AAA, the yield of diagnostic whole exome sequencing (WES) of a panel of aneurysm genes in AAA patients is presented. As part of an ongoing genetic AAA study consecutively diagnosed patients at the department of Vascular Surgery were invited for genetic counseling. In this way WES DNA testing was performed in 858 AAA patients (738 unique families, 345 with familial AAA). A pathogenic (PV) variant occurred in 20 (3%) AAA index cases and 7 family members and 301 AAA patients (41%) had a variant of unknown clinical significance (VUS). PV occurred in FBN1 ( n = 4), COL3A1 ( n = 6), DSCH1 ( n = 1), TGFBR2 ( n = 3), NOTCH1 ( n = 1), PLOD1 ( n = 1), MYLK ( n = 1), MFAP5 ( n = 1) SMAD6 ( n = 1) and SLC2A10 ( n = 1, heterozygous). VUS were observed in FBN2 , COL5A1 , MYH11 , PLOD1 , TGFB2 , COL1A1 and COL1A2 . There was no difference between familial and sporadic AAA in the WES yield. The outcome was comparable to that found in thoracic aneurysm patients. The conclusion is that DNA diagnostics may improve stratification of familial risk for aortic aneurysms and distinguish relatives who benefit most from close surveillance. Funding Lijf en Leven Foundation Conflicts of interest: none R.L. Lechner: None. A.S. IJpma: None. D. Heijsman: None. J.W. Roos-Hesselink: None. H.J.M. Verhagen: None. S. ten Raa: None. R.M.W. Hofstra: None. D.F. Majoor-Krakauer: None. P05.03.C ADAMTS19 associated heart valve defects: novel genetic variants consolidating a recognizable cardiac phenotype

S. Massadeh 1,2 , A. AlHashem 3 , I. van de Laar 4 , F. Alhabshan 5 , N. Ordonez 6 , S. Alawbathani 6 , S. Khan 6 , M. Kabbani 5 , F. Chaikhouni 5 , A. Sheereen 1 , I. Almohammed 1,2 , B. Alghamdi 1 , S. Ahmad 7 , I. Frohn-Mulder 8 , C. Beetz 6 , A. Rolfs 6,9 , P. Bauer 6 , M. Wessels 4 , M. Alaamery 1,2 , A. Bertoli-Avella 6

1 Developmental Medicine Department, King Abdullah International Medical Research Center, King Saud Bin Abdulaziz University for Health Sciences, King Abdulaziz Medical City, Ministry of National Guard Health Affairs, Riyadh, Saudi Arabia, 2 The Joint Center of Excellence for Biomedicine Between King Abdulaziz City for Science and Technology (KACST) and Brigham & Women’s Hospital (BWH), Joint Centers of Excellence Program, King Abdulaziz City for Science and Technology (KACST), Riyadh, Saudi Arabia, 3 Division of Pediatric Genetics, Department of Pediatrics, Prince Sultan Military Medical City, Riyadh, Saudi Arabia, 4 Department of Clinical Genetics, Erasmus University Medical Center, Rotterdam, Netherlands, 5 Cardiac Sciences Department, Ministry of the National Guard - Health Affairs, King Abdullah International Medical Research Center, King Saud bin Abdulaziz University for Health Sciences, Riyadh, Saudi Arabia, 6 CENTOGENE AG, Rostock, Germany, 7 Cardiac Center, Prince Sultan Military Medical City, Riyadh, Saudi Arabia, 8 Department of Pediatric Cardiology, Erasmus University Medical Center, Rotterdam, Netherlands, 9 University of Rostock, Rostock, Germany
Recently, ADAMTS19 was identified as a novel causative gene for heart valve disease (HVD), affecting mainly the aortic and pulmonary valves as described in four patients from two families and supported by a mouse knockout model. Exome sequencing and data repository (CentoMD®) analyses were performed to identify patients with ADAMTS19 variants (two families). A third family was recognized based on cardiac phenotypic similarities and SNP array homozygosity. Three novel loss of function (LoF) variants were identified in six patients from three families. Clinically, all patients presented anomalies of the aortic/pulmonary valves, which included thickening of valve leaflets, stenosis and insufficiency. Three patients had (recurrent) subaortic membrane, suggesting that ADAMTS19 is the first gene identified related to discrete subaortic stenosis. In contrast to previous publication, none of these patients were reported as having a bicuspid aortic valve. However, one case presented a bi-commissural pulmonary valve. All patients displayed some degree of atrioventricular valve insufficiency. Other cardiac anomalies included atrial/ventricular septal defects, persistent ductus arteriosus, and mild dilated ascending aorta. Our findings confirm that biallelic LoF variants in ADAMTS19 are causative of a specific and recognizable cardiac phenotype. We recommend considering ADAMTS19 genetic testing in all patients with multiple semilunar valve abnormalities, particularly in the presence of subaortic membrane. ADAMTS19 screening in patients with semilunar valve abnormalities is needed to estimate the frequency of the HVD related phenotype, which might be not so rare. S. Massadeh: None. A. AlHashem: None. I. van de Laar: None. F. Alhabshan: None. N. Ordonez: A. Employment (full or part-time); Significant; CENTOGENE AG. S. Alawbathani: A. Employment (full or part-time); Significant; CENTOGENE AG. S. Khan: A. Employment (full or part-time); Significant; CENTOGENE AG. M. Kabbani: None. F. Chaikhouni: None. A. Sheereen: None. I. Almohammed: None. B. Alghamdi: None. S. Ahmad: None. I. Frohn-Mulder: None. C. Beetz: None. A. Rolfs: A. Employment (full or part-time); Significant; CENTOGENE AG. P. Bauer: A. Employment (full or part-time); Significant; CENTOGENE AG. M. Wessels: None. M. Alaamery: None. A. Bertoli-Avella: A. Employment (full or part-time); Significant; Centogene AG. P05.04.A Aortic aneurysm and dissection, an underestimated finding in JAG1 mutation carriers

I. Van Gucht 1 , J. R. Bento 1 , A. Van Berendoncks 2 , E. Van Craenenbroeck 2 , J. Saenen 3 , S. Laga 3 , J. Meester 1 , A. Verstraeten 1 , N. Boeckx 1 , L. Van Laer 1 , B. Loeys 1

1 Center of Medical Genetics Antwerp, University of Antwerp, University Hospital Antwerp, Edegem, Belgium, 2 Department of Cardiac Surgery, University Hospital Antwerp, University of Antwerp, Edegem, Belgium, 3 Department of Cardiology, University Hospital Antwerp, University of Antwerp, Edegem, Belgium
Loss-of-function Jagged-1 mutations classically lead to Alagille syndrome (AGS), a dominantly inherited disorder affecting liver, heart, eye, face and skeleton. Pulmonary artery stenosis, present in two thirds of AGS patients, is considered as a hallmark of the condition. Other vascular anomalies are identified in up to 10% of AGS patients. We describe a unique family with a JAG1 missense variant that segregates with a wide range of cardiovascular anomalies. Whole exome sequencing in a fetus with left hypoplastic heart and left renal agenesis revealed a missense variant (c.2242T>C; p.Cys748Arg) in JAG1 , which was absent from gnomAD, predicted likely pathogenic by prediction programs and maternally inherited. The mother underwent surgery for aortic coarctation at age 10. The JAG1 variant was present in the mother’s monozygotic twin-sister and brother but absent in another sister. Subsequent familial clinical evaluation revealed normal eye exam (no embryotoxon), normal ultrasound of kidneys and absence of hemivertebrae in all mutation carriers. Echocardiographic evaluation revealed a normal aorta in the mother, borderline aortic dilatation (40 mm) in her monozygotic twin-sister and a huge aortic sinus aneurysm (58 mm) in the brother. The brother also has scoliosis. Remarkably, in literature another JAG1 variant affecting a Cys-residu has been linked to severe congenital heart disease (Tetralogy of Fallot). Aortic aneurysm was previously reported in four patients, three of which were only diagnosed on autopsy after fatal dissection. Our family data warrant regular echocardiographic follow-up in JAG1 mutation carriers as severe aortic aneurysms remain asymptomatic and predispose to fatal aortic dissection. I. Van Gucht: None. J.R. Bento: None. A. Van Berendoncks: None. E. Van Craenenbroeck: None. J. Saenen: None. S. Laga: None. J. Meester: None. A. Verstraeten: None. N. Boeckx: None. L. Van Laer: None. B. Loeys: None. P05.06.C Expanding the genetic and phenotypic spectrum of ACTA2 -related vasculopathies in a Dutch cohort

L. van den Bersselaar 1 , E. Rompen 1 , S. Kurul 1 , M. Kempers 2 , A. Houweling 3 , E. Overwater 3 , Y. Hilhorst 4 , D. Barge-Schaapveld 4 , I. Krapels 5 , E. Dulfer 6 , M. Wessels 1 , B. Loeys 2 , J. Bekkers 1 , H. Bruggenwirth 1 , A. Maugeri 3 , J. Roos-Hesselink 1 , J. Verhagen 1 , I. van de Laar 1

1 Erasmus Medical Center, Rotterdam, Netherlands, 2 Radboud University Medical Center, Nijmegen, Netherlands, 3 Amsterdam University Medical Center, Amsterdam, Netherlands, 4 Leiden University Medical Center, Leiden, Netherlands, 5 Maastricht University Medical Center, Maastricht, Netherlands, 6 University Medical Center Groningen, Groningen, Netherlands
Introduction: Heterozygous variants in the ACTA2 gene, encoding the smooth muscle cell specific isoform of alpha-actin, confer a high risk for thoracic aortic aneurysms and aortic dissections. The purpose of this retrospective multicenter study was to review the clinical and genetic data of 47 new individuals with a pathogenic or likely pathogenic ACTA2 variant and elucidate the clinical outcome of ACTA2 -related vasculopathies. Material and Methods: Index patients and relatives with a pathogenic or likely pathogenic variant in the ACTA2 gene were included in this study. Data were collected through retrospective review of available medical records using a standardized questionnaire. Results: Forty-seven individuals from 26 families participated in our study. Mean age at last evaluation was 50.8 ± 17.0 years. A total of 21 different ACTA2 variants were detected in our cohort, including 19 missense variants, 1 splice site variant and 1 frameshift variant. Four variants are novel. Aortic events occurred in 30/47 (63.8%) of patients. Mean age at first aortic event was 55.6 ± 13.7 years. Male gender and the presence of hypertension emerged as significant predictors of aortic events. Men were also at higher risk of aortic aneurysms compared to women. Other clinical findings included congenital heart disease in 6/47 and iris flocculi in 6/47. Conclusions: We provide further insight into the genotype and phenotype of ACTA2 -related vasculopathies. Male gender and hypertension are predisposing factors for aortic events in individuals with a pathogenic ACTA2 variant. Supported by Dutch Heart Foundation (2014T007) and Erasmus University Rotterdam Fellowship to IvdL. L. van den Bersselaar: None. E. Rompen: None. S. Kurul: None. M. Kempers: None. A. Houweling: None. E. Overwater: None. Y. Hilhorst: None. D. Barge-Schaapveld: None. I. Krapels: None. E. Dulfer: None. M. Wessels: None. B. Loeys: None. J. Bekkers: None. H. Bruggenwirth: None. A. Maugeri: None. J. Roos-Hesselink: None. J. Verhagen: None. I. van de Laar: None. P05.11.B Prevalence of TTR mutations associated with transthyretine amyloidosis (ATTR) estimated from national exome sequencing data .

D. Monies 1,2 , D. Mohty 3,4,5 , B. Fadel 3,4 , B. Meyer 1,2 , I. Alayary 6 , M. Abouelhoda 1,2

1 Department of Genetics, King Faisal Specialist Hospital and Research Centre, Riyadh, Saudi Arabia, 2 Saudi Human Genome Program, King Abdulaziz City for Science and Technology, Riyadh, Saudi Arabia, 3 Heart center, King Faisal Specialist Hospital & Research Center, Riyadh, Saudi Arabia, 4 Al-Faisal University, College of Medicine, Affiliate Harvard Medical School International, Riyadh, Saudi Arabia, 5 CHU, Limoges, France, 6 Pfizer Inc, Jeddah, Saudi Arabia
Introduction: Amyloidosis is a rare, frequently under-diagnosed systemic disease due to protein deposition that develops when a native protein misfolds into insoluble amyloid fibrils. These fibril deposits alter function of most affected organs, including the myocardium. Transthyretin amyloidosis (ATTR) is one of the two main causes of cardiac amyloidosis and occurs as a result of mutations in TTR . Under or misdiagnosis of affected individuals and incomplete penetrance make estimates of prevalence challenging. While worldwide prevalence is estimated to be around 50,000 individuals, with TTR V142I being the most common cardiac ATTR associated mutation, no data on TTR mutations or their frequency in Middle Eastern countries are available to date. Materials and Methods: Blinded from clinical information, we undertook data mining of 13,906 exomes of unrelated Saudi individuals, sequenced between 2015 and 2019 as part of the Saudi Human Genome Project (SHGP). Pathogenic or highly pathogenic TTR variants were identified following filtration to remove common, synonymous, non-coding and other benign variants. Results and conclusion: We identified seven missense variants (known or predicted pathogenic) in TTR , three of which have been previously reported in amyloidosis patients worldwide. The most frequent variants were c.424G>A:p.V142I (1:435) and c.370C>T:p.R124C (1:2,318). Prevalence of TTR variants in the general Saudi population was much higher than the actual estimated rate of ATTR cases nationally (1:10,000), which may be explained by incomplete penetrance or sub-clinical manifestation of the disease. D. Monies: None. D. Mohty: None. B. Fadel: None. B. Meyer: None. I. Alayary: None. M. Abouelhoda: None. P05.13.A Investigation of NOTCH1mutation frequency in bicuspid aortic valve disease

S. E. Hamby 1,2 , R. Debiec 1,2,3 , M. Nath 1,2 , P. D. Jones 1,2 , S. Coolman 1,2 , M. Asiani 1,2 , S. Kharodia 1,2 , G. J. Skinner 3 , N. J. Samani 1,2 , A. Bolger 1,2,3 , T. R. Webb 1,2

1 University of Leicester Department of Cardiovascular Sciences, Leicester, United Kingdom, 2 NIHR Leicester Biomedical Research Centre, Leicester, United Kingdom, 3 East Midlands Congenital Heart Centre, Glenfield Hospital, Leicester, United Kingdom
Background: Bicuspid Aortic valve (BAV) is a common congenital heart defect present in around 1% of the population. Familial BAV has an autosomal dominant pattern of inheritance with reduced penetrance, however, the majority of BAV cases are sporadic. Pathogenic and likely pathogenic NOTCH1 variants have been reported in familial, as well as up to 4% of sporadic cases. Aim: To investigate the frequency of causative NOTCH1 mutations in a large cohort of patients of sporadic and familial BAV disease recruited to the Bicuspid aoRtic vAlVe gEnetic research (BRAVE) study. Methods: Whole exome sequencing (WES) was performed in 12 families (30 affected and 25 unaffected family members). Targeted NOTCH1 sequencing was carried out in 383 sporadic BAV cases. NOTCH1 variants were filtered by gnomAD allele frequency (< 0.001) and potential deleterious effect (Combined Annotation Dependent Depletion (CADD) score>20). We also compared cumulative frequency of rare NOTCH1 variants between sporadic BAV patients and 1102 apparently healthy controls. Results: One BAV family had a truncating NOTCH1 variant (c.873C>G/p.Tyr291*). There were no candidate NOTCH1 variants in any of the other 11 families. We identified seven rare and potentially deleterious NOTCH1 variants in the sporadic patient group. Among which, there were no protein truncating mutations. There was no enrichment of NOTCH1 variants between cases and controls (1.83% vs 3.09%, p -value 0.18). Conclusions: Our data suggest that whilst occasionally responsible for familial cases of BAV, NOTCH1 mutations are an infrequent cause of sporadic forms of the disease. S.E. Hamby: None. R. Debiec: None. M. Nath: None. P.D. Jones: None. S. Coolman: None. M. Asiani: None. S. Kharodia: None. G.J. Skinner: None. N.J. Samani: None. A. Bolger: None. T.R. Webb: None. P05.14.B Bicuspid aortic valve: a pilot candidate gene-based approach in a representative Czech cohort

P. Votýpka 1 , P. Peldová 1 , A. Krebsová 2 , V. Zoubková 1 , M. Balaščaková 1 , M. Macek, Jr. 1

1 Department of Biology and Medical Genetics, 2nd Faculty of Medicine, Charles University and Motol, Prague, Czech Republic, 2 Department of Cardiology, Institute for Clinical and Experimental Medicine, Prague, Czech Republic
Aim: Analysis of the genetic basis in a representative cohort of Czech patients with familiar bicuspid aortic valve (BAV) with / without aortic aneurysm (MIM# 109730). Materials and Methods: Altogether 100 unrelated cases were clinically examined, underwent genetic counselling and positive cases were subjected to “cascade screening” of first-degree relatives. Massively parallel sequencing (MiSeq platform; Illumina.com) was utilised for a custom-made panel comprising either 136, 229 or 100 cardiac/aortic conditions-related candidate genes (Sophia Genetics.com). Presence of pathogenic variants was validated by Sanger sequencing and via their segregation in respective families. Results: Pathogenic/likely pathogenic variant (ACMG Class ≥ 4) were found in 3/100 patients (3 %) within TGFB2, SMAD6 and FBN1 . Variants of unknown significance (Class 3) were detected in 9/100 patients (9 %) comprising genes FLNA, FBN1, MYH11, TAB2, TGFB2 and GATA5 . A family with concurrent familial dilated cardiomyopathy, BAV and ectopia lentis was analysed as well. Likely causative DNA variants in MYBPC3 and FBN1 were identified, but FBN1 variants did not co-segregate with BAV. Conclusions: The yield of genetic testing in familial forms of BAV is rather low in our cohort. Moreover, the identification of pathogenic variant in BAV-related gene is not a guarantee of their causality, hence detected variants should be carefully interpreted. The genetic basis for the familial forms of BAV will be further studied, e.g. by whole exome analysis together with improved clinical stratification of our patient cohort. Supported by Ministry of Health of the Czech Republic, grant Nr. NV18-02-00237 P. Votýpka: None. P. Peldová: None. A. Krebsová: None. V. Zoubková: None. M. Balaščaková: None. M. Macek, Jr.: None. P05.16.A Electrophysiological characterization of a Brugada syndrome SCN5A Belgian founder mutation in induced pluripotent stem cell cardiomyocytes

E. Simons 1 , A. Nijak 1 , B. Vandendriessche 1 , D. Van de Sande 2 , E. Sieliwończyk 1 , A. J. Labro 2 , D. Snyders 2 , D. Schepers 1 , B. Loeys 1,3 , M. Alaerts 1

1 Centre of Medical Genetics, faculty of Medicine and Health Sciences, University of Antwerp & Antwerp University Hospital, Antwerp, Belgium, 2 Laboratory of Molecular Biophysics, Physiology & Pharmacology, Department of Biomedical Sciences, University of Antwerp, Antwerp, Belgium, 3 Department of Human Genetics, Radboud University Medical Centre, Nijmegen, Netherlands
Introduction: SCN5A encodes the α-subunit of voltage-gated cardiac sodium channel Na v 1.5. Mutations in SCN5A are identified in about 20% of patients with Brugada syndrome (BrS), an inherited cardiac arrhythmia. We have identified an SCN5A founder mutation (c.4813+3_4813+6dupGGGT), leading to a loss-of-function of Na v 1.5 in 25 different families. Mutation carriers show variable expression of the phenotype: from asymptomatic to syncopes and SCD. We used induced pluripotent stem cell derived cardiomyocytes (iPSC-CM) to investigate the underlying pathophysiology. Material & Methods: Dermal fibroblasts of three patients with different severity, and one unrelated healthy control were reprogrammed using the CytoTune™-iPS 2.0 Sendai Reprogramming Kit. iPSC-CMs were differentiated following a published protocol. T3 hormone addition was tested to improve maturity of the cells. We performed several differentiation rounds and investigated expression of cardiac markers using qPCR and immunocytochemistry and electrophysiological properties using patch-clamping and calcium imaging. Results: All iPSC-CMs expressed tested markers. We observed reduction in sodium current density in patient iPSC-CMs, together with shortening of the action potential and calcium transient duration compared to the control. However, our data display variability between the differentiation batches, as well as between clones generated from one donor. Addition of T3 improved organization of the sarcomeres and decreased result variability. Conclusions: We established iPSC-CM models for a unique Belgian SCN5A founder mutation. Despite observed variability, we could detect expected differences in electrophysiological properties of patient cells compared to controls. We prove that T3 addition improves the maturity of the cells, with a positive impact on the variability. E. Simons: None. A. Nijak: None. B. Vandendriessche: None. D. Van de Sande: None. E. Sieliwończyk: None. A.J. Labro: None. D. Snyders: None. D. Schepers: None. B. Loeys: None. M. Alaerts: None. P05.18.C Network-based identification of novel Shox2-dependent genes in cardiac rhythm control

S. Hoffmann 1,2 , S. Schmitteckert 1 , K. Raedecke 1,2 , D. Rheinert 1 , S. Diebold 3 , R. Roeth 1 , B. Weiss 1 , M. Granzow 4 , B. Niesler 1,2 , A. Griesbeck 1 , V. Eckstein 5 , W. H. Zimmermann 6,7 , S. Just 3 , G. A. Rappold 1,2

1 Department of Human Molecular Genetics, Institute of Human Genetics, University of Heidelberg, Heidelberg, Germany, 2 DZHK, German Center for Cardiovascular Research, Partner Site Heidelberg/Mannheim, Heidelberg, Germany, 3 Department of Internal Medicine II, University of Ulm, Ulm, Germany, 4 Department of Human Genetics, Institute of Human Genetics, University of Heidelberg, Heidelberg, Germany, 5 FACS Core Facility, Department of Medicine V, University Hospital Heidelberg, Heidelberg, Germany, 6 Institute of Pharmacology, University Medical Center Goettingen, Goettingen, Germany, 7 DZHK, German Centre for Cardiovascular Research, Partner Site Goettingen, Goettingen, Germany
The transcription factor SHOX2 is involved in the development and function of the sinoatrial node (SAN), which is crucial for initiating and controlling heart rhythm. SHOX2 has been associated with cardiac arrhythmias such as atrial fibrillation (AF) and sinus node dysfunction. Yet, our knowledge on co-regulatory networks associated with cardiac rhythm control in health and disease still remains limited. To dissect the role of Shox2-dependent networks, we utilized a murine embryonic stem cell (ESC) model that allowed us to investigate molecular pathways involving Shox2 in SAN-like cardiomyocytes. Differential RNA-seq-based expression profiling of Shox2 +/+ and Shox2 -/- ESCs revealed 94 regulated transcripts in Shox2 -/- SAN-like cells. Fifteen putative target genes were selected for further validation based on comparative expression analysis of our dataset with publicly available RNA-seq data of SAN and right atria enriched genes at different mouse embryonic stages. Network-based analyses integrating data from the Mouse Organogenesis Cell Atlas and the Ingenuity pathway analysis tool as well as validation in mouse and zebrafish confirmed a species conserved regulation for Shox2 target genes in cardiac rhythm control. Our results indicate that co-regulatory networks involving SHOX2 participate in the development of conduction traits through regulatory mechanisms, thereby affecting the expression of multiple genes. This work was supported by grants from the DFG (RA 380/14-4) and DZHK (81X2500110 and 81X2500171). S. Hoffmann: None. S. Schmitteckert: None. K. Raedecke: None. D. Rheinert: None. S. Diebold: None. R. Roeth: None. B. Weiss: None. M. Granzow: None. B. Niesler: None. A. Griesbeck: None. V. Eckstein: None. W.H. Zimmermann: None. S. Just: None. G.A. Rappold: None. P05.19.A Diagnostic yield of targeted versus broad next generation sequencing data analysis in inherited cardiomyopathy and arrhythmia syndromes

K. Kolokotronis 1 , N. Pluta 1 , E. Klopocki 1 , S. Störk 2 , S. Rost 1 , B. Gerull 2

1 Institute of Human Genetics, University of Wuerzburg, Wuerzburg, Germany, 2 Comprehensive Heart Failure Center and Department of Medicine I, University Hospital Würzburg, Wuerzburg, Germany
Introduction: Although next generation sequencing has drastically changed cardiogenetics, the fact that many cases with positive family history remain unsolved after routine panel diagnostics suggests that some genetic causes remain to be discovered. In this study we examined the diagnostic yield of targeted gene panels compared to whole exome data analysis. Methods: The cohort consisted of 61 consecutive patients with a diagnosis of cardiomyopathy or primary arrhythmia syndrome, with DCM being the most common diagnosis. The genetic analysis involved whole exome sequencing with subsequent stepwise bioinformatic analysis starting from virtual gene panels till whole exome data. Genetic variants were classified according to ACMG-criteria. Results: Overall, in 39 out of 61 (64%) patients a variant of interest was detected. The detection rate was highest in patients with a diagnosis of HCM (7/9; 77%) or DCM (25/36; 69%). In DCM the diagnostic yield was better compared to current literature. The majority of variants of interest were found in the disease-specific panel (46%; 28/61), while the further analysis of an extended panel and whole exome data led to an additional diagnostic yield of 13% and 5%, respectively. Whole exome data analysis also detected variants in thus far not well characterized candidate genes whose expression and function profile suggested a probable pathogenetic role. Conclusions: Targeted gene panel analysis led to the identification of the genetic cause in the majority of solved cases. The additional diagnostic yield of whole exome data analysis was low, enabled though the detection of potentially causal variants in candidate genes. K. Kolokotronis: None. N. Pluta: None. E. Klopocki: None. S. Störk: None. S. Rost: None. B. Gerull: None. P05.20.B Diagnostic yield of core panels for cardiomyopathy- and arrhythmia genes versus a larger panel of heart-disease related genes

D. M. E. I. Hellebrekers 1 , B. J. C. van den Bosch 1 , G. R. F. Claes 1 , I. Roozen 1 , Y. E. G. Barrois 1 , N. Vergoossen 1 , A. T. M. Hendrickx 1 , W. van Dijk 1 , C. L. M. Marcelis 2 , M. Kempers 2 , Y. M. Hoedemaekers 2 , A. T. J. M. Helderman-van den Enden 1 , E. K. Vanhoutte 1 , I. P. C. Krapels 1 , A. van den Wijngaard 1

1 Maastricht University Medical Center, Maastricht, Netherlands, 2 Radboud University Medical Center, Nijmegen, Netherlands
Introduction: We evaluated the prevalence of (likely) pathogenic variants in our cardiomyopathy- and arrhythmia core panels in patients with cardiac disease. Furthermore, the additional diagnostic value of a larger panel of heart disease-associated genes was assessed. Methods: This study included a large population of ~2000 patients suspected of a heritable cardiac condition who underwent cardiogenetic testing at the laboratory of the Clinical Genetics Department in Maastricht. Sequencing a diagnostic panel of 46 cardiomyopathy genes and/or 27 arrhythmia genes was performed using single-molecule molecular inversion probes (smMIPs). For 347 of these patients, whole exome sequencing was performed for the analysis of a panel of 350 heart disease-associated genes. Results: A (likely) pathogenic variant was found in 20% (336/1719) and 8% (34/408) of patients tested for the cardiomyopathy or arrhythmia gene panel, respectively. The majority of these variants were found in the MYBPC3, MYH7, LMNA, TNNT2, MYL2 and PKP2 genes for cardiomyopathy and SCN5A, KCNQ1, RYR2 and KCNH2 genes for arrhythmia. Sequencing the larger panel of 350 genes in 347 patients identified an additional pathogenic variant explaining the clinical phenotype in only 3 patients. Conclusions: Our data support parallel sequencing of core genes as the preferred strategy for genetic testing of patients suspected of having a genetic cardiomyopathy or arrhythmia. The incremental diagnostic yield of a larger panel of heart disease-associated genes is minimal. Therefore, focus should not be on analysing more genes, but on developing functional assays to predict pathogenicity of variants of unknown significance (VUS’s) in the core panel genes. D.M.E.I. Hellebrekers: None. B.J.C. van den Bosch: None. G.R.F. Claes: None. I. Roozen: None. Y.E.G. Barrois: None. N. Vergoossen: None. A.T.M. Hendrickx: None. W. van Dijk: None. C.L.M. Marcelis: None. M. Kempers: None. Y.M. Hoedemaekers: None. A.T.J.M. Helderman-van den Enden: None. E.K. Vanhoutte: None. I.P.C. Krapels: None. A. van den Wijngaard: None. P05.21.C Dominant mild Carvajal syndrome: arrhythmogenic cardiomyopathy caused by the heterozygous p.R1113* nonsense variant in the DSP gene .

A. T. J. M. Helderman-vd Enden 1 , J. G. Post 2 , K. J. A. F. van Kaam 1 , R. H. Lekanne Deprez 3 , D. Dooijes 4 , A. A. M. Wilde 5 , I. P. C. Krapels 1

1 Department of Clinical Genetics, Maastricht University Medical Center, Maastricht, Netherlands, 2 Department of Clinical Genetics, University Medical Center Utrecht, Utrecht, Netherlands, 3 Department of Clinical Genetics, Amsterdam University Medical Center, Amsterdam, Netherlands, 4 Department of Clinical Genetics, University Medical Center Utrecht, Utrecht, Netherlands, 5 Department of Cardiology, Amsterdam University Medical Center, Amsterdam, Netherlands
Introduction: Arrhythmogenic cardiomyopathy (ACM) is a dominant inherited cardiac disease characterized by progressive fibrofatty replacement of the myocardium that predisposes to ventricular tachycardia and sudden death. Carvajal syndrome is a rare recessive form of ACM associated with woolly hair and palmo-plantar keratoderma and pathogenic variants in the desmoplakin (DSP) gene. Some families have been described with a dominantly inherited DSP variant with features of Carvajal. Materials and Methods: In a four generation family seggregation analysis was done for a dominantly inherited DSP p.R1113* variant. The phenotypes are described and the results are compared to literature. Results: In the large sibship (4M + 8F) at least three siblings are obligate carrying the DSP pathogenic variant and have offspring with cardiac abnormalities. Five family members died suddenly, 2/5 obligate carriers, 3/5 the status is unknown. The curly hair is co-seggregating with the variant. Not all carriers have keratoderma. A number of individuals with the variant have no cardiac complaints so far, cardiological investigations are planned. An overview will be presented at the conference and compared to the six families described by Maruthappu et al in 2019, one of these families also has the p.R1113* variant. Conclusion: The p.R1113* variant in the DSP gene leads to a well recognizable phenotype of mild Carvajal syndrome. Drawing the attention to this phenotype by presenting this large four generation family may help in identifying other families with this disease and in preventing sudden death. A.T.J.M. Helderman-vd Enden: None. J.G. Post: None. K.J.A.F. van Kaam: None. R.H. Lekanne Deprez: None. D. Dooijes: None. A.A.M. Wilde: None. I.P.C. Krapels: None. P05.22.A Chromosome 22q11 copy number variations in paediatric and adult patients with congenital heart defects

G. K. ZODANU , D. Nagy, M. Széll, M. Oszlánczi, K. Havasi, A. Kalapos, M. Katona

Introduction: Congenital heart defect (CHD) is the most common birth defect, affects ~1% of newborns. Chromosome 22q11 microdeletion syndrome is often associated with CHDs and is the most common human microdeletion, occurs in ~1/4000 live births in the general population and in 2.8-14.9% of CHD patients. 22q11 copy number variants (CNVs) are caused by non-allelic homologous recombination events in the flanking low copy repeat regions (LCRs), labeled A-H. Materials and Methods: Our aim was to evaluate the prevalence of 22q11 CNVs in Hungarian CHD patients. 175 blood samples were obtained from children and adults with different isolated and combined CHDs. Samples were analyzed using multiplex ligation-dependent probe amplification (MLPA) to detect CNVs in the given chromosomal region. Positive MLPA results were confirmed with locus-specific FISH. Genotype-phenotype comparison and family screening was also performed. Results: CNVs were detected in overall 10 cases (5.7%). Six cases with typical microdeletion (between LCRs A and D) also known as DiGeorge syndrome, 2 cases with microduplication and 2 cases with combined microdeletion and microduplication were detected. The phenotypical appearance of patients was highly variable. CNVs were familiar in 3 cases. Conclusions: Until present this was the largest systemic genetic screening of Hungarian CHD patients. Since the clinical diagnosis of 22q11 microdeletion/microduplication syndrome may be challenging due to its variable manifestation, systematic genetic testing may be of utmost benefit in the early diagnosis and proper management of these patients. Funding grant: GINOP-2.3.2-15-2017-00039 G.K. Zodanu: None. D. Nagy: None. M. Széll: None. M. Oszlánczi: None. K. Havasi: None. A. Kalapos: None. M. Katona: None. P05.23.B Genetic variation in participants of the 100,000 Genomes Project with congenital heart disease

S. L. Baross 1 , S. G. Williams 1 , K. E. Hentges 2 , A. D. Sharrocks 3 , B. D. Keavney 1

1 Division of Cardiovascular Sciences, University of Manchester, Manchester, United Kingdom, 2 Division of Evolution and Genomic Sciences, University of Manchester, Manchester, United Kingdom, 3 Division of Molecular and Cellular Function, University of Manchester, Manchester, United Kingdom
Congenital heart disease is the most common birth defect, affecting approximately 1% of live births globally. Congenital heart disease shows a high degree of heritability but the genetic causes are still poorly understood. The 100,000 Genomes Project provides whole genome sequences for patients with rare disease or cancer and their families. Over 600 participants were recruited to the project under the congenital heart disease arm of the project, almost all of whom have no known causative variant. In addition, analysis of phenotyping and clinical data identified an additional ~2,500 participants with congenital heart disease who were recruited to the project for other conditions. We will present rare, likely pathogenic single nucleotide, short insertions/deletions and structural variants, both transmitted and de novo, from this cohort of 3,100 participants with congenital heart disease. Short variants are filtered for variants with moderate or high impact on protein function; CADD (combined annotation dependent depletion) score ≥ 20; and absent in gnomAD v3. This research was made possible through access to the data and findings generated by the 100,000 Genomes Project ( http://www.genomicsengland.co.uk ). Grant reference: BHF FS/16/58/32734. S.L. Baross: None. S.G. Williams: None. K.E. Hentges: None. A.D. Sharrocks: None. B.D. Keavney: None. P05.24.C Mutation spectrum of congenital heart disease in 73consanguineous Turkish families

W. Dong 1 , H. Kaymakcalan 2 , N. Diab 1 , S. Chih Jin 1 , C. Tanıdır 3 , A. Yalcin 2 , S. Mane 1 , K. Bilguvar 1 , M. Brueckner 1 , R. Lifton 1

1 Yale University, New Haven, CT, United States, 2 Demiroglu Bilim University, Istanbul, Turkey, 3 Mehmet Akif Ersoy Hospital, istanbul, Turkey
Mutation spectrum of congenital heart disease in 73 consanguineous Turkish families Introduction: Congenital heart disease (CHD) is the most frequent defect among live births and the leading cause of mortality from a birth defect. Nearly 90% of CHD cases have a suspected genetic etiology, but in 56% of cases the genetic etiology is unknown. Few gene discovery efforts have been made with consanguineous CHD cohorts. We recruited 74 CHD patients from 73 consanguineous families in Turkey and used whole exome sequencing to identify affected genes in these patients. In 13.7% of the cohort, we could identify a genetic etiology and we discovered four new genes that are putatively causative for CHD. This represents an important advance in our understanding of the genetic consequences of consanguinity in CHD. Materials and methods: 74 samples from 73 families were recruited from two pediatric cardiology clinics in Istanbul, Turkey. Cardiac phenotypes were classified into 5 major categories and whole exome sequencing was performed at the Yale Center for Genomic Analysis using Illumina. Result: 7 out of 73 (9.6%) patients carried damaging homozygous variants in known CHD genes and 3 out of 73 (4.1%) of them harbored other types of CHD-related genomic alterations, which explained 13.7% of the cohort. Conclusions: Our findings is consistent with the hypothesis that high consanguinity is more likely to be associated with recessive cardiac abnormalities. Additionally, it fits the perception that the heritability for severe defects is higher than milder malformations. Grant: Yale-National Institutes of Health Center for Mendelian Genomics W. Dong: None. H. Kaymakcalan: None. N. Diab: None. S. Chih Jin: None. C. Tanıdır: None. A. Yalcin: None. S. Mane: None. K. Bilguvar: None. M. Brueckner: None. R. Lifton: None. P05.26.B Altered microRNA and target gene expression related to Tetralogy of Fallot

M. Grunert, S. Appelt, S. Rickert-Sperling

Charité - Universitätsmedizin Berlin, Berlin, Germany
MicroRNAs (miRNAs) play an important role in guiding development and maintaining function of the human heart. Dysregulation of miRNAs has been linked to various congenital heart diseases including Tetralogy of Fallot (TOF), which represents the most common cyanotic heart malformation in humans. Several studies have identified dysregulated miRNAs in right ventricular (RV) tissues of TOF patients. In the study presented here, we profiled genome-wide the whole transcriptome and analyzed the relationship of miRNAs and mRNAs of RV tissues of a homogeneous group of 22 non-syndromic TOF patients. Observed profiles were compared to profiles obtained from right and left ventricular tissue of normal hearts. To reduce the large list of predicted target genes of dysregulated miRNAs, we applied a stringent target prediction pipeline integrating probabilities for miRNA-mRNA interaction. The final list of disease-related miRNA-mRNA pairs comprises novel as well as known miRNAs including miR-1 and miR-133, which are essential to cardiac development and function by regulating KCNJ2, FBN2, SLC38A3 and TNNI1. Overall, our study provides additional insights into post-transcriptional gene regulation of malformed TOF hearts. M. Grunert: None. S. Appelt: None. S. Rickert-Sperling: None. P05.28.A Circulating lncRNA ANRIL a potential biomarker in Tanzanian patients with coronary artery disease

G. AKAN 1 , E. Nyawawa 2 , B. Nyangasa 2 , M. K. Turkcan 3 , E. Mbugi 1 , O. Ozgen 4 , M. Janabi 2 , F. Atalar 1

1 MUHAS Genetics Laboratory, Biochemistry Department, School of Medicine, Muhimbili University of Health and Allied Science, Dar es Salaam, Tanzania, United Republic of, 2 Muhimbili National Hospital Jakaya Kikwete Heart Institute, Dar es Salaam, Tanzania, United Republic of, 3 Department of Electrical Engineering, Columbia University,, New York, NY, United States, 4 Molecular Medicine Department, Aziz Sancar Experimental Medicine Research Institute, Istanbul University, Istanbul, Turkey
Background: 9p21.3 locus was identified as a hot spot locus associated with coronary artery disease(CAD) and it contains CDKN2A and CDKN2B genes which encode the cell cycle regulators and a non-coding RNA ANRIL gene. Role of 9p21.3 locus variations in molecular mechanisms of CAD pathogenesis and the expression of neighbour-genes remain unknown. The study aims to assess of expressions of CDKN2A, CDKN2B, ANRIL and ANRIL splices variants association with CAD susceptibility, and to investigate the impact of 9p21.3 locus variations on the expression of studied genes in peripheral blood, Epicardial Adipose Tissue(EAT), Mediastinal Adipose tissue(MAT) and Subcutaneous Adipose Tissue(SAT) in Tanzanian CAD patients. Methods&Results: Expression of CDKN2A, CDKN2B, ANRIL, and its splice variants (circANRIL, NR003529, EU741058 and DQ485454)were measured by QRT-PCR in peripheral blood, EAT, MAT and SAT of 25 patients undergoing coronary artery bypass grafting due to CAD and 25 non-CAD patients undergoing heart valve surgery. ANRIL gene expression levels were significantly up-regulated while expression levels of circANRIL were significantly down-regulated in peripheral blood of CAD patients. Moreover, increased expression levels of circANRIL were associated with less severity of CAD. ROC analysis data has also confirmed that circANRIL could act as a potential biomarker for detecting CAD. Conclusions: Our results are the first data presenting the expressions of ANRIL and its splice variants in cardiac adipose tissues of CAD patients. We demonstrate circANRIL has a synergistic effect with ANRIL and plays a protective role in CAD. circANRIL could possibly be used as a biomarker for CAD in Tanzania. G. Akan: None. E. Nyawawa: None. B. Nyangasa: None. M.K. Turkcan: None. E. Mbugi: None. O. Ozgen: None. M. Janabi: None. F. Atalar: None. P05.29.B Members of subgingival plaque bacterial complexes are present in blood of patients with coronary artery disease

A. Suarez-Molina 1 , C. Fong 2 , L. Cifuentes-C 2 , S. Guauque-Olarte 2

1 Instituto Departamental de Salud de Nariño, Pasto, Colombia, 2 Universidad Cooperativa de Colombia, Pasto, Colombia
Introduction: It has been hypothesized that oral bacteria can migrate, through the blood, from the mouth to the artery plaques exacerbating atherosclerosis. This study compared the bacteria present in peripheral blood of individuals with and without coronary artery disease. Materials and Methods: RNA sequences from blood downloaded from GEO (GSE58150) correspond to 8 cases with coronary artery calcium (CAC) >514 and 8 controls (CAC=0). After quality controls, the sequences were aligned to the hg38 reference genome using Hisat2. The unmapped sequences were fed into Kraken to determinate bacterial taxa. Ecological indices were calculated using Vegan. The counts of bacteria between groups was compared using a Mann-Whitney U test. Results: The mean number of species was different between cases (433.4) and controls (226.1) ( p -value = 0.0001). A total of 129 species were found only in cases and 74 were exclusive of controls. According to the Jaccard similarity index 16% of the species identified is not shared between cases and controls (p=0.001). The Alcaligenaceae family was more frequent in cases than in controls (FDR: 0.048). The red complex bacteria Treponema denticola was detected only in 2 cases. The mean count of bacteria belonging to subgingival plaque bacterial complexes was higher in cases (573.5) than in controls (249) ( p -value = 0.0206). Conclusions: This study identified periodontopathogens in blood of patients with coronary artery disease. The list of microorganisms presented only in cases will be key to identify new periodontopathogens that can relate periodontitis and this cardiovascular disease. SGO received funding by CONADI-Universidad Cooperativa de Colombia, grant 1839. A. Suarez-Molina: None. C. Fong: None. L. Cifuentes-C: None. S. Guauque-Olarte: None. P05.30.C Molecular genetic screening in patients with spontaneous coronary artery dissection

M. Antonutti 1 , F. Baldan 2 , L. Spedicato 1 , D. Zanuttini 1 , T. Bisceglia 1 , E. Favaretto 1 , C. Tioni 1 , D. Sut 1 , M. Ghidina 1 , C. Lanera 3 , D. Gregori 3 , D. Fabbro 1 , G. Damante 1 , A. Proclemer 1

1 ASUFC, Udine, Italy, 2 University of Udine, Udine, Italy, 3 DCTVPH, Padova, Italy
Spontaneous coronary artery dissection (SCAD) is increasingly recognized as an important cause of myocardial infarction, particularly in women often with few cardiovascular risk factors. SCAD is defined as a spontaneous tear in the coronary arterial wall that is not traumatic or iatrogenic. Studies are still underway to investigate the role of genetics in promoting SCAD; genetic diseases frequently associated with SCAD are connective tissue diseases. Considering the role of SCAD for sudden cardiac death and the increased risk for future cardiac events, an early diagnosis is crucial. We enrolled 60 patients with nonatherosclerotic angiographic diagnosis of SCAD. All patients’ genomic DNA was analyzed by NGS with gene panel involved in collagenopathies and arteriopathies ( ACTA2, CBS, COL3A1, COL5A1, COL5A2, ELN, FBN1, FBN2, LTBP2, MYLK, NOTCH1, MYH11, SMAD3, SMAD4, SLC2A10, TGFB2, TGFB3, TGFBR1, TGFBR2, PLOD1 ). Seven patients result to carry potential SCAD-related mutations. In particular, we observed a pathological COL3A1 mutation; 3 different VUS mutations in: LTBP2 (two patients), MYLK and NOTCH1 . Moreover, we found a new nonsense mutation in SMAD3 and a missense alteration in COL5A2; both not enlisted in public database, but that could be categorized as pathogenetic and VUS, respectively, according to the ACMG classification. These data support the use of sequencing of genes inclusive of collagenopathies and arteriopathies as an approach to evaluate genetically mediated SCAD. However,, considering that only a minority of patients have clear SCAD-causative mutations, these results remark the need of further studies to define genetic mechanisms of SCAD. M. Antonutti: None. F. Baldan: None. L. Spedicato: None. D. Zanuttini: None. T. Bisceglia: None. E. Favaretto: None. C. Tioni: None. D. Sut: None. M. Ghidina: None. C. Lanera: None. D. Gregori: None. D. Fabbro: None. G. Damante: None. A. Proclemer: None. P05.32.B Comparison of variant detection rate in 100 candidate genes between two representative cohorts of Czech living patients versus victims of sudden cardiac death with clinical. or post mortem diagnosis of dilated-, arrhythmogenic- and hypertrophic cardiomyopathy .

A. Krebsova 1 , P. Votýpka 2 , P. Peldová 2 , K. Ruecklova 3 , V. Zoubkova 2 , S. Pohlova-Kucerova 4 , A. Pilin 5 , M. Kulvajtova 6 , M. Kubanek 1 , M. Bilek 5 , T. Tavacova 7 , J. Petrkova 8 , M. Dobias 9 , P. Tomasek 10 , M. Macek Sr. 2 , M. Macek Jr. 2 , J. Janousek 7 , J. Kautzner 1

1 Institute for Clinical and Experimental Medicine, Prague, Czech Republic, 2 Department of Biology and Medical Genetics, 2nd Faculty of Medicine, Charles University and Motol University Hospital, Prague, Czech Republic, 3 Paediatric Department, 3rd Faculty of Medicine, Charles University, Prague, Czech Republic, 4 Department of Forensic Medicine, Faculty of Medicine in Hradec Králové, Charles University, Hradec Kralove, Czech Republic, 5 Institute for forensic medicine and toxicology, 1st Faculty of Medicine, Charles University, Prague, Czech Republic, 6 Institute for Forensic Medicine, 3rd Faculty of Medicine, Charles University, Prague, Czech Republic, 7 Children´s Heart Centre, 2nd Faculty of Medicine, Charles University and Motol University Hospital, Prague, Czech Republic, 8 Department of Cardiology, Faculty Hospital Olomouc and Palacký University, Olomouc, Czech Republic, 9 Institute for Forensic Medicine, Faculty Hospital Olomouc, Olomouc, Czech Republic, 10 Institute for Forensic Medicine, Hospital Bulovka, 2nd Faculty of Medicine, Charles University, Prague, Czech Republic
Introduction: Hereditary cardiomyopathy is associated with risk of sudden cardiac death (SCD). Genetic aetiology of SCD in a representative Czech cohort with post mortem diagnosis of cardiomyopathy was studied and results compared to those in living cases. Patients and Methods: Altogether 47 victims of SCD with post mortem diagnosis of hypertrophic- (HCM; 18/47), arrhythmogenic- (ACM; 19/47) and dilated cardiomyopathy (DCM; 10/47) were ascertained. Concurrently, genetic testing was performed in 114 living patients (HCM 54/114, ACM 22/114, DCM 38/114). NGS (MiSeq platform; Illumina.com) was utilised for a custom-made panel comprising 100 candidate genes (Sophia Genetics, Switzerland). Results: The causative detection rate (according to ACMG.net classes 4 or 5) in SCD victims with DCM was 60 % (6/10) and in living patients with DCM 47.4 % (18/38). Variants in TTN , RBM20 , DES and FLNC prevailed. Detection rate in ACM was 5 % (1/19 in SCN5A gene) in SCD victims and 31.8 % (7/22) [JJ1] in living patients. The detection rate in SCD victims with HCM was 16 % (3/18) and in living patients 35 % (19/54). The most prevalent gene was MYBPC3 , while PRKAG2 was detected in SCD victim and in cardiac arrest survivor. Conclusion: Post-mortem genetic analysis in DCM yields a high detection rate and allows effective primary prevention of SCD in at risk relatives. In contrast, molecular autopsy of HCM and ACM renders a much lower yield which could reflect more complex genetic aetiology in SCD. Supported by Ministry of Health of the Czech Republic, grant Nr. NV18-02-00237 and 00064203/6003 A. Krebsova: None. P. Votýpka: None. P. Peldová: None. K. Ruecklova: None. V. Zoubkova: None. S. Pohlova-Kucerova: None. A. Pilin: None. M. Kulvajtova: None. M. Kubanek: None. M. Bilek: None. T. Tavacova: None. J. Petrkova: None. M. Dobias: None. P. Tomasek: None. M. Macek Sr.: None. M. Macek Jr.: None. J. Janousek: None. J. Kautzner: None. P05.33.C Genome wide association analysis in dilated cardiomyopathy revealed two new susceptibility loci for systolic heart failure

S. Garnier 1,2 , M. Harakalova 3,4 , S. Weiss 5,6 , M. Mokry 3,7,8 , J. van Setten 3 , C. Proust 9 , L. Duboscq-Bidot 1,2,10 , A. Boland 11,12 , J. Deleuze 11,12,13 , M. Dörr 6,14 , F. Asselbergs 3,15,16 , F. Cambien 9 , E. Villard 1,2,10 , D. Trégouët 9 , P. Charron 1,2,10,17

1 Sorbonne Université, INSERM, UMR-S1166, research unit on cardiovascular disorders, metabolism and nutrition, Team Genomics & Pathophysiology of Cardiovascular Diseases, Paris, France, 2 ICAN Institute for Cardiometabolism and Nutrition, Paris, France, 3 Department of Cardiology, Division Heart & Lungs, University Medical Center Utrecht, Utrecht University, Utrecht, Netherlands, 4 Regenerative Medicine Utrecht (RMU), University Medician Center Utrecht, Utrecht, Netherlands, 5 Interfaculty Institute for Genetics and Functional Genomics, Department of Functional Genomics, University Medicine Greifswald, Greifswald, Germany, 6 DZHK (German Centre for Cardiovascular Research), partnersite Greifswald, Greifswald, Germany, 7 Laboratory of Clinical Chemistry and Haematology, University Medical Center, Heidelberglaan 100, Utrecht, Netherlands, 8 Laboratory for Experimental Cardiology, University Medical Center Utrecht, Heidelberglaan 100, Utrecht, Netherlands, 9 INSERM UMR_S 1219, Bordeaux Population Health Research Center, University of Bordeaux, Bordeaux, France, 10 APHP, Pitié-Salpêtrière Hospital, Cardiology Department, Paris, France, 11 Centre National de Recherche en Génomique Humaine (CNRGH), Institut de Biologie François Jacob, CEA, Université Paris-Saclay, Evry, France, 12 Laboratory of Excellence GENMED (Medical Genomics), Paris, France, 13 Centre d’Etude du Polymorphisme Humain, Fondation Jean Dausset, Paris, France, 14 Department of Internal Medicine B, University Medicine Greifswald, Greifswald, Germany, 15 Institute of Cardiovascular Science, Faculty of Population Health Sciences, University College London, London, United Kingdom, 16 Health Data Research UK and Institute of Health Informatics, University College London, London, United Kingdom, 17 AP-HP, Département de génétique, Centre de Référence Maladies Cardiaques Héréditaires, Hôpital Pitié-Salpêtrière, Paris, France
We conducted the largest Genome Wide Association Study performed so far in Dilated Cardiomyopathy (DCM), a leading cause of systolic heart failure and cardiovascular death. Using a discovery phase of 2,719 cases and 4,440 controls and a replication phase of 584 independent cases and 966 controls, we identified and replicated two new DCM-associated loci one on chromosome 3p (meta-analysis p = 5.3 10 −13 ) and the second on chromosome 22q (meta-analysis p = 5.0 10 −10 ) while confirming the two previously identified DCM loci on chromosome 10 and 1, BAG3 and HSPB7 for an estimated heritability of 31% ± 8%. The genetic risk score constructed from the number of lead risk-alleles at these 4 loci revealed a 27% risk increased in individuals with 8 risk-alleles compared to the 5 risk alleles reference group. The two association signals were then fine-mapped by combining in silico and functional genomics investigations. While a few genes remain candidates at the second locus and deserve further investigations, our work clearly identified one gene as responsible for the association at the first locus whose role in the pathophysiology of DCM is supported by recent observations in human and mice. As the biological pathway in which this gene is involved is a potential target for pharmacological agents, our finding opens novel therapeutic perspectives for treating or preventing heart failure. These results provide new findings that add both on the understanding of the genetic architecture of heart failure and on potential new players involved in the pathophysiology of this devastating disease. S. Garnier: None. M. Harakalova: None. S. Weiss: None. M. Mokry: None. J. van Setten: None. C. Proust: None. L. Duboscq-Bidot: None. A. Boland: None. J. Deleuze: None. M. Dörr: None. F. Asselbergs: None. F. Cambien: None. E. Villard: None. D. Trégouët: None. P. Charron: None. P05.34.A Utility of genetics for risk stratification in paediatric dilated cardiomyopathy

J. C. Herkert 1 , M. H. van der Meulen 2 , S. L. den Boer 2 , G. J. du Marchie Sarvaas 3 , N. Blom 4,5 , A. D. J. ten Harkel 4 , H. M. P. J. Breur 6 , L. A. J. Rammeloo 7 , R. Tanke 8 , C. Marcelis 9 , I. M. B. H. van de Laar 10 , J. M. A. Verhagen 10 , A. Baas 11 , A. Sammani 11 , I. Christiaans 1 , D. Q. C. M. Barge-Schaapveld 12 , R. H. Lekanne dit Deprez 13 , J. P. van Tintelen 11 , M. Dalinghaus 2

1 University of Groningen, University Medical Center Groningen, Department of Genetics, Groningen, Netherlands, 2 Erasmus MC, University Medical Center Rotterdam, Department of Paediatric cardiology, Rotterdam, Netherlands, 3 University of Groningen, University Medical Center Groningen, Department of Paediatric cardiology, Groningen, Netherlands, 4 University of Leiden, Leiden University Medical Center, Department of Paediatric cardiology, Leiden, Netherlands, 5 Amsterdam University Medical Center, location AMC, Department of Paediatric cardiology, Amsterdam, Netherlands, 6 University of Utrecht, Wilhelmina Children’s Hospital, University Medical Center Utrecht, Department of Paediatric cardiology, Utrecht, Netherlands, 7 Amsterdam University Medical Center, location Free University Medical Center, Department of Paediatric cardiology, Amsterdam, Netherlands, 8 Radboud University Medical Center, Department of Paediatric cardiology, Nijmegen, Netherlands, 9 Radboud University Medical Center, Department of Genetics, Nijmegen, Netherlands, 10 Department of Clinical Genetics, Erasmus MC, University Medical Center Rotterdam, Rotterdam, Netherlands, 11 University of Utrecht, University Medical Center Utrecht, Department of Genetics, Utrecht, Netherlands, 12 University of Leiden, Leiden University Medical Center, Department of Clinical Genetics, Leiden, Netherlands, 13 Amsterdam University Medical Center, location AMC, Department of Clinical Genetics, Amsterdam, Netherlands
Introduction: Dilated cardiomyopathy (DCM) in children may have an underlying genetic cause in a substantial number of cases. We describe the current practice and results of genetic evaluation in children with DCM and evaluate genotype-phenotype correlations that may guide prognosis. Methods: We performed a multicentre prospective observational study in children diagnosed with DCM from 2010-2017. Results: One hundred forty-four patients were included. Initial diagnostic categories consisted of idiopathic DCM in 67 children (47%), familial in 18 (13%), neuromuscular in 7 (5%), inborn error of metabolism in 4 (3%), malformation syndrome in 2 (1%), myocarditis in 23 (16%) and ‘other’ in 23 (16%). Median follow-up time was 2.1 years [IQR 1.0-4.3]. Hundred-seven patients (74%) underwent genetic testing. A likely pathogenic (LP) or pathogenic (P) variant was found in 39 children (36%); the majority in MYH7 ( n = 9). Three patients had more than one LP/P variant. In at least 5/39 patients (13%) the variant occurred de novo. During the study, 39 patients (27%) reached a study endpoint (SE: all-cause death or heart transplantation). Transplant-free survival was significantly lower in patients with a LP/P variant ( P = 0.005). Children who carried a LP/P variant were 2.8 times more likely to reach a SE compared to children without, while clinical characteristics at diagnosis did not differ (hazard ratio 2.8; 95%CI 1.3-5.8, P = 0.007). Conclusions: Genetic testing is useful to predict clinical outcome in children with DCM and should be incorporated into the initial work-up. Patients with a LP/P variant have a poorer prognosis than patients without LP/P variant. J.C. Herkert: None. M.H. van der Meulen: None. S.L. den Boer: None. G.J. du Marchie Sarvaas: None. N. Blom: None. A.D.J. ten Harkel: None. H.M.P.J. Breur: None. L.A.J. Rammeloo: None. R. Tanke: None. C. Marcelis: None. I.M.B.H. van de Laar: None. J.M.A. Verhagen: None. A. Baas: None. A. Sammani: None. I. Christiaans: None. D.Q.C.M. Barge-Schaapveld: None. R.H. Lekanne dit Deprez: None. J.P. van Tintelen: None. M. Dalinghaus: None. P05.35.B Non-segregation of truncating TTN variants in families with dilated cardiomyopathy (DCM)

A. G. Fleming , L. Brett, S. Wilkinson, S. John, L. Briggs, M. Edwards, D. J. Morris-Rosendahl

Clinical Genomics Laboratory, London, United Kingdom
DCM is a highly heterogeneous condition with over 30 genes implicated in inherited forms, including TTN . TTN truncating variants are found in ~2% of the general population (Roberts et al . (2015) Sci Transl Med . 7(270); 270) but there is a higher association with DCM for truncating variants in cardiac isoforms of titin; therefore, only truncating variants in exons which are spliced into >90% of TTN cardiac transcripts are reported as potentially pathogenic in DCM by our laboratory. We have identified TTN truncating variants that have been classified as either likely pathogenic or pathogenic in 82 of 613 DCM patients in our cohort. Due to the prevalence of TTN truncations in healthy individuals, predictive testing for truncating TTN variants is offered with caution, and instead segregation testing in affected relatives is first undertaken. Within our cohort we have found several variants which do not segregate with DCM. Out of a total of 36 individuals, across 25 families, we have 5 cases of non-segregation of truncating TTN variants; 3 frameshift, 1 splice site and one exonic deletion. All of these non-segregating variants are absent from populations databases and occur in exons which are spliced into 100% of cardiac TTN isoforms; and were therefore initially classified as likely pathogenic. These findings highlight the importance of a cautious approach when interpreting TTN truncating variants, as well as the risks of predictive testing; especially if this results in clinical discharge for individuals who do not carry the familial truncating TTN variant. A.G. Fleming: None. L. Brett: None. S. Wilkinson: None. S. John: None. L. Briggs: None. M. Edwards: None. D.J. Morris-Rosendahl: None. P05.37.A Optimization and refinement of the diagnosis of genetic dyslipidemias by targeted NGS

L. Zuurbier , O. R. F. Mook, M. A. Haagmans, J. C. Defesche

Amsterdam University Medical Centers, Amsterdam, Netherlands
Introduction: Genetic and acquired dyslipidemias are very frequent in the general population and strongly associated with premature atherosclerosis, myocardial infarctions, arterial complications and sudden death at young age. Genetic lipoprotein abnormalities can be explained by a limited number of genes. For optimal clinical management, risk assessment and prevention of cardiovascular disease an accurate diagnosis is an absolute requirement, but the interconnection between the metabolic pathways of different lipoprotein fractions jeopardizes this when only lipid levels are assessed. Materials and Methods: In order to pinpoint the culprit lipoprotein fraction of clinical dyslipidemia in 4277 patients, we evaluated NGS and CNV data, including 29 dyslipidemia-associated genes that enabled to examine all separate lipoprotein components simultaneously. With this approach 10 types of dyslipidemia can be discriminated. Moreover, this panel includes a genetic risk factor for statin intolerance. Results: A genetic cause for dyslipidemia was found in 32% of the patients. Of these, 4% carried a CNV. The specific initial clinical diagnoses of hypercholesterolemia or hypertriglyceridemia were confirmed in only 17 and 35% of patients, respectively. However, unsolicited findings causal for a second or an unexpected type of dyslipidemia, were encountered in 17% of cases, and suggest a broader role than initially anticipated for variants causing dysbetalipoproteinemia or hypertriglyceridemia. Conclusions: The use of the NGS-Dyslipidemia gene panel accurately separated genetic from acquired dyslipidemias and within the detected genetic dyslipidemias the diagnosis was refined based on unsolicited findings in a substantial number of patients. This allows targeted and optimal treatment to reduce cardiovascular risk. L. Zuurbier: None. O.R.F. Mook: None. M.A. Haagmans: None. J.C. Defesche: None. P05.38.B Hypermethylation of NRG1 gene correlates with the presence of heart defects in Down’s syndrome

A. Dobosz , A. Grabowska, M. Bik-Multanowski

Department of Medical Genetics, Jagiellonian University Medical College, Krakow, Poland
Congenital heart defects can decrease the quality of life and life expectancy in affected individuals, and constitute a major burden for the health care systems. Endocardial cushion defects are among the most prevalent heart malformations in the general population, and are extremely frequent (approximately a 100-fold higher prevalence) in children with Down syndrome. Several genes have been proposed to be involved in the pathogenesis of these malformations, but no common pathogenic DNA variants have been identified so far. Here, we focussed on constitutive, epigenetic alterations of function of selected genes, potentially important for endocardial cushion development. We used two types of microarrays, dedicated for assessment of gene promoter methylation and whole genome expression. First, we compared the gene promoter methylation profiles between two groups of Down syndrome patients, with and without heart defects of endocardial cushion-type. Then, to determine the functional role of the detected methylation alterations, we assessed the expression of the genes of interest. We detected significant hypermethylation of the NRG1 gene promoter region in children with heart defects. NRG1 is a key factor in maturation of endocardial cushions. Supplementary gene expression assessment revealed significantly decreased activity of the ERBB3, SHC3 and SHC4 genes in children with heart defects. The above three genes are closely related to the NRG1 gene and are crucial elements of the NRG/ErbB pathway. The results of this pilot study show that hypermethylation of the NRG1 gene promoter can reflect the functional genome alteration contributing to development of congenital heart defects of endocardial cushion-type. A. Dobosz: None. A. Grabowska: None. M. Bik-Multanowski: None. P05.39.C How Effective is Genetic Testing for Familial Hypercholesterolaemia Patients in Tayside?

C. Leung , E. Gellatly, J. Berg

Ninewells Hospital and Medical School, University of Dundee, Dundee, United Kingdom
Background: DNA analysis for Familial Hypercholesterolaemia (FH), including the LDLR , PCSK9 and ApoE genes, is available to Scottish individuals with high cholesterol who fulfil Simon Broome criteria. The aim of mutation identification is to allow more effective management of the proband, as well as cascade testing and identification of at risk individuals. The ultimate aim is early statin treatment of affected individuals with prevention of cardiovascular morbidity. Method: 60 index patients with causative LDLR mutations were identified between 2010 and 2018 in NHS Tayside (Scotland). We used record linkage to assess the effect of genetic testing on cholesterol levels on probands and the number of subsequent cascade tests performed on family members. The main endpoint we used was achieving a 50% reduction of LDL cholesterol levels, as recommended by the UK National Institute of Clinical Excellence (NICE). Results: 49/189 (25.9%) eligible first degree relatives received cascade testing. Where data was available, 6/22 (27.3%) patients met the 50% reduction target in LDL-C levels. There was no demonstrable relationship between cholesterol reduction and attendance at a cardiovascular risk clinic. Conclusions: The number of cascade tests following diagnosis in a proband is low. Even if a causative LDLR mutation was identified, there was limited impact on the clinical improvement of cholesterol levels, with only a minority of patients achieving recommended cholesterol lowering following diagnosis. A better mechanism for cascade testing and management of FH is required to gain the maximum clinical benefit from DNA analysis in Familial Hypercholesterolaemia. C. Leung: None. E. Gellatly: None. J. Berg: None. P05.41.A Mutation spectrum and polygenic score in German patients with familial hypercholesterolemia

L. Rieck 1 , F. Bardey 1 , T. Grenkowitz 1 , L. Bertram 2 , J. Helmuth 3 , C. Mischung 3 , J. Spranger 1 , T. Bobbert 1 , U. Kassner 1 , I. Demuth 1

1 Charité Universitätsmedizin Berlin, Berlin, Germany, 2 Lübeck Interdisciplinary Platform for Genome Analytics, Institutes of Neurogenetics and Cardiogenetics, Lübeck, Germany, 3 Department Molecular Diagnostics, Labor Berlin, Berlin, Germany
Introduction: Autosomal-dominant familial hypercholesterolemia (FH) is a genetic disorder characterized by increased plasma concentrations of LDL-Cholesterol and a substantially elevated risk for cardiovascular disease. Mutations in three major genes are known to cause FH: the LDL receptor gene ( LDLR ), the apolipoprotein B gene ( APOB ) and the proprotein convertase subtilisin/kexin 9 gene ( PCSK9 ). Our aim is to analyze the mutation spectrum and to determine a polygenic risk score in a cohort of hypercholesterolemic patients. Methods: We screened 336 patients who were clinically assessed by the Dutch Lipid Clinic Network (DLCN) score and suspected to have FH for disease causing variants and genotyped six single nucleotide polymorphisms (SNPs) to calculate the polygenic risk score for the FH patients and a control group of 1,946 healthy participants. Results: We identified an FH causing variant in 117 of the 336 patients (12 novel variants) from the FH cohort. Most sequence variants were found in the LDLR gene (84.9%) out of which 44.4% were of the missense type. The discriminatory power of the DLCN score distinguished FH mutation carriers from non-carriers with an AUC = 0.76 ( p < .001) in a receiving operator characteristic (ROC) curve, similar to the value for native LDL-C (AUC = 0.77 ( p < .001)). Similar to previous studies, the mean polygenic risk score was significantly higher in FH mutation negative subjects than in FH mutation positive patients ( p < .05) and healthy controls ( p < .001). Conclusions: This study substantiates the known clinical and genetic variability of FH for German hypercholesterolemic patients and examines a possible polygenic aetiology. L. Rieck: None. F. Bardey: None. T. Grenkowitz: None. L. Bertram: None. J. Helmuth: None. C. Mischung: None. J. Spranger: None. T. Bobbert: None. U. Kassner: None. I. Demuth: None. P05.43.A FLNC gene in Hypertrophic Cardiomyopathy: an effort to accurate variant classification

E. Cuesta-Llavona , R. Lorca, B. Díaz-Molina, J. Lambert, B. Alonso, S. Iglesias-Álvarez, J. Reguero, E. Coto, J. Gómez

Hospital Universitario Central de Asturias, Oviedo, Spain
Hypertrophic Cardiomyopathy (HCM) is the most common inherited heart disease. Recent studies have identified the Filamin C ( FLNC ) gene as a HCM-associated gene. The aim of this study is to assign the percentage of HCM cases which could be relationated with FLNC variants. We sequenced 924 index cases of HCM for a comprehensive cardiovascular gene panel of 194 genes, in an Ion GeneStudio S5 Sequencer. We identified 365 patients (39%) with variants in the sarcomeric and FLNC genes. Among them, 330 (90%) were carriers of sarcomeric variants, and 35 (10%) harbored rare FLNC variants (either not described or ≤ 10 cases in gnomAD). Eleven patients were carriers of both sarcomeric and FLNC variant, thus their were excluded to the further analysis and variant classification since their pathogenicity is complex to measure. In this way, we finally selected 24 carriers of 20 different FLNC variants. Family studies were performed in 16/24 cases. In total, we identified 55 relatives who harbor a FLNC variant, thirty nine of them have a HCM diagnosis (71% penetrance). According to the ACMG criteria, and the performed family studies, seven variants (35%) have been classified as pathogenic or likely pathogenic, twelve as variants of unknown significance (60%), and a variant classified as benign or likely benign (5%). In conclusion, at least 1.2% of HCM index cases harbor a FLNC likely pathogenic variant. However, further co-segregation and functional studies have to be performed in order to accomplish an accurate variant classification, specially in VUS variants. E. Cuesta-Llavona: None. R. Lorca: None. B. Díaz-Molina: None. J. Lambert: None. B. Alonso: None. S. Iglesias-Álvarez: None. J. Reguero: None. E. Coto: None. J. Gómez: None. P05.44.B A founder truncating mutation in GDF1 causes autosomal-recessive right isomerism in multiplex Arab kindreds .

D. Yagel 1,2 , Y. Bolkier 3,2 , O. Barel 4 , A. Vardi 3,2 , D. Mishali 3,2 , A. Veber 1 , N. Shalva 1 , U. Katz 3,2 , S. Abudi 1,2 , N. Kol 4 , A. Raas-Rothschild 5,2 , G. Rechavi 4,2,6 , B. Pode-Shakked 1,7,2 , Y. Anikster 1,2,6

1 Metabolic Disease Unit, Edmond and Lily Safra Children’s Hospital, Sheba Medical Center, Ramat Gan, Israel, 2 Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv, Israel, 3 Department of Pediatric Cardiac Intensive Care, Edmond Safra International Congenital Heart Center, Edmond and Lily Safra Children’s Hospital, Sheba Medical Center, Ramat Gan, Israel, 4 Sheba Cancer Research Center, Sheba Medical Center, Ramat Gan, Israel, 5 The Institute for Rare Diseases, The Danek Gertner Institute of Human Genetics, Sheba Medical Center, Ramat Gan, Israel, 6 The Wohl Institute for Translational Medicine, Sheba Medical Center, Ramat Gan, Israel, 7 Talpiot Medical Leadership Program, Sheba Medical Center, Ramat Gan, Israel
The genetic basis of congenital heart malformations associated with disruption of left-right (L-R) asymmetry is broad and heterogenous, with variants in over twenty five genes implicated thus far. Of these, deleterious mutations in the Growth/Differentiation Factor 1 ( GDF1 ) gene have recently been shown to cause heterotaxy with varied complex heart malformations of left-right patterning, in 23 individuals reported to date, either in monoallelic or biallelic state. We report three unrelated individuals exhibiting heterotaxy with congenital heart defects, each originating from consanguineous kindreds of Arab-Muslim descent. Using Whole exome sequencing, a shared novel homozygous truncating c.608G>A (p.W203*) mutation in the GDF1 gene, was revealed as the molecular basis of their disease. Subsequently, targeted sequencing of this variant showed full segregation with the disease in these families, with over 15 reportedly affected individuals, enabling genetic counselling, prenatal diagnosis and planning of future pregnancies. Our findings further confirm the association of GDF1 variants, heterotaxy and congenital heart defects of left-right patterning, and expand the previously described phenotypic spectrum and mutational profile. Finally, given the high rate of consanguinity and high frequency of the p.W203* mutation in the Arab-Muslim population residing near the city of Hebron, we suggest targeted screening for this variant in relevant clinical circumstances. D. Yagel: None. Y. Bolkier: None. O. Barel: None. A. Vardi: None. D. Mishali: None. A. Veber: None. N. Shalva: None. U. Katz: None. S. Abudi: None. N. Kol: None. A. Raas-Rothschild: None. G. Rechavi: None. B. Pode-Shakked: None. Y. Anikster: None. P05.46.A Pathogenic variationin the copper binding domain of lysyl oxidase causes aortic dilation modified by sex and mechanical forces

K. Tsang 1 , R. H. Knutsen 1 , C. J. Billington Jr 1 , E. Lindberg 1 , H. Steenbock 2 , A. Wardlaw-Pickett 1 , C. K. E. Bleck 1 , J. Brinckmann 2 , B. A. Kozel 1

1 National Institutes of Health, Bethesda, MD, United States, 2 University of Luebeck, Luebeck, Germany
Mutations in lysyl oxidase ( LOX ) contribute to familial thoracic aortic aneurysm. GnomAD reveals variation throughout LOX , with more variants reported in the region encoding the N-terminal pro-domain. While ClinVar showed disease-associated stopgain and frameshift variants throughout LOX , the majority of pathogenic missense mutations were identified near the copper binding domain, within the relatively variant-poor C-terminal catalytic domain. We then studied the Lox b 2b370.2Clo (c.G854T; p.C285F) mouse whose mutationfalls within the copper binding domain. The Lox C285F protein was stably secreted but exhibited decreased enzymatic activity and altered elastin crosslinks. Structural analysisby two photon and scanning electron microscopyrevealed holes in the elastic lamellar sheets that increased with age and blood pressure. As in humans, the rate of aortic dilation was higher in males, but was also amplified by pregnancy. Lox mutants showed altered transcription of matrisome genes and their aortas showed increased susceptibility to elastase. Taken together, these findings suggest that missense variation surrounding the LOX copper binding domain increases risk for aortic aneurysm while missense variation in the region encoding the N-terminal pro-domain appears largely tolerated. Novel gene-first studies are underway to test this hypothesis in a large hospital-based cohort. Our mouse studies confirm that copper binding domain LOX variants cause disease through the production of a dysfunctional extracellular matrix that is unusually susceptible to proteolytic damage. In both humans and mice, the progressive weakening of the connective tissue leads to worsening aortic disease, modified by sex and blood pressure. Funding: NIH HL006244, USA K. Tsang: None. R.H. Knutsen: None. C.J. Billington Jr: None. E. Lindberg: None. H. Steenbock: None. A. Wardlaw-Pickett: None. C.K.E. Bleck: None. J. Brinckmann: None. B.A. Kozel: None. P05.47.B Rare variants in HCN4 identified in the general population are associated with complete atrioventricular (AV) block, 1. degree AV block and bundle branch block, results from 50.000 exomes

J. S. Jensen 1,2 , O. B. Vad 1,2 , C. Paludan-Müller 2,1 , P. R. Lundegaard 1 , J. H. Svendsen 2,3 , S. Haunsø 2,3 , H. Bundgaard 2,3 , M. S. Olesen 1,2

1 Department of Biomedical Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark, 2 Laboratory for Molecular Cardiology, Department of Cardiology, Centre for Cardiac, Vascular, Pulmonary and Infectious Diseases, Rigshospitalet, Copenhagen University Hospital, Copenhagen, Denmark, 3 Department of Clinical Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
Introduction: Cardiac conduction disorders cover disorders such as atrioventricular (AV) block and bundle branch block (BBB). Genome-wide association studies have identified more than 100 genetic loci for atrial fibrillation, including HCN4 loci associated with duration of the PR interval, a proxy for AV dysfunction. Recent candidate studies suggest an association between HCN4 variants and AV-block. We seek to determine the contribution of rare genetic variants in HCN4 to complete AV-block, 1. degree AV-block and BBB in the general population. Materials/methods: The UK Biobank is a population-based study of 500 000 individuals including a subset with genome-wide genotyping and exome sequencing ( n = 45,596). In this case-control study, we included persons of genetically determined white-European ancestry and analyzed their exome data. Analyses were performed using a logistic mixed-effects model. A gene-based burden analysis and single variant test were performed to examine the relationship between HCN4 variants and complete AV-block, 1. degree AV-block and BBB in the general population. Results: The study included 43,371 persons. In an analysis of the HCN4 genes a significant association between rare variants (MAF < 0.01) in HCN4 gene was found (complete AV-block, P=2.5 x 10 −5 , 1. degree AV-block, P=1.3 x 10 −3 and BBB, P=0.01). The association to complete AV block was mostly driven by the variants Ser835Phe (P=2.7e −3 ), Glu153Gly (P=3.5e −3 ) and Arg378Cys (P=6.3e −3 ). Conclusions: Rare HCN4 variants contribute to the risk of complete AV-block, 1. degree AV-block and BBB in the general population. These HCN4 variants seem to confer a substantial penetrance. Clinical screening for some of these variants seems appropriate. J.S. Jensen: None. O.B. Vad: None. C. Paludan-Müller: None. P.R. Lundegaard: None. J.H. Svendsen: None. S. Haunsø: None. H. Bundgaard: None. M.S. Olesen: None. P05.49.A MicroRNAs as biomarkers of heart failure

A. M. Peterlin 1 , K. Pocivavsek 2 , D. Petrovic 1 , B. Peterlin 3

1 Institute of Histology and Embriology, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia, 2 Department of Cardiovascular Surgery, University Medical Centre Ljubljana, Ljubljana, Slovenia, 3 Clinical Institute of Genomic Medicine, University Medical Centre Ljubljana, Ljubljana, Slovenia
Introduction: MicroRNAs are highly investigated for their role in the pathogenesis of cardiovascular diseases. Nevertheless, evidence for clinical implementation is still lacking. In our systematic review, we evaluated the potential of microRNA as pathophysiological and diagnostic biomarkers of heart failure. Materials and methods: The literature search was conducted in the PubMed database up to August 2019. Newcastle-Ottawa quality assessment scale was used to assess the quality of included research papers. Using miRTarBase, we identified all known gene targets for selected miRNAs that were differentially expressed in research papers included in the systematic review. We then comprised the list of all target genes and performed gene set enrichment analysis using Enrichr. Results from KEGG, BioPlanet, and Panther databases were analyzed. Results: We identified 72 differentially expressed microRNA molecules among groups of heart failure patients and control groups. There was no substantial overlap of differentially expressed miRNAs among different studies, only five miRNAs (miR-1228, miR-122, miR-423-5p, miR-142-3p, and exosomal miR-92b-5p) were differentially expressed in more than one included study. Gene set enrichment analysis, based on the gene targets of microRNAs presented in included studies, showed that gene targets of differentially expressed miRNAs were enriched in MAPK signaling pathway, TGFβ signaling pathway, PI3K-Akt signaling pathway, IL-2 signaling pathway, apoptosis pathway, p53 activity regulation, angiogenesis pathway, and PDGF signaling pathway. Conclusions: Results of our systematic review show that currently, there is insufficient support for the use of any of the presented miRNAs as a pathophysiological or prognostic biomarker in the clinical setting. A.M. Peterlin: None. K. Pocivavsek: None. D. Petrovic: None. B. Peterlin: None. P05.51.C Germline variants in transcription factor HEY2 functional domains lead to congenital heart defects and thoracic aortic aneurysms

E. S. van Walree 1,2 , G. Dombrowsky 3 , I. E. Jansen 2 , M. Umićević Mirkov 2 , R. Zwart 4 , A. Ilgun 4 , D. Guo 5 , S. B. Clur 6 , A. S. Amin 7 , J. E. Savage 2 , A. C. van der Wal 8 , Q. Waisfisz 9 , A. Maugeri 9 , A. Wilsdon 10 , F. A. Bu’lock 11 , M. E. Hurles 12 , S. Dittrich 13 , F. Berger 14 , E. Audain Martinez 3 , M. Hitz 3 , D. M. Milewicz 5 , D. Posthuma 2 , H. Meijers-Heijboer 1 , A. V. Postma 1,4 , I. B. Mathijssen 1

1 Department of Clinical Genetics, Amsterdam UMC, University of Amsterdam, Meibergdreef 9, Amsterdam, Netherlands, 2 Department of Complex Trait Genetics, Center for Neurogenomics and Cognitive Research, Amsterdam Neuroscience, Vrije Universiteit Amsterdam, de Boelelaan 1085, Amsterdam, Netherlands, 3 Department of Congenital Heart Disease and Pediatric Cardiology, Universitätsklinikum Schleswig-Holstein Kiel, Kiel, Germany, 4 Department of Medical Biology, Amsterdam UMC, University of Amsterdam, Meibergdreef 9, Amsterdam, Netherlands, 5 Department of Internal Medicine, McGovern Medical School, University of Texas Health Science Center at Houston, 6431 Fannin, Houston, TX, United States, 6 Department of Pediatric Cardiology, Emma Children’s Hospital, Amsterdam UMC, University of Amsterdam, Meibergdreef 9, Amsterdam, Netherlands, 7 Department of Clinical and Experimental Cardiology, Amsterdam UMC, University of Amsterdam, Meibergdreef 9, Amsterdam, Netherlands, 8 Department of Pathology, Amsterdam UMC, University of Amsterdam, Meibergdreef 9, Amsterdam, Netherlands, 9 Department of Clinical Genetics, Amsterdam UMC, Vrije Universiteit Medisch Centrum, de Boelelaan 1118, Amsterdam, Netherlands, 10 School of Life Sciences, University of Nottingham, Queen’s Medical Centre, Nottingham, United Kingdom, 11 East Midlands Congenital Heart Centre and University of Leicester, Glenfield Hospital, Leicester, United Kingdom, 12 Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, United Kingdom, 13 Department of Pediatric Cardiology, University of Erlangen-Nürnberg, Loschgestrasse 15, 91054, Erlangen, Germany, 14 German Heart Center Berlin, Department of Congenital Heart Disease, Pediatric Cardiology, Berlin, Germany
Background: Affecting approximately 1% of all newborns, cardiovascular defects (CVDs) are the most common congenital anomaly worldwide. Here, we present a large family from a genetically isolated population presenting with CVDs, including congenital heart defects (CHD) and familial thoracic aortic aneurysms (FTAA), caused by a loss-of-function mutation in the Notch target gene HEY2 , which hasn’t been associated with hereditary CVDs in humans previously. Methods: The index patient, who has severe CVD, underwent trio WES. The identified variant was subsequently screened in 30 members of this patient’s family, which has a high prevalence of CVDs. Additionally, 3011 unrelated cases and 4940 controls from two datasets (one with CHD and one with FTAA) were screened for rare risk variants in HEY2 . To investigate how the various identified variants influence gene function, the regulatory activity of mutated and wildtype HEY2 on a target gene was measured with luciferase assays and qPCR. Results: A homozygous frameshift variant (p.G108*) in HEY2 was identified in three individuals with life-threatening CVDs. Twenty family members were found to be heterozygous for this variant, of whom 80% had CVD. Two additional CVD datasets show enrichment for rare, likely deleterious variants in HEY2 after meta-analysis. Lastly, we demonstrate that these variants induce failure of repression of HEY2 target genes. Conclusions: We find that rare HEY2 variants predispose to a broad spectrum of CVDs and advise clinicians to consider HEY2 as a causative gene in unsolved CVD patients. Identifying a potentially deleterious variant in HEY2 warrants clinical follow-up. E.S. van Walree: None. G. Dombrowsky: None. I.E. Jansen: None. M. Umićević Mirkov: None. R. Zwart: None. A. Ilgun: None. D. Guo: None. S.B. Clur: None. A.S. Amin: None. J.E. Savage: None. A.C. van der Wal: None. Q. Waisfisz: None. A. Maugeri: None. A. Wilsdon: None. F.A. Bu’lock: None. M.E. Hurles: None. S. Dittrich: None. F. Berger: None. E. Audain Martinez: None. M. Hitz: None. D.M. Milewicz: None. D. Posthuma: None. H. Meijers-Heijboer: None. A.V. Postma: None. I.B. Mathijssen: None. P05.52.A The Unknown Genetic Architecture of Severe Hypercholesterolemia in Finland

N. Junna 1 , S. Ruotsalainen 1 , S. Ripatti 1,2 , E. Widén 1

1 FIMM, Helsinki, Finland, 2 Department of Public Health, Clinicum, Faculty of Medicine, University of Helsinki, Helsinki, Finland
While severe hypercholesterolemia is a significant risk factor for ischemic heart disease (IHD), Finnish patients remain inadequately identified and only a part of their mutation load is known. To elucidate the genetic architecture of severe hypercholesterolemia in Finland, we have screened participants of the GeneRISK-study ( n = 7 342) originating from South-eastern Finland for novel mutations and utilized the FinnGen-cohort ( n = 110 654) to assess the mutation prevalence, impact, and nationwide distribution. Five percent ( n = 366) of GeneRISK-participants had severe hypercholesterolemia (LDL ≥ 5 mmol/l) and exome-sequencing revealed 3 carriers of previously known mutations ( LDLR R594Q, LDLR P309Kfs and APOB R3527Q, which has not previously been reported in Finland). We also identified a novel, likely pathogenic mutation, LDLR R574L. We assessed the geographical distribution of the mutation and the cardiometabolic status of mutation carriers in the nationwide FinnGen-cohort and compared them with 5 previously described hypercholesterolemia-mutations ( LDLR R594Q, LDLR G844D, LDLR L401H, LDLR D579N, LDLR C352W). The novel LDLR R574L-mutation was unevenly distributed in the country with a high prevalence among individuals born in the Southeast (the ceded Karjala region) and prevalence of cardiovascular disease among mutation carriers was elevated (22% had been diagnosed with IHD and 14% with stroke). The data further showed that less than half of mutation carriers (both the novel and previously known mutations) were on statin therapy. Our data show that the genetic spectrum of severe hypercholesterolemia in Finland is more diverse than previously anticipated, and further highlight the urgent need for better recognition and treatment of this disease. N. Junna: None. S. Ruotsalainen: None. S. Ripatti: None. E. Widén: None. P05.53.B Mendelian randomisation identifies pleiotropy between uric acid and cardiovascular diseases

E. M. Wigmore 1 , O. Chazara 1 , . FinnGen Consortium 2 , K. Carss 1 , A. Matakidou 1 , C. Haefliger 1

1 AstraZeneca, Cambridge, United Kingdom, 2 University of Helsinki, Helsinki, Finland
Elevated levels of uric acid (UA), hyperuricaemia, leads to the formation of urate crystals that cause gout. Hyperuricaemia has also been linked to cardiovascular and metabolic diseases but, unlike gout, causality has not been ascertained. Here we utilised biomarker data from the UK Biobank (UKB, n = ~500,000) and health outcome data from a Finnish biobank, FinnGen ( n = ~135,000) to explore the causal relationship between biomarkers and disease. Genome-wide association studies (GWAS) of 48 available biomarkers were conducted in UKB and FINEMAP was applied to ascertain putative causal variants. We assessed the genetic correlation of these biomarkers with 1,485 disease endpoints in FinnGen using LD score regression. Those with a significant correlation following FDR correction were assessed for causal associations using Mendelian randomisation (MR). GWAS of UA corrected for allopurinol treatment (a UA-lowering medication) was completed on 389,114 individuals and FINEMAP identified 320 putative causal signals. Significant genetic correlations were found with multiple diseases ( n = 183) including hypertensive diseases (rg=0.25, P FDR =6.2x10 −38 ), type 2 diabetes (rg=0.29, P FDR =6.2x10 −38 ), cardiovascular disease (rg=0.23, P FDR =9.6x10 −29 ) and gout (rg=0.70, P FDR =8.3x10 −21 ). Results from MR analyses confirmed the known causal association of UA with gout (IVW estimate=1.6, P FDR =7.8x10 −62 ) as well as with polyarthropathies (IVW estimate=0.37, P FDR =1.9x10 −12 ) but not with other diseases. A significant causal association was found with hypertension (IVW estimate=0.20, P FDR =0.0015) but horizontal pleiotropy was also detected (MR-Egger intercept P= 0.0069). Our finding suggests that although MR does not support the direct association between UA and cardiovascular disease, we observe pleiotropic effects which could support therapies targeting shared pathways. E.M. Wigmore: A. Employment (full or part-time); Significant; AstraZeneca. O. Chazara: A. Employment (full or part-time); Significant; AstraZeneca. .. FinnGen Consortium: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Significant; AstraZeneca, Abbvie, Biogen, Celgene, Genentech, GSK, Merck, Pfizer, Sanofi. K. Carss: A. Employment (full or part-time); Significant; AstraZeneca. A. Matakidou: A. Employment (full or part-time); Significant; AstraZeneca. C. Haefliger: A. Employment (full or part-time); Significant; AstraZeneca. P05.55.A Pathogenic variant in the MYBPC3 gene with founding effect in Galician population

A. Blanco-Verea 1,2 , R. Cruz 3,2 , M. Álvarez-Barredo 4,1,5 , B. López-Abel 6,1 , R. Gil 1,2 , E. Ramos-Luis 1,2 , L. Martínez-Campelo 1,2 , I. Quintela 7,2 , J. González-Juanatey 4,5 , Á. Carracedo 8,2,3,7 , M. Brion 1,2,5

1 Xenética Cardiovascular, Instituto de Investigación Sanitaria de Santiago, Santiago de Compostela, Spain, 2 Grupo de Medicina Xenómica, Universidade de Santiago de Compostela, Santiago de Compostela, Spain, 3 CIBER Enfermedades Raras, Santiago de Compostela, Spain, 4 Complejo Hospitalario Universitario de Santiago, Servicio de Cardiología, Santiago de Compostela, Spain, 5 CIBER Cardiovascular, Santiago de Compostela, Spain, 6 Complejo Hospitalario Universitario de Santiago, Servicio de Pediatría, Santiago de Compostela, Spain, 7 Centro Nacional de Genotipado (CEGEN-PRB3-ISCIII), Santiago de Compostela, Spain, 8 Fundación Pública Galega de Medicina Xenómica, Santiago de Compostela, Spain
Introduction: The Myosin Binding Protein C3 gene (MYBPC3) is one of the main genes in which mutations involved in familial hypertrophic cardiomyopathy (HCM) appear. One of these pathogenic variants is the deletion rs397515926 that appears in the gnome AD database in only one individual of Latino origin (Allele Frequency=0.000004913). However, we have found 18 unrelated patients with HCM from Galicia with this variant, which has led us to think about a possible founding effect of this variant in the Galician population. Materials and Methods: Trying to unmask a possible founding effect or a distant family relationship between patients, we made a genotyping with the Applied Biosystems TM Axiom TM Spain Biobank Array in 6 of these patients and in a cohort of healthy Galician and Spanish population. Principal components analysis and discriminant analysis of principal components were performed in Plink and adegenet package of R software. Results: discriminant analysis of principal components taking into account rare variants of the array and comparing our patients with the general Spanish and Galician population clearly differentiate our patients with respect to the rest. Conclusions: Considering the results of the analysis of the genotypes between our cases and the general population, the practically null bibliographic mention and very scarce presence in databases of this variant, we could assume a possible common origin for the presence of this variant in the Galician population, highlighting a possible founding effect. Founding: Instituto de Salud Carlos III and Fondo Europeo de Desarrollo Regional (PI16/00903, CB16/11/00226, CB03/07/0088). A. Blanco-Verea: None. R. Cruz: None. M. Álvarez-Barredo: None. B. López-Abel: None. R. Gil: None. E. Ramos-Luis: None. L. Martínez-Campelo: None. I. Quintela: None. J. González-Juanatey: None. Á. Carracedo: None. M. Brion: None. P05.56.B The potential diagnostic value of circulating serum-derived miRNAs in ischemic heart disease (IHD) as a complication of type 2 diabetes mellitus (T2DM) .

A. Bielska 1 , W. Bauer 1,2 , I. Sidorkiewicz 1 , A. Szalkowska 1 , J. Raczkowska 1 , D. Ostrowski 1 , P. Prokopczuk 3 , S. Dobrzycki 3 , M. Niemira 1 , A. Kretowski 1,4

1 Clinical Research Centre, Medical University of Bialystok, Bialystok, Poland, 2 Clinical Research Centre, Medical University of Bialystok, Białystok, Poland, 3 Department of Invasive Cardiology, Medical University of Bialystok, Bialystok, Poland, 4 Department of Endocrinology, Diabetology and Internal Medicine, Medical University of Bialystok, Białystok, Poland
Introduction: Currently, IHD is the most common cause of death in patients with diabetes and it develops much earlier before clinical diagnosis. Therefore, there is an urgent need for the detection of sensitive and specific early biomarkers to predict the risk of the IHD development. We postulate that non-coding miRNAs may have higher diagnostic value for early detection of IHD than inflammatory mediators like chemokine CXCL12 and macrophage migration-inhibitory factor (MIF). Materials and Methods: We obtained serum samples from 43 T2DM patients (24 with IHD and 19 without IHD). The levels of 798 miRNAs were analysed using NanoString nCounter Technology. Additionally, we predicted the key miRNA regulatory network and miRNAs targets and pathways that may be critical in IHD development using the Ingenuity Pathway Analysis software. The concentration of MIF and CXCL12 was estimated by enzyme-linked immunosorbent assay. Receiver operating characteristic analysis was used to assess the diagnostic value of each miRNA. Results: Our data showed 9 miRNAs (miRNA-1224-5p, miRNA-1303, miRNA-3147, miRNA-4455, miRNA-498, miRNA-548b-3p, miRNA-548d-3p, miRNA-615-3p, miRNA-651-5p) significantly upregulated in T2DM IHD group compared to T2DM noIHD (FC>1.4 and FDR < 0.05). Furthermore, all miRNAs showed high diagnostic value (AUC>0.85). Targeted genes were identified in canonical pathways involved in the pathology of the cardiovascular system. No statistical differences were observed in the concentration of MIF and CXCL12 between tested groups. Conclusions: Our findings suggest that circulating miRNAs might have a pivotal role in the development of IHD in T2DM patients and may be used as non-invasively biomarkers for early diagnosis. A. Bielska: None. W. Bauer: None. I. Sidorkiewicz: None. A. Szalkowska: None. J. Raczkowska: None. D. Ostrowski: None. P. Prokopczuk: None. S. Dobrzycki: None. M. Niemira: None. A. Kretowski: None. P05.57.C Derivation and validation of a polygenic risk score associated with ischemic heart failure in multiethnic cohorts

I. Paranjpe 1 , J. De Freitas 1 , N. Tsao 2 , R. Judy 2 , K. Chaudhary 3 , I. Forrest 4 , CBIPM Genomics Team, Regeneron Genomics Team, S. Coca 5 , J. Narula 6 , B. Glicksberg 1 , R. Do 3,4 , S. Damrauer 2 , G. Nadkarni 1,3,7

1 The Hasso Plattner Institute for Digital Health at Mount Sinai, New York, NY, United States, 2 Department of Surgery, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States, 3 The Charles Bronfman Institute for Personalized Medicine, Icahn School of Medicine at Mount Sinai, New York, NY, United States, 4 Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, United States, 5 Division of Nephrology, Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, NY, United States, 6 Division of Cardiology, Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, NY, United States, 7 Renal Program, James J Peters VA Medical Center at Bronx, New York, NY, United States
Introduction: Mortality from heart failure (HF) secondary to coronary artery disease (CAD) is 50% and risk-stratification approaches are limited. We aimed to stratify risk of progression to HF in CAD patients by developing a polygenic risk score (PRS). Methods: Using summary statistics from a recent GWAS for HF secondary to CAD (ischemic HF), we computed risk scores by varying the rho parameter in LDPred and varying the p -value threshold and r 2 . We selected the PRS that maximized the association with ischemic HF (PRS iHF ) from the multiethnic Mount Sinai Bio Me Biobank(N=6276). We validated the association in Penn Medicine BioBank (PMBB; N=6674). We performed association analysis using logistic regression adjusted for age, sex, smoking history, hypertension, BMI, type 2 diabetes, and ten genetic principal components. Results: In Bio Me , one standard deviation (SD) increase in PRS iHF was significantly associated with HF (OR=1.1; p=0.001; 2530 cases, 3746 controls HF risk increased monotonically with PRS iHF in a subset of individuals with CAD but no evidence of obstructive CAD on coronary catheterization (N=4775). In PMBB, PRS iHF was associated with ischemic HF in European Americans (adjusted OR=1.07; p=0.02; 2692 cases, 2472 controls) but not African Africans (adjusted OR=0.94; p=0.3; 921 cases, 589 controls). Conclusions: We derived and validated a PRS for ischemic HF in two multiethnic cohorts and demonstrated association with and without obstructive CAD. PRS iHF replicated in patients of European ancestry, but not in African ancestry. Although studies on clinical utility and in more diverse populations are needed, our results suggest application in early risk stratification. I. Paranjpe: None. J. De Freitas: None. N. Tsao: None. R. Judy: None. K. Chaudhary: None. I. Forrest: None. S. Coca: None. J. Narula: None. B. Glicksberg: None. R. Do: None. S. Damrauer: None. G. Nadkarni: None. P05.58.A Identifying Long QT syndrome (LQTS) patients in primary care, an approach for rare diseases .

W. R. H. Evans 1 , S. F. Weng 1 , R. K. Akyea 1 , A. D. Simms 2 , J. Kai 1 , N. Qureshi 1

1 University of Nottingham, Nottingham, United Kingdom, 2 Department of Cardiology, Leeds Teaching Hospital NHS Trust, Leeds, United Kingdom
Introduction: LQTS is a rare (~1 in 2000 births) inherited cardiac arrhythmia that predisposes patients to syncope and sudden cardiac death. Identification and treatment can substantially reduce this risk. We aimed to identify the clinical features that precede the diagnosis of LQTS in primary care and use these to develop a predictive model. Methods: 1495 patients diagnosed with LQTS and 7475 propensity-score matched controls were identified in a database of primary care electronic records (CPRD). A range of clinical features that occurred at greater frequency in cases than control (p <0.05) before diagnosis were identified and incorporated into a multivariable logistic regression model. Results: 18 clinical features were incorporated into the final model, including: epilepsy (1.70 (1.12, 2.56)), palpitations (2.22 (1.80, 2.74)), collapse (1.636 (1.32, 2.03), mitral valve disease (2.64 (1.41, 4.91)), atrial fibrillation (1.91 (1.41, 2.60)) and IBS (1.78 (1.41, 2.26)) (OR (95% CI)). The discriminative accuracy of the model assessed by area under the curve (AUC) was 0.74, (95% CI 0.73 to 0.75), with comparable performance in two more tightly phenotyped subgroups: LQTS diagnosis <45 years and those commenced on a betablocker &/or fitted with an Implantable Cardioverter Defibrillator (ICD) post-diagnosis. Conclusion: This study demonstrates that it is possible to develop a predictive model for LQTS that performs with similar accuracy to other widely adopted cardiovascular risk models. Furthermore, it shows the potential of primary care records to phenotypically identify patients that are at an increased risk of having a rare disease. W.R.H. Evans: D. Speakers Bureau/Honoraria (speakers bureau, symposia, and expert witness); Modest; Intrabio inc. F. Consultant/Advisory Board; Modest; Mendelian.co. S.F. Weng: D. Speakers Bureau/Honoraria (speakers bureau, symposia, and expert witness); Modest; Amgen. R.K. Akyea: None. A.D. Simms: None. J. Kai: None. N. Qureshi: D. Speakers Bureau/Honoraria (speakers bureau, symposia, and expert witness); Modest; Amgen. P05.60.C Functional investigation of possible pathogenic EPHB4 variants associated with lymphatic related fetal hydrops and hereditary haemorrhagic telangiectasia

E. Sackey, S. Martin-Almedina, K. Ogmen, D. Grigoriadis, S. Mansour, P. Ostergaard

St. George’s University, London, United Kingdom
The use of next generation sequencing (NGS) technologies has increased the capacity of rare and novel variant detection. However, this brings challenges to confirm pathogenicity of the identified variants and evaluate them functionally, both needed for precise clinical diagnostics. This study investigates nine novel possible pathogenic nonsynonymous variants in the EPHB4 (Ephrin Receptor B4) gene. EPHB4 mutations have been associated with lymphatic related fetal hydrops (LRFH) and hereditary haemorrhagic telangiectasia (HHT) both with autosomal dominant inheritance. Non-immune fetal hydrops is characterised by fluid accumulation in at least two extravascular fetal compartments and about 15% of the cases result from a lymphatic abnormality. HHT presents commonly with arteriovenous malformations (AVMs) in the liver and lung, in connection with cutaneous and mucosal telangiectasias. Functional analysis including immunoprecipitation, western blotting, and immunofluorescence assays were performed on nine novel variants on EPBH4 to investigate their pathogenicity. This study demonstrates the pathogenic effect of eight variants out of nine. Most of the LRFH-associated EPHB4 variants presented similar protein expression levels compared to the wildtype protein but reduced tyrosine kinase activity while HHT-associated EPHB4 variants showed a reduction or total absence of protein expression. Based on these results, we suggest there could be two distinct EPHB4-associated disease phenotypes distinguished by two distinctive molecular mechanisms, but further research is necessary to elucidate this. E. Sackey: None. S. Martin-Almedina: None. K. Ogmen: None. D. Grigoriadis: None. S. Mansour: None. P. Ostergaard: None. P05.61.A Identifying metabolomic fingerprints of microRNAs in cardiovascular disorders

R. Mustafa 1 , M. Mens 2 , R. Pinto 1 , I. Karaman 1 , G. Roshchupkin 3,4 , J. Huang 1 , P. Elliott 1 , M. Evangelou 5 , A. Dehghan 1 , M. Ghanbari 2

1 Department of Epidemiology and Biostatistics, Imperial College London, London, United Kingdom, 2 Department of Epidemiology, Erasmus Medical Center, Rotterdam, Netherlands, 3 Department of Radiology and Nuclear Medicine, Erasmus Medical Center, Rotterdam, Netherlands, 4 Department of Medical Informatics, Erasmus Medical Center, Rotterdam, Netherlands, 5 Department of Mathematics, Imperial College London, London, United Kingdom
Introduction: microRNAs (miRNAs) are small non-coding RNAs that regulate gene expression. Despite an increasing evidence that supports miRNAs as potential biomarkers in cardiovascular disorders, the underlying mechanisms remain to be elucidated. We aimed to identify metabolites that mediate the effect of miRNAs on cardiovascular disorders. Methods: We performed two steps of two-sample Mendelian Randomization (MR) to study the causal association between miRNAs and metabolites, and between the metabolites and cardiovascular disorders. We conducted genome-wide association studies (GWAS) on miRNA expression data (N=1,687) in the Rotterdam Study to identify genetic instruments for miRNAs. MR was performed in a hypothesis-free manner between 591 miRNAs and 886 metabolites measured by the Metabolon platform. The association of the genetic instruments with metabolites were examined in the Airwave Study (N=1,942). We further performed MR to investigate the causal role of identified metabolites on cardiovascular disorders using publicly available GWAS summary results. Results: Our analysis indicated causal associations between 16 miRNAs and 21 metabolites after multiple testing correction. Among the identified metabolites, nominally significant associations were found between androsterone sulfate, cysteine sulfinic acid, and N-palmitoylglycine with stroke, and 3-hydroxy-5-cholestenoic acid with coronary artery disease. Taken together, we were able to identify metabolites that might link miR-1273h-5p, miR-3937, and miR-4753-5p with stroke, and miR-181a-2-3p with coronary artery disease. Conclusions: Our study has added insight into understanding the role of metabolites in the link between miRNAs and cardiovascular disorders. This approach could offer a way for finding candidate biomarkers and therapeutic targets in cardiovascular disorders. R. Mustafa: None. M. Mens: None. R. Pinto: None. I. Karaman: None. G. Roshchupkin: None. J. Huang: None. P. Elliott: None. M. Evangelou: None. A. Dehghan: None. M. Ghanbari: None. P05.62.B The detection of mosaic mutationsin hereditary aortic disease through next-generation sequencing

Z. G. Yan , H. Yang, Y. H. Zhang, Z. Zhou

State Key Laboratory of Cardiovascular Disease, Beijing Key Laboratory for Molecular Diagnostics of Cardiovascular Diseases, Diagnostic Laboratory Service, Fuwai Hospital, National Center for Cardiovascular Diseases, Chinese Academy of Medical Sciences, Beijing, China
Background: The mosaic carriers have been clinically normal or had only minor manifestations when the proportion of mutated cells is low, which makes it difficult to be detected. However, if the mutations were transmitted from asymptomatic carriers to their children, it will cause unexpected occurrence of diseases. So genetic tests are often required. Methods: We choosed 563 patients with suspected marfan syndrome (MFS) or thoracic aortic aneurysm/dissection, who had been detected by Panel. Among them, we selected 56 patients who carried likely pathogenic (pathogenic) mutations and were not de novo, their parents who harbored the same mutation were clinically normal. For these Trio families, we used the Life PGM TM DX platform to perform deep sequencing of single amplicon (approximately 5000×) on DNA samples extracted from whole blood. Results: We detected two mosaic mutations in these Trio families. The first patient AD700-1 who had typical MFS features carried the mutation FBN1 , C1305F. After deep sequencing,23% mutated alleles were detected in healthy father who harbored the same mutation. The second mutation FBN1 , c.2167+1G>A was identified in patient AD1833, who was suspected to be MFS. The mutation was also carried by healthy father, who was found to carry 17% of the mutant allele through deep sequencing. Conclusions: Mosaic mutations could be a more common phenomenon in aortic diseases than has been realized so far. Therefore, whenever children are identified with harmful mutations, the parents who lack obvious phenotypic characteristics should be detected by genetic testing, which will provide more accurate guidance for genetic counseling. Z.G. Yan: None. H. Yang: None. Y.H. Zhang: None. Z. Zhou: None. P05.63.C Familial aortic aneurysm syndrome caused by a +3 variant at a donor splice site of MYH11 .

B. Chesneau 1,2 , A. Plancke 1 , G. Rolland 1 , B. Marcheix 3 , Y. Dulac 2 , T. Edouard 2 , M. Aubert-Mucca 2,4 , S. Julia 4 , M. Langeois 2,4 , V. Gaston 4 , P. Khau Van Kien 1

1 Unité de génétique médicale et cytogénétique, Hôpital Carémeau, CHU de Nîmes, Nîmes, France, 2 Centre de Référence du syndrome de Marfan et des syndromes apparentés, Hôpital des Enfants, CHU de Toulouse, Toulouse, France, 3 Département de chirurgie cardiaque, Hôpital Universitaire de Rangueil, Toulouse, France, 4 Service de génétique médicale, Hôpital Universitaire de Purpan, Toulouse, France
Pathogenic variants in the myosin smooth muscle heavy chain 11 ( MYH11 ) are associated with familial aortic aneurysm (FAA) and patent ductus arteriosus (PDA) syndrome. We report a family with two siblings who presented with type A aortic dissections. The sister made an aortic dissection at 45 years-old. The brother made an aortic dissection at 22 years-old treated with an aortic graft procedure, at 46 years-old he had revision surgery because of pseudoaneurysms complications. His daughter was diagnosed with a patent ductus arteriosus . Their father suddenly died at 28 years-old. Molecular analysis of a panel of 15 aortic aneurysm genes in the brother revealed only the variant c.4599+3A>C at the 5’ donor splice site (5’ss) of the exon 33 of MYH11 (NM_00104114.1) with a potential splicing impact. This variant was also present in the affected sister. It was absent from GnomAD and internal variant database (>1000 patients) and never reported in the literature. Interestingly, a single-nucleotide polymorphism is described at this position: c.4599+3A>G (rs143288748). Interpretation of variations at the position +3 of the 5’ss is difficult because data of the literature are conflicting with variations reported to induce aberrant splicing in some cases but not in others. We performed transcript analysis with RNA extracted from an aortic biopsy during the brother’s second intervention. RT PCR revealed a skipping of the exon 33 of MYH11 predicting a truncated myosin tail and a dominant negative effect. This report confirms genotype-phenotype correlations for MYH11 and FAA/PDA. B. Chesneau: None. A. Plancke: None. G. Rolland: None. B. Marcheix: None. Y. Dulac: None. T. Edouard: None. M. Aubert-Mucca: None. S. Julia: None. M. Langeois: None. V. Gaston: None. P. Khau Van Kien: None. P05.64.A A founder pathogenic variant in MYH7 causing hypertrophic cardiomyopathy in Catalonia

P. Fernández-Álvarez 1 , M. Codina 1 , J. Limeres 2 , C. Serra-Juhé 3 , M. Masas Castro 1 , A. Cordero Lopez 1 , E. García-Arumí 1,4 , E. Tizzano 1,4

1 Department of Clinical and Molecular Genetics and Medicine Genetics Group, VHIR, University Hospital Vall d’Hebron, Barcelona, Spain, 2 Department of Cardiology, Hospital Universitari Vall d’Hebron, Barcelona, Spain, 3 Department of Genetics, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain, 4 CIBERER, Centro de Investigación Biomédica en Red de Enfermedades Raras, ISCIII, Madrid, Spain
Pathogenic variants in MYH7 are the second most common cause of inherited hypertrophic cardiomiopathy (HCM). 262 unrelated HCM probands were screened using the Illumina TruSightCardio kit. Twenty-nine patients were heterozygous for MYH7 pathogenic variants, 6 (21%) of them had the same variant c.2674C>A p.(Gln892Lys). This variant has been previously described as a VUS in a HCM patient which harbored another pathogenic variant in MYH7 and in ClinVar, and is absent in GnomAD. In all our six patients p.(Gln892Lys) was the only probably pathogenic variant found. Patients (4 males/2 females) were of Catalan origin and had been diagnosed of HCM around 30 years-old. Familial sudden death before the fifth decade of life were reported. We extended the genetic and cardiologic study to 20 relatives of the probands. Twelve were carriers, 9 of them symptomatic and p.(Gln892Lys) co-segregated with the HCM in all the families. We studied the possibility of a founder effect by haplotype analysis using the SNP data from the TruSightCardio kit. We used 14 SNPs, flanking an area of 49,35 kb before MYH6 and after MYH7 . A common haplotype with a calculated haplotype frequency in Iberia population of 0.0187 was shared in the 6 patients and was not found in our remaining 256 patients with HCM. Our results indicate a possible founder effect of this MYH7 pathogenic variant in HCM patients of Catalan origin. P. Fernández-Álvarez: None. M. Codina: None. J. Limeres: None. C. Serra-Juhé: None. M. Masas Castro: None. A. Cordero Lopez: None. E. García-Arumí: None. E. Tizzano: None. P05.65.B Biallelic variants in MYL3 cause autosomal recessive cardiomyopathy

D. P. S. Osborn 1 , L. Emrahi 2 , M. T. Tabrizi 3 , M. Yazdchi 3 , N. Mazaheri 4 , A. Mitchell 5 , C. Hesse 6 , H. Goullée 7 , D. Burkardt 5 , N. Laing 7 , A. Y. B. Wan 1 , H. Galehdari 4 , R. Maroofian 1 , G. Shariati 4 , H. Tajsharghi 8 , Y. Jamshidi 1

1 Genetics Centre, London, United Kingdom, 2 Tarbiat Modares University, Tehran, Iran, Islamic Republic of, 3 Tabriz University of Medical Sciences, Tabriz, Iran, Islamic Republic of, 4 Shahid Chamran University of Ahvaz, Ahvaz, Iran, Islamic Republic of, 5 Case Western Reserve University, Cleveland, OH, United States, 6 University of Gothenburg, Gothenburg, Sweden, 7 The University of Western Australia and the Harry Perkins Institute of Medical Research, Nedlands, Australia, 8 University of Skovde, Skovde, Sweden
MYL3 encodes the slow-twitch specific myosin essential light chain (ELC), and pathogenic variants in this gene are a rare cause of hypertrophic cardiomyopathy. The majority of cases reported in the literature involve MYL3 missense variants transmitted in an autosomal dominant mode of inheritance. Here, through exome sequencing of four unrelated consanguineous families we identify biallelic MYL3 sequence variants. Affected individuals present with hypertrophic or dilated cardiomyopathy of variable severity from early- to late-onset of cardiomyopathy, and sudden cardiac death in 10 individuals. We report homozygous and compound heterozygous missense variants p.Ala57Asp, p.Met173Lys and p.Glu177Gly, a novel homozygous translational stop codon variant p.Glu36Ter, and a homozygous splice variant c.482-1G>A in MYL3 . G0 zebrafish larvae with CRISPR/Cas9 gene disruption or transient knockdown of the major ELC orthologue in zebrafish, cmlc1, display significantly larger ventricular chamber volume and compromised cardiac function, which cannot be rescued by re-introduction of human mutant RNA. Our genetic and functional data expand the mutational and phenotypic spectrum of MYL3 variants, identifying for the first time a homozygous loss of function variant, and highlighting the role of MYL3 in recessive hypertrophic and dilated cardiomyopathy with occurrence of sudden cardiac death. D.P.S. Osborn: None. L. Emrahi: None. M.T. Tabrizi: None. M. Yazdchi: None. N. Mazaheri: None. A. Mitchell: None. C. Hesse: None. H. Goullée: None. D. Burkardt: None. N. Laing: None. A.Y.B. Wan: None. H. Galehdari: None. R. Maroofian: None. G. Shariati: None. H. Tajsharghi: None. Y. Jamshidi: F. Consultant/Advisory Board; Modest; DiNAQOR AG. P05.66.C Impact of abdominal obesity on postprandial lipid concentrations

M. R. Christiansen 1 , T. I. A. Sorensen 1,2 , T. Hansen 1 , T. O. Kilpeläinen 1

1 Novo Nordisk Foundation Center for Basic Metabolic Research, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark, 2 Department of Public Health, Section of Epidemiology, University of Copenhagen, Copenhagen, Denmark
Introduction: Humans spend most of the day in a post-absorptive state, and thus prolonged and high postprandial lipemia can have major implications for the risk of cardiovascular disease. Abdominal adiposity may influence postprandial lipemia by influencing lipid metabolism after a meal. To date, >400 genetic loci have been discovered for association with abdominal adiposity, assessed by waist-hip ratio adjusted for body mass index (WHRadjBMI). Whether these variants are associated with postprandial lipid concentrations, remains unknown. We thus examined whether genetic risk score for increased waist-hip ratio adjusted for body mass index (WHRadjBMI) is associated with lipid concentrations at fasting state and after a high-fat meal. Materials and Methods: 771 individuals of European ancestry and with obesity from the NUGENOB study consumed a liquid high-fat meal (95% fat of the energy content). The postprandial concentrations of HDL cholesterol, LDL cholesterol, free fatty acids, glycerol and triglycerides were measured at fasting state and postprandially for 3 hours. The associations between the WHRadjBMI score and lipid concentrations were examined using linear models. Results: At fasting state, the genetic risk score for WHRadjBMI was associated with higher fasting triglycerides (P=0.0013) and lower HDL cholesterol (P=0.0059). Postprandially, the genetic risk score was not associated with changes in triglycerides or HDL cholesterol (P>0.05), but was significantly associated with elevated glycerol concentrations (P=0.012). Conclusions: Genetic predisposition to abdominal adiposity is associated with elevated blood glycerol concentration after a high-fat meal. M.R. Christiansen: None. T.I.A. Sorensen: None. T. Hansen: None. T.O. Kilpeläinen: None. P05.68.B Familial pulmonary arterial hypertension by KDR heterozygous loss of function

M. EYRIES 1 , D. Montani 2 , B. Girerd 2 , N. Favrolt 3 , M. Riou 4 , L. Faivre 5 , G. Manaud 6 , F. Perros 6 , S. Gräf 7 , N. Morrell 8 , M. Humbert 2

1 Département de génétique, Hôpitaux Universitaires Pitié Salpêtrière - Charles Foix,, Paris, France, 2 Service de Pneumologie, Centre de Référence de l’Hypertension Pulmonaire, Hôpital Bicêtre, Le Kremlin Bicêtre, France, 3 Service de Pneumologie et Soins Intensifs Respiratoires, Centre de référence constitutif des maladies pulmonaires rares de l’adulte, Centre de compétence de l’hypertension pulmonaire, CHU Dijon-Bourgogne, Dijon, France, 4 Service de Pneumologie, Nouvel hôpital civil, Strasbourg, France, 5 Centre de génétique, FHU TRANSLAD, Institut GIMI et UMR INSERM 1231, CHU de Dijon et Université de Bourgogne, Dijon, France, 6 UMR_S 999, Université Paris-Saclay, INSERM, Hôpital Marie Lannelongue, Le Plessis Robinson, France, 7 NIHR Bioresource – Rare Diseases, Department of Medicine, Department of Haematology, Cambridge Biomedical Campus, Cambridge, United Kingdom, 8 Department of Medicine, University of Cambridge School of Clinical Medicine, Addenbrooke’s and Royal Papworth Hospitals, Cambridge, United Kingdom
Introduction: Pulmonary arterial hypertension (PAH) is a rare and devastating disease, resulting from progressive obliteration of small caliber pulmonary arteries leading to cardiac failure. PAH can occur in the context of family history or genetic mutations causing heritable PAH, or is considered as idiopathic in the absence of an identified predisposing factor. Beyond the major gene BMPR2, a gene encoding a type 2 receptor of the BMP signaling pathway several new genes predisposing to PAH have been identified during the last decade. Recently, preliminary evidence of the involvement of the KDR gene was found in a large genetic association study. Methods: We prospectively analysed the KDR gene by targeted panel NGS sequencing in a series of 311 PAH patients referred to a clinical molecular laboratory for genetic diagnosis of PAH. Results: Two index cases with severe PAH were found to carry a loss-of-function mutation in the KDR gene. These two patients were clinically characterized by low diffusing capacity for carbon monoxide adjusted for hemoglobin (DLCOc) and radiological evidence of parenchymal lung disease. Segregation analysis in one family revealed that all KDR mutations carriers have a low DLCOc that is more pronounced in affected subjects compared to non-affected Conclusions: Our study provided genetic evidence for considering KDR as a newly identified PAH causing gene since we describe the segregation of KDR mutations with PAH in two families. Furthermore, KDR mutations are associated with a particular form of PAH characterized by low DLCOc and parenchymal lung disease. M. Eyries: None. D. Montani: None. B. Girerd: None. N. Favrolt: None. M. Riou: None. L. Faivre: None. G. Manaud: None. F. Perros: None. S. Gräf: None. N. Morrell: None. M. Humbert: None. P05.70.A PHACTR1 mRNA expression is affected by rs9349379 in PBMC of patients six months after the first MI

J. Kuveljic 1 , T. Djuric 1 , M. Dekleva 2 , N. Markovic Nikolic 2 , A. Stankovic 1 , D. Alavantic 1 , M. Zivkovic 1

1 Vinca Institute of Nuclear Sciences, Laboratory for Radiobiology and Molecular Genetics, University of Belgrade, Belgrade, Serbia, Belgrade, Serbia, 2 Department of Cardiology, University Clinical Center “Zvezdara”, Belgrade, Serbia; Faculty of Medicine, University of Belgrade, Belgrade, Serbia, Belgrade, Serbia
Aim: Phosphatase and actin regulator 1 (PHACTR1) was at first associated with early onset myocardial infarction (MI). Later on, it was associated with other vascular disorders, but still the mechanism of its effect is unknown. Rs9349379 is an eQTL for PHACTR1 in coronary arteries, where A allele leads to higher expression. Also PHACTR1 mRNA expression is elevated in the heart after MI, indicating its up-regulation in pathological conditions. The aim of our study was to investigate the association of rs9349379 with the first MI in Serbian population and its effect on PHACTR1 mRNA expression in PBMC of MI patients and controls. Methods: Blood samples from 171 controls and 156 patients with the first MI were obtained for DNA extraction. For RNA extraction, we collected 32 control samples and 65 samples of patients six months after the first MI. Genotyping and expression analysis were performed with Taqman technology. Statistical analyses were done by Statistica 8 software. Results: The association of rs9349379 with the first MI was not significant (p=0.13). PHACTR1 mRNA expression was significantly higher in MI patients compared to controls (p=0.02). We have found that rs9349379 has no effect on PHACTR1 mRNA expression in control group (p=0.90), while AA genotype led to higher expression in patient group (p=0.04). Conclusions: Rs9349379 affects PHACTR1 mRNA expression in PBMC of MI patients, six months after the MI. In order to accurately estimate its association with MI, further analysis and larger sample size are needed. Funded by Serbian Ministry, grant III41028. J. Kuveljic: None. T. Djuric: None. M. Dekleva: None. N. Markovic Nikolic: None. A. Stankovic: None. D. Alavantic: None. M. Zivkovic: None. P05.71.B Polygenic risk scores for hypertension risk stratification

O. Borisov 1 , E. Salvi 2 , L. Citterio 3 , C. Lanzani 3 , C. Barlassina 4 , N. Glorioso 5 , P. Krawitz 1 , D. Cusi 4 , P. Manunta 3 , C. Maj 1

1 Institute for Genomic Statistics and Bioinformatics, Bonn, Germany, 2 Neuroalgology Unit, Fondazione IRCCS Istituto Neurologico “Carlo Besta”, Milan, Italy, 3 Genomics of Renal Diseases and Hypertension Unit, Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS) San Raffaele Scientific Institute; Vita-Salute San Raffaele University, Milan, Italy, 4 Dept. Of Health Sciences, University of Milan, Milan, Italy, 5 Hypertension and related diseases Centre Department of Clinical and Experimental Medicine University of Sassari Italy, Sassari, Italy
Introduction: Hypertension (HTN) is a major cardiovascular risk factor and responsible for ~50% of cardiovascular morbidity and mortality. Blood pressure (BP) is also a complex genetic trait with heritability estimates of 30-50%. Genome-wide association studies (GWAS) have identified a high number of genetic loci associated with HTN and BP traits, indicating underlying polygenic architectures. This study proposes to compute polygenic risk scores (PRS) for HTN and BP for potential genetic risk stratification. Materials and Methods: We investigated the additive effect of common genetic variants on HTN and BP by means of regression models based on PRS. We used genotyping data of 561 cases and 731 controls from Hypergenes cohort. We computed PRS based on clumping and thresholding method using GWAS summary statistics of systolic/diastolic BP measurements from GERA cohort (99,785 health records) and the HTN case-control cohort of the UK Biobank (144,793 cases and 313,761 controls). Results: PRS for HTN and diastolic/systolic BP are strongly associated with hypertensive status and BP measurements. The highest significance is achieved for the case-control status by considering UK Biobank effect sizes (p=6.4e-28, R2=0.09). The area under the curve is 0.68. The model remains significant after adjusting for sex and body mass index (p=4.1e-27). Conclusions: PRS modeling shows a strong polygenic component both for HTN status and for BP levels. Despite the limited predictive accuracy expected for BP, which is largely influenced by environmental factors, the strength of associations suggests that PRS could be potentially used for genetic risk stratification. O. Borisov: None. E. Salvi: None. L. Citterio: None. C. Lanzani: None. C. Barlassina: None. N. Glorioso: None. P. Krawitz: None. D. Cusi: None. P. Manunta: None. C. Maj: None. P05.72.C Biallelic variants in PPP1R13L cause paediatric dilated cardiomyopathy

H. K. Robinson 1 , E. Zaklyazminskaya 2 , I. Povolotskaya 3 , Y. Surikova 4 , L. Mallin 1 , C. Armstrong 5 , M. R. Chrisant 6 , P. J. Benke 7 , M. McDonald 8 , C. C. Marboe 9 , K. McWalter 10 , G. Douglas 10 , K. Agre 11 , D. Deyle 11 , K. M. Balashova 3 , V. Kaimonov 3 , N. Shirokova 3 , E. Pomerantseva 3 , C. Turner 12 , S. Ellard 1,13

1 Exeter Genomics Laboratory, Exeter, United Kingdom, 2 Center of Syncope and Cardiac Arrhythmias in Children and Adolescents, Moscow, Russian Federation, 3 Centre of Genetics and Reproductive Medicine “Genetico”, Moscow, Russian Federation, 4 B.V. Petrovsky Russian National Centre of Surgery, Moscow, Russian Federation, 5 Paediatric Cardiac Service, Bristol Royal Hospital for Children, Bristol, United Kingdom, 6 Joe DiMaggio Children’s Hospital, Hollywood, FL, United States, 7 Charles E. Schmidt College of Medicine, Florida Atlantic University, Hollywood, FL, United States, 8 Department of Pediatrics, Division of Medical Genetics, Duke University Medical Center, Durham, NC, United States, 9 Department of Pathology and Cell Biology, Columbia University Medical Centre, New York, NY, United States, 10 GeneDx Inc., Gaithersburg, MD, United States, 11 Mayo Clinic, Department of Clinical Genomics, Rochester, MN, United States, 12 Peninsula Clinical Genetics Service, Department of Clinical Genetics, Royal Devon and Exeter NHS Foundation Trust, Exeter, United Kingdom, 13 Institute of Biomedical and Clinical Science, College of Medicine and Health, Exeter, United Kingdom
Introduction: Childhood DCM is a leading cause of heart failure requiring cardiac transplantation, with ~5% of cases resulting in sudden death. Knowledge of the underlying genetic cause can aid prognostication and clinical management and enables accurate recurrence risk counselling. Here our aim was to identify the causative genetic variant(s) in families with children affected by severe DCM. Materials and Methods: Exome/genome sequencing was performed on leukocyte DNA from seven children affected with severe DCM and their unaffected parents from five independent families, followed by inheritance-based gene-agnostic variant filtering. Families were brought together by an international collaborative effort facilitated by GeneMatcher. Results: DCM presented early (range: 3 months – 9 years) and was progressive, resulting in death ( n = 3) or transplant ( n = 3), with one child currently awaiting transplant. Biallelic variants in PPP1R13L were identified in the affected children in each family. PPP1R13L encodes inhibitor of apoptosis-stimulating protein of p53 protein (iASPP). iASPP has roles in apoptosis, acts as a regulator of desmosomes and has been implicated in inflammatory pathways. Spontaneous biallelic loss-of-function variants in PPP1R13L orthologues have been shown to underlie bovine and murine models of DCM and biallelic variants were previously reported in a single consanguineous family with paediatric DCM. Conclusions: Gene-agnostic exome/genome sequencing is valuable for the identification of novel and emerging candidate genes. The identification of these five families now provides sufficient evidence to support a robust gene-disease association between PPP1R13L and severe paediatric DCM. PPP1R13L is therefore recommended for inclusion in panel-based genetic testing for paediatric DCM. H.K. Robinson: None. E. Zaklyazminskaya: None. I. Povolotskaya: None. Y. Surikova: None. L. Mallin: None. C. Armstrong: None. M.R. Chrisant: None. P.J. Benke: None. M. McDonald: None. C.C. Marboe: None. K. McWalter: A. Employment (full or part-time); Significant; GeneDx Inc. G. Douglas: A. Employment (full or part-time); Significant; GeneDx Inc.. K. Agre: None. D. Deyle: None. K.M. Balashova: None. V. Kaimonov: None. N. Shirokova: None. E. Pomerantseva: None. C. Turner: None. S. Ellard: None. P05.75.C Identification of ANGPT2 mutations as a novel cause of primary lymphedema

V. Leppänen 1 , P. Brouillard 2 , E. Korhonen 1 , N. Revencu 3 , G. Koh 4 , P. Saharinen 1 , K. Alitalo 1 , M. Vikkula 2,5

1 Wihuri Research Institute, Biomedicum Helsinki, Helsinki, Finland, 2 Human Molecular Genetics, de Duve Institute, UClouvain, Brussels, Belgium, 3 Center for Human Genetics, Cliniques universitaires Saint-Luc, UCLouvain, Brussels, Belgium, 4 Center for Vascular Research, Korea Advanced Institute of Science and Technology (KAIST), Daejoen, Korea, Republic of, 5 Walloon Excellence in Lifesciences and Biotechnology (WELBIO), UCLouvain, Brussels, Belgium
Purpose: Primary lymphedema (PLE) is caused by developmental and functional defects of the lymphatic system resulting in accumulation of protein-rich fluid in tissues. The 28 currently known genes causing lymphedema explain <30% of cases. We search for additional genes to better understand pathophysiology. Methods: We used WES to screen 543 patients within our cohort of >900 index patients. We filtered variants using our in-house developed Highlander software. In vitro and in vivo studies were performed to validate functional effects. Results: We identified a complete angiopoietin-2 ( ANGPT2) gene deletion in one family, and four variants of interest in four other families. All are extremely rare or absent in controls and databases, and predicted to be pathogenic by several algorithms. Functional analyses of the substitution-mutants unraveled that 3/4 showed decreased ANGPT2 secretion and even inhibited secretion of the wild-type ANGPT2, suggesting that they have a dominant-negative effect. The fourth mutant, located in the dimerization interphase, showed reduced integrin-α5 binding and its expression in mouse skin promoted hyperplasia and dilation of cutaneous lymphatic vessels. Conclusions: We identified five mutations in the TIE2-ligand ANGPT2 as a novel cause of primary lymphedema. The mutations alter ANGPT2 structure and/or function. The data underscore loss of ANGPT2 function as the mechanism leading to primary lymphedema, and provide new insights into TIE2 signaling. The ANGPT2 gene is now included in the diagnostic panel used in our hospital to perform diagnostic testing for primary lymphedema. These studies were supported by Walloon Excellence in Lifesciences and Biotechnology (WELBIO). V. Leppänen: None. P. Brouillard: None. E. Korhonen: None. N. Revencu: None. G. Koh: None. P. Saharinen: None. K. Alitalo: None. M. Vikkula: None. P05.76.A Novel variants in ABIN2 and TRAF2 implicated in pathogenesis of pulmonary arterial hypertension

N. Gallego 1 , D. Condon 2 , S. Pienkos 2 , V. de Jesús Pérez 2 , I. Hernández 3 , C. Pérez-Olivares 4 , N. Ochoa 4 , P. Spanish Consortium 5 , P. Lapunzina 6 , P. Escribano 7 , J. Tenorio 6

1 Instituto de Genética Médica y Molecular, Madrid, Spain, 2 Stanford University, Standford, CA, United States, 3 Hospital Universitario Fundación Jiménez Díaz, Madrid, Spain, 4 Hospital Universitario Doce de Octubre, Madrid, Spain, 5 Instituto de Genética Médica y Molecular, Centro de Investigación Biomédica en Red de Enfermedades Raras, Madrid, Spain, 6 Instituto de Genética Médica y Molecular, Centro de Investigación Biomédica en Red de Enfermedades Raras, Madrid, Spain, 7 Hospital Universitario Doce de Octubre, Centro de Investigación Biomedica en Red en Enfermedades Cardiovasculares, Madrid, Spain
Introduction: Pulmonary Arterial Hypertension (PAH) is an infrequent cardiopathy with poor prognosis despite therapeutic advances. Mutation in several known risk genes predispose to disease however, some cases remain genetically undefined. Increasing knowledge of the underlaying molecular mechanisms may lead to a better understanding of the disease and the opening of new drug targets that can improve the treatment of patients. The advance in massive paralleled sequencing technologies has allowed improving the characterization of the molecular profiling of the patients and a better clinical characterization. Material and methods: The aim of this project is to study 20 patients and available relatives by whole exome sequencing (WES) technology in order to identify new genes and regions that can be involved in PAH. Variant prioritization was performed through bioinformatic tools and a custom algorithm. In addition, to stablish mechanistic significance, a series of in vitro studies were performed. Results: Several candidate genes were identified after the variant prioritization. We have identified two genes ( TRAF2 and TNIP2 ) in two unrelated families, which are related to the NFƙβ pathway, associated with the progression of PAH. In vitro studies demonstrate a possible impairment of NFƙβ transcription factors localization of a modulation of NFƙβ-inducible genes after inhibition of ABIN2 and TRAF2. Conclusions: Although these results have to be validated through more different functional assays, they suggested an association between disease and new genes which may also be considered for inclusion in genetic screening for PAH patients. Grants: FIS-PI18/01233, Janssen unrestricted grant, FCHP . N. Gallego: None. D. Condon: None. S. Pienkos: None. V. de Jesús Pérez: None. I. Hernández: None. C. Pérez-Olivares: None. N. Ochoa: None. P. Spanish Consortium: None. P. Lapunzina: None. P. Escribano: None. J. Tenorio: None. P05.77.B Association of two novel SCN5A intragenic duplications with epilepsy, ventricular tachycardia, fibrillation and Brugada-like Syndrome

N. Marziliano 1 , A. Medoro 2 , D. Fiscella 3 , A. Fiscella 4 , L. Tursi 1 , L. Greco 1 , C. Reverberi 5 , S. Folzani 6 , M. Intrieri 2

1 ASL TARANTO, Taranto, Italy, 2 Università degli Studi del Molise, Campobasso, Italy, 3 Ospedale Garibaldi, Catania, Italy, 4 Fondazione Floresta Longo, Catania, Italy, 5 Centro Clinico Gemini, Parma, Italy, 6 Poliambulatorio Città di Collecchio, Collecchio, Italy
In the last years, the increasing evidences about the co-existence of genetically based cardiac arrhythmias and epilepsy brings out the emerging concept of cardio-cerebral channelopathy. In this view, we describe two unrelated cases presenting cardiac and epileptic phenotypes in which two novel intragenic duplications in the SCN5A gene (c.1627_1653dup, p.Phe543_Ala551dup and c.1846_1863dup, p.Ser616_Pro621dup, respectively) were identified. The SCN5A gene encodes for the α-subunit of the Na v 1.5 channel and mutations in this gene are mainly involved in electrical channelopathies that lead to life-threatening arrhythmias (Brugada syndrome, Long-QT syndrome, Sudden Infant Death syndrome, etc.) or structural changes in the myocardium, such as in the dilated cardiomyopathy (DCM). With the aim of characterizing the biological effects of such genetic defects, we studied the biophysical properties of the Na v 1.5 channels revealing that duplication might prompt for a loss-of-function of the Na v 1.5-mediated current in a zebrafish model without alterations in the heart structure and function (at least for the first mutation). Taking together, clinical and biochemical data support the idea that the intragenic in-frame duplications within the SCN5A gene could be responsible for both cardiac and epileptic phenotypes. N. Marziliano: None. A. Medoro: None. D. Fiscella: None. A. Fiscella: None. L. Tursi: None. L. Greco: None. C. Reverberi: None. S. Folzani: None. M. Intrieri: None. P05.78.C Clonal occurrence of the Hutchinson-Gilford progeria syndrome lamin A mutation in chronic kidney disease

G. Revechon 1 , H. Helgadottir 1 , N. Viceconte 1 , A. Witasp 2 , A. Sola Carvajal 1 , D. Whisenant 1 , D. McGuinness 3 , G. Artiach 4 , E. Wallen Arzt 1 , A. Thorell 5 , A. Babler 6 , S. Ziegler 6 , R. Kramann 6 , M. Bäck 4 , P. G. Shiels 3 , A. Wernerson 2 , P. Stenvinkel 2 , M. Eriksson 1

1 Karolinska Institute, Dept of Biosciences and Nutrition, Huddinge, Sweden, 2 Karolinska Institute, Dept of CLINTEC, Huddinge, Sweden, 3 University of Glasgow, Institute for Cancer Sciences, Glasgow, United Kingdom, 4 Karolinska Institute, Dept of Medicine, Stockholm, Sweden, 5 Karolinska Institute, Dept of Clinical Sciences, Dept of Surgery, Ersta hospital, Stockholm, Sweden, 6 University hospital RWTH Aachen, Aachen, Germany
Somatic mutagenesis occurs continuously across the life course, resulting in tissues composed of clones with different genetic makeups. While the majority of mutations have no functional significance, some can contribute to disease and aging. The nature of the arterial wall, with its media being composed of clonal smooth muscle cells, makes this tissue particularly sensitive to somatic mutagenesis. Here we analyzed progerin, the protein responsible for the premature aging disorder Hutchinson-Gilford progeria syndrome (HGPS), for its possible involvement in the accelerated vascular aging process observed in chronic kidney disease (CKD), due to similarities in vascular phenotypes. We provide evidence for expression of progerin, and the occurrence of the HGPS-causing LMNA c.1824C>T mutation as a somatic mutation in CKD patient arteries. Clonal populations of vascular cells with the 1824C>T mutation was supported by clusters of progerin positive cells in the media and a high frequency of the mutant allele in several CKD arteries. Further analysis of proliferation and DNA damage in the CKD arteries indicated regeneration and progerin-induced genomic damage. Our results implicate somatic mutations in the functional decline of the vascular wall and support that somatic mutations may become a risk factor during extensive tissue damage. G. Revechon: None. H. Helgadottir: None. N. Viceconte: None. A. Witasp: None. A. Sola Carvajal: None. D. Whisenant: None. D. McGuinness: None. G. Artiach: None. E. Wallen Arzt: None. A. Thorell: None. A. Babler: None. S. Ziegler: None. R. Kramann: None. M. Bäck: None. P.G. Shiels: None. A. Wernerson: None. P. Stenvinkel: None. M. Eriksson: None. P05.79.A Enrichment of rare variants in Loeys-Dietz syndrome genes in spontaneous coronary artery dissection but not in severe fibromuscular dysplasia

J. Meester 1 , A. Verstraeten 1 , M. Perik 1 , L. Van Den Heuvel 1 , M. Kempers 2 , I. Krapels 3 , L. Van Laer 1 , N. Samani 4 , F. Van Dijk 5 , A. Persu 6 , D. Adlam 4 , B. Loeys 1

1 Centre of Medical Genetics, University of Antwerp, Antwerp University Hospital, Antwerp, Belgium, 2 Department of Human Genetics, Radboud University Nijmegen Medical Center, Nijmegen, Netherlands, 3 Department of Clinical Genetics, Maastricht University Medical Centre, Maastricht, Netherlands, 4 Department of Cardiovascular Sciences and National Institute for Health Research Leicester Biomedical Research Centre, Glenfield Hospital, Leicester, United Kingdom, 5 Ehlers-Danlos Syndrome, National Diagnostic Service, Northwick Park and St. Mark’s Hospitals, Harrow, United Kingdom, 6 Pole of Cardiovascular Research, Institut de Recherche Expérimentale et Clinique, Université Catholique de Louvain, and Division of Cardiology, Cliniques Universitaires Saint-Luc, Université Catholique de Louvain, Brussels, Belgium
Spontaneous coronary artery dissection (SCAD) is characterized by separation of the inner intimal lining of the coronary artery from the outer vessel wall. It is the prime cause of acute myocardial infarction in women below the age of 50. Fibromuscular dysplasia (FMD) presents with condensed areas of arterial stenosis, aneurysm, tortuosity and dissection. It has become clear that FMD is prevalent in SCAD cohorts and vice versa. However, the genetic etiology of both conditions remains largely elusive. We aimed to investigate the contribution of rare genetic variants in vascular Ehlers-Danlos and the Marfan, Loeys-Dietz (LDS), Shprintzen-Goldberg syndrome genes to the etiology of SCAD and FMD in a large multi-center patient cohort. Haloplex-based gene panel sequencing of COL3A1 , FBN1 , TGFB2/3, SMAD2/3, TGFBR1/2 and SKI was performed in 191 SCAD patients with or without FMD and 111 isolated FMD patients. Subsequent variant filtering was performed. The gnomAD database was used as an independent control dataset and filtered identically. Case-control burden analyses were performed. Rare variants in SMAD2 are significantly enriched in SCAD patients compared to controls (p=0.001). A combined LDS-gene burden analysis was performed, revealing a highly significant association (4.5% vs 1.5%; p=0.000003). When performed for FMD, none of the analyses yielded a significant p -value or showed a trend. We here provide evidence for an important contribution of rare genetic variants in LDS genes to the etiology of SCAD. Our findings suggest a strong role for the TGFbeta signaling in the pathogenesis of SCAD but not FMD. J. Meester: None. A. Verstraeten: None. M. Perik: None. L. Van Den Heuvel: None. M. Kempers: None. I. Krapels: None. L. Van Laer: None. N. Samani: None. F. Van Dijk: None. A. Persu: None. D. Adlam: None. B. Loeys: None. P05.80.B Genetic association study of eight steroid hormones and implications for sexual dimorphism of coronary artery disease

J. Pott 1,2,3 , Y. J. Bae 4,2 , K. Horn 1,2 , A. Teren 2,5 , A. Kühnapfel 1,2,3 , H. Kirsten 1,2 , U. Ceglarek 4,2 , M. Loeffler 1,2 , J. Thiery 4,2 , J. Kratzsch 4,2 , M. Scholz 1,2,3

1 Institute of Medical Informatics, Statistics und Epidemiology, University of Leipzig, Leipzig, Germany, 2 LIFE Research Center for Civilization Diseases, University of Leipzig, Leipzig, Germany, 3 IFB AdiposityDiseases, University Hospital, Leipzig, Germany, 4 Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, University Hospital, Leipzig, Germany, 5 Heart Center Leipzig, Leipzig, Germany
Introduction: Steroid hormones act as important regulators of physiological processes in the body. Our aim is to identify genetic factors that influence steroid hormone metabolism, to detect genetic sexual dimorphisms and to investigate possible causal implications for atherosclerotic vascular disease phenotypes. Materials and Methods: In two independent cohorts, we performed genome-wide sex-specific meta-association studies of eight steroid hormones: progesterone, 17-hydroxyprogesterone (17-OHP), androstenedione, testosterone, estradiol, dehydroepiandrosterone sulfate (DHEA-S), cortisol, and aldosterone (LIFE-Adult, LIFE-Heart, max. n = 7667). Genome-wide significant loci (p < 5x10 −8 ) were tested for sex differences in effect size. The causal relationships between hormone levels and atherosclerotic vascular diseases were investigated using sex-specific eQTL analyses and Mendelian Randomization (MR) approaches. Results: We detected 22 genome-wide significant loci for seven steroid hormones of our panel, of which nine displayed significant sexual dimorphisms. Eight loci relate to genes coding for enzymes involved in steroid biosynthesis, almost completing the list of genes of this pathway with genetic associations. Other loci correspond, e.g., to the WNT4/β-catenin pathway. MR revealed that cortisol, androstenedione, 17-OHP and DHEA-S had sex-specific causal effects on coronary artery disease. Conclusions: Our study greatly improves insight into genetic regulation of steroid hormones and their dependence on sex. The significant MR estimates support the hypothesis that steroid hormones influence the development of atherosclerosis, which partly explains the higher risk for men. These results could serve as a basis for analyzing sex-dimorphisms in other complex diseases. This project was supported by the HI-MAG Project Funding for Young Scientists of the Medical Faculty of the University Leipzig. J. Pott: None. Y.J. Bae: None. K. Horn: None. A. Teren: None. A. Kühnapfel: None. H. Kirsten: None. U. Ceglarek: None. M. Loeffler: None. J. Thiery: None. J. Kratzsch: None. M. Scholz: None. P05.81.C Genetic post-mortem in drug users with sudden cardiac death

H. Massey , J. Dean, L. Deboys, D. O’Sullivan

Aberdeen royal infirmary, Aberdeen, United Kingdom
Aim: To quantify our ability to make a genetic diagnosis in drug users with sudden cardiac death (SCD). Background: While there are European recommendations for genetic testing in SCD1, these guidelines do not extend to drug users with positive toxicology at post-mortem. We wanted to gain a better understanding of the genetic pick up rate in this population. Method: We undertook a 10-year retrospective study looking at genetic diagnoses in drug related deaths discussed at the joint pathology-genetic MDT. This was compared to age and provisional aetiology matched controls. Results: 60 drug related deaths were identified, and 40 patients underwent genetic testing. 7 variants were found of which 3 were classified as benign and 4 VUS. The most common reason for genetic testing was enlarged heart at post mortem; however the genetic pick up in this group was 0. Patients with structurally normal hearts had the most variants detected. This would coincide with the majority of variants being found in arrhythmogenic genes. Family history did not influence genetic pick up. In the control group variants in arrhythmogenic genes again predominated correlating with the highest genetic pick up in patients with normal post-mortems. However in the control group 17 variants were detected of which 3 were pathogenic. Conclusions: Our ability to make a genetic diagnosis in drug users with SCD is low. We need to undertake a larger study to characterise those drug users with a positive genetic diagnosis so testing can be rationalised. H. Massey: None. J. Dean: None. L. Deboys: None. D. O’Sullivan: None. P05.82.A Insights into the genetic basis of ventricular tachycardia in Kazakhstani patients. Targeted next generation sequencing

A. R. Akilzhanova 1 , C. Guelly 2 , Z. Abilova 1 , S. Rakhimova 1 , A. Akhmetova 1 , U. Kairov 1 , S. Trajanoski 2 , G. Akilzhanova 3 , M. Bekbosynova 4 , Z. Zhumadilov 1

1 Center for life sciences, National Laboratory Astana, Nazarbayev University, Nur-Sultan, Kazakhstan, 2 Center for Medical Research, Medical University of Graz, Graz, Austria, 3 Medical University of Semey, Pavlodar branch, Pavlodar, Kazakhstan, 4 National Research Cardiac Surgery Center, Nur-Sultan, Kazakhstan
Introduction: Recent advances in genetic screening and the potential of therapeutic intervention in patents with cardiac arrhythmias have garnered this group of disorders much attention in the scientific community. The purpose of the study was to identify the genetic basis of ventricular tachycardia (VT) in Kazakhstani patients. Material and Methods: using predeveloped a targeted panel of 96 known cardiac disease genes, associated with cardiomyopathy and arrhythmia we screened 92 individuals, diagnosed with VT and dilated cardiomyopathy (VT DCM) or idiopathic VT (iVT). Results. Targeted sequencing and stepwise filtering of the annotated variants identified 307 unique variants in 74 genes totaling up in 456 variants for the overall study group. Variants included one in/del, four splice-site and 451 single-nucleotide variants (SNV) within the coding exonic regions. Seven (0.15%) of the SNVs were unique stop-gain variants, three of those residing in the TTN gene. 168 HGMD mutations (61 unique) were observed in 37 genes. According to ACMG all variants were classified as 9 pathogenic (KCNJ2 R218Q and TTN R5338X in iVT patients, KCNQ1 c.477+1G>A, LMNA Q353X, MYH7 F244L, TTN L17465X and W21011X, DSG2 c.2334+1G>A, GAA W746C in VT DCM patients), 11 likely pathogenic, 97 variants with uncertain significance and rest as benign. Conclusions: Individuals with VT either secondary to DCM or of idiopathic etiology carry multiple rare mutations and potentially pathogenic sequence variants in cardiac risk genes in a similar pattern and at a comparable frequency. Study support: grant from the Ministry Education and Science, Republic of Kazakhstan (AP05134683). A.R. Akilzhanova: None. C. Guelly: None. Z. Abilova: None. S. Rakhimova: None. A. Akhmetova: None. U. Kairov: None. S. Trajanoski: None. G. Akilzhanova: None. M. Bekbosynova: None. Z. Zhumadilov: None. P05.83.B Identification of the major genetic contributors to Tetralogy of Fallot

R. M. Monaghan 1 , D. J. Paige 2 , S. G. Williams 1 , B. D. Keavney 1

1 University of Manchester, Manchester, United Kingdom, 2 Manchester Metropolitan University, Manchester, United Kingdom
Tetralogy of Fallot (TOF) is the most common cyanotic congenital heart defect. Rare genetic variants have been identified as important contributors to the risk of congenital heart disease, but relatively small numbers of TOF cases have been studied. We used whole exome sequencing to assess the prevalence of unique, deleterious variants in the largest cohort of nonsyndromic TOF patients reported to date. The presence of such variants was defined by their absence in the Genome Aggregation Database and bioinformatic prediction of their deleterious effect on function. The enrichment of variants in two genes, NOTCH1 and FLT4 , surpassed thresholds for exome-wide significance ( P < 5×10-8) after correction for multiple comparisons. NOTCH1 was most frequently found to harbour unique, deleterious variants (4.5%; 95% CI, 3.2%-6.1%). Three NOTCH1 variants were subjected to functional evaluation, and two showed a reduction in Jagged1-induced NOTCH signalling. FLT4 variants were found in 2.4% (95% CI, 1.6%-3.8%) of our cohort. In addition, variants in the well-established TOF gene, TBX1 , and the genes were also significantly enriched. Our study underlines the importance of sequencing large cohorts of CHD cases in order to further discover their genetic basis. Grant reference: BHF (RG/15/12/31616) R.M. Monaghan: None. D.J. Paige: None. S.G. Williams: None. B.D. Keavney: None. P05.84.C In search of genetic modifiers that explain the phenotypic variability in SMAD3 -related aortopathy

J. D. Velchev 1 , M. Perik 1 , I. M. B. H. van de Laar 2 , J. Richer 3 , A. Verstraeten 1 , M. Alaerts 1 , B. L. Loeys 1,4

1 Center of Medical Genetics, University of Antwerp and Antwerp University Hospital, Antwerp, Belgium, 2 Department of Clinical Genetics, Erasmus MC, University Medical Center Rotterdam, Rotterdam, Netherlands, 3 Department of Medical Genetics, Children’s Hospital of Eastern Ontario, Children’s Hospital of Eastern Ontario Research Institute, Ottawa, ON, Canada, 4 Department of Human Genetics, Radboud University Nijmegen Medical Center, Nijmegen, Netherlands
Introduction: Thoracic aortic aneurysm and dissection (TAAD) is a frequent cause of mortality in the Western world. Loeys-Dietz syndrome (LDS), an early-onset TAAD syndrome is caused by mutations in genes coding for components of the TGFbeta signaling, including SMAD3 . Remarkably, a fraction of SMAD3 mutation carriers remains cardiovascularly unaffected throughout life, suggesting the existence of genetic modifiers for aortopathy. This study implements a combination of linkage analysis and whole-genome sequencing (WGS) in order to identify genetic modifiers that explain the variability in SMAD3 -related aortopathy. Material and Methods: In two large LDS families ( n = 41 mutation carriers) segregating SMAD3 missense mutations and presenting variable TAAD expressivity genome-wide SNP-based linkage analysis was performed. LOD scores were obtained using MERLIN and SUPERLINK and linkage was complemented with WGS of six affected and six unaffected mutation carriers. Results: In the first family linkage analysis identified a chromosomal region (chr2:149,604,077-155,607,398) with a LOD score of 2.68, suggesting the presence of an aggravating modifier. Linkage peak was observed in the second family with a LOD score of 2.86 (chr18:43,013,157-57,021,550). Additionally, the WGS data analysis of the second family resulted in the discovery of a rare variant in TGFB2 , a known TAA causative gene, exclusively present in affected mutation carriers. Conclusions: The obtained data indicate that variants in known TAAD genes may play a role in modulating a primary causative mutation; chromosomal regions segregate with disease severity suggesting a presence of genetic modifiers and that distinct families may bear different genetic modifiers as indicated by the linkage analysis. J.D. Velchev: None. M. Perik: None. I.M.B.H. van de Laar: None. J. Richer: None. A. Verstraeten: None. M. Alaerts: None. B.L. Loeys: None. P05.85.A Identification of Clinically Relevant Variants by Whole Exome Sequencing in Chinese Patients with Sporadic Non-syndromic Type A TAAD

J. Guo , C. Hao, X. Hu, F. Leng, X. Liu

Genetics and Birth Defects Control Center, Beijing Children’s Hospital, Beijing, China
Introduction: Thoracic aortic aneurysm and dissection (TAAD) is a life-threatening disease, of which genetic abnormalities are considered as important risk factors. The present research aims at identifying causal variants in Chinese patients with sporadic non-syndromic type A TAAD (ATAAD). Materials and Methods: Whole exome sequencing (WES) was performed on 73 sporadic Chinese patients with ATAAD, 30 TAAD associated genes were curated for bioinformatic analyses. Clinical differences were compared between patients with and without causal variants. Results: 15 pathogenic/likely pathogenic variants were identified (8 novel and 7 previously described) in 4 known TAAD-causal genes ( FBN1 , TGFBR2 , SMAD3 and ACTA2 ) in 15 individuals, including 11 variants in FBN1 (7 missense, 3 truncating, and 1 splicing variants), 2 missense variants in TGFBR2 , 1 ACTA2 frameshift variant and 1 SMAD3 frameshift variant. Significant clinical differences were found between patients with and without causal variants. Patients with TAAD-causal variants proved to have an earlier onset age, a more dilated aorta, and relatively intractable subtypes. Even without risk factor like hypertension, they might still suffer from TAAD with TAAD-causal variants. Conclusions: The variants identified in our research might not only result in the occurrence of ATAAD, but also add complexities and difficulties to the clinical practice. Our data demonstrated that WES was an effective tool for determining genetic etiologies of non-syndromic ATAAD and could be helpful in genetic counseling for ATAAD patients and their at-risk family members. J. Guo: None. C. Hao: None. X. Hu: None. F. Leng: None. X. Liu: None. P05.87.C Investigating the role of pathogenic TTN variants in heart failure by whole-exome sequencing

O. Chazara 1 , G. Povysil 2 , S. Deevi 1 , J. Armisen 1 , D. B. Goldstein 2 , C. Haefliger 1

1 Astrazeneca, Melbourn, United Kingdom, 2 Institute for Genomic Medicine, Columbia University Medical Center, New York, NY, United States
Heart failure (HF) affects ~40 million people globally. Estimates of the genetic predisposition of developing HF is 1.69 if one parent is affected and 1.92 if both parents have HF (Framingham cohort study, Lopez et al. 2013). Recent studies suggest higher cardiovascular genetic risk in patients with and without diagnosed heart failure if they carry a pathogenic loss of function variant in the gene encoding titin (TTN). Here, we assess the role of pathogenic TTN variants by whole-exome sequencing in 5916 patients with heart failure from two clinical studies CHARM and CORONA, and 268,451 unrelated Europeans from the UK Biobank prospective cohort study, including 4634 individuals with ICD-10 codes for heart failure (HF, based on hospital in-patient diagnoses). In both types of cohorts, we detect a significant enrichment of pathogenic TTN variants in subjects with HF diagnosis. Furthermore, after restricting the analysis to the variants located in exons that are predominantly spliced in the heart, the genetic risk was increased: from 2.54 (95% CI, 1.96-3.31) to 4.52 (95% CI, 3.10-6.68) in the clinical studies, and from 2.95 (95% CI, 2.42-3.57) to 4.97 (95% CI, 3.94-6.19) in the UKBB. The enrichment observed is similar in clinical trial data and routine care health records. As expected, individuals with a diagnosis of cardiomyopathy had the highest rate of TTN mutations, but we also observed TTN variants in up to 5% of HF patients due to other aetiologies, such as ischaemic and hypertensive disease. This result highlights the importance of TTN variants beyond cardiomyopathies. O. Chazara: A. Employment (full or part-time); Significant; Astrazeneca. G. Povysil: None. S. Deevi: A. Employment (full or part-time); Significant; Astrazeneca. J. Armisen: A. Employment (full or part-time); Significant; Astrazeneca. D.B. Goldstein: F. Consultant/Advisory Board; Modest; Astrazeneca. C. Haefliger: A. Employment (full or part-time); Significant; Astrazeneca. P05.88.A Non-amyloidotic transthyretin cardiomyopathy

J. Gimeno Blanes 1 , M. Sabater Molina 1 , I. San Roman 1 , L. Polo 2 , I. Garrido 3 , C. Salas 4 , F. Lopez Andreu 5

1 Inherited Cardiac Disease Unit. Universitary Hospital Virgen de la Arrixaca., Murcia, Spain, 2 Hospital Universitario Virgen Arrixaca, Department of Pathological Anatomy., Murcia, Spain, 3 Department of Cardiology Universitary Hospital Virgen de la Arrixaca., Murcia, Spain, 4 Hospital Puerta de Hierro, Department of Pathological Anatomy, Madrid, Spain, 5 Hospital General Reina Sofia, Service of Internal Medicine, Murcia, Spain
Introduction: We aim to present an unsolved case of a young patient with unclassified cardiomyopathy associated with a genetic TTR variant predicted to cause a dramatic change in protein structure. Clinical course went from asymmetric hypertrophy with restrictive phenotype to cardiac dilatation and systolic dysfunction. Pulmonary pressures were strikingly elevated. Case: A 33 years old woman was admitted in 1997 with limiting dyspnoea. Echocardigram showed non-obstructive asymmetrical septal hypertrophy of 17mm maximum at the septum and restrictive physiology. Sarcoid cardiomyopathy was ruled out with negative scintigraphy and unspecific biopsy in the absence of other extracardiac features. Atrio-ventricular ablation and permanent pacing were required in 2002. Clinical deterioration led to frequent heart failure admissions and one episode of syncope. Implantable cardioverter defibrillator was implanted. Emergency transplant was performed with a postoperative complicated course in 2006. Patient remained stable to date. Study of the explanted heart demonstrated macro and microscopic hypertrophy, extensive fibrosis, unspecific vacuolization and disarray. There were no signs of amyloid with usual stains and immunohistochemical typing. Sequencing of the TTR gene led to identification of a de novo frameshift mutation p.S132fs*25(g.6903_6906delinsGTAGGG) causing original stop codon lost, results in prolonged protein. Cardiac evaluation of her brother, sister and parents ruled out any sign of cardiomyopathy. All relatives were free for the TTR variant. Conclusions: Case of cardiomyopathy associated with a TTR variant. Severe right and left heart failure clinical course with very particularly severe pulmonary hypertension ended up in heart transplant. Mechanisms of disease in this case remain unknown. J. Gimeno Blanes: None. M. Sabater Molina: None. I. San Roman: None. L. Polo: None. I. Garrido: None. C. Salas: None. F. Lopez Andreu: None. P05.89.B Searching for genes associated with primary varicose veins using targeted next-generation sequencing

E. Strauss 1,2 , M. Zmyslowski 3 , B. Budny 4 , L. Dzieciuchowicz 3 , E. Wrotkowska 4 , M. Ruchala 4 , K. Ziemnicka 4 , G. Oszkinis 5 , Z. Krasinski 3

1 Institute of Human Genetics of the Polish Academy of Sciences in Poznan, Poznan, Poland, 2 Department of Vascular, Endovascular Surgery, Angiology and Phlebology Poznan University of Medical Sciences, Poznań, Poland, 3 Department of Vascular, Endovascular Surgery, Angiology and Phlebology Poznan University of Medical Sciences, Poznan, Poland, 4 Department of Endocrinology, Metabolism and Internal Medicine, Poznan University of Medical Sciences, Poznan, Poland, 5 Department of General and Vascular Surgery, Poznan University of medical Sciences, Poznan, Poland
Introduction: The aim of this study was to search for genes involved in the pathogenesis of the primary varicose veins (PVV) using targeted next-generation gene sequencing. Sporadic cases with the onset of the first symptoms of disease below or equal 40 years of age were considered. Methods: A cohort of 66 PVV patients was collected and exome sequencing of the 35 candidates´ genes was performed, which focused on the genes encoding structural elements of the vessel wall, metalloproteinases, and their protein inhibitors, proteins involved in the homeostasis of the coagulation system and regulation of sex hormones system, as well as genes previously associated with VV development. Results: Most of the patients (91.9%) had a positive family history of VV, considering first-, second- or third-degree relatives. 26 potentially pathogenic changes: 12 mutations in 9 genes (7 known and 5 novel), 5 rare SNPs and 9 common SNPs were found. 40% of the patients had a potentially causative mutation in the candidate genes: 33% of them had 1 mutation, 3% had 2 mutations and 1.5% had 3 mutations. The association between one SNP in the MMP-9 gene and the age of the onset of PVV symptoms was also observed. Conclusions: This report provides evidence for the heterogeneity and complexity of the genetic background of PVV in sporadic cases and shows the influence of genetic factors on the age of the onset of this disease. It also reveals attractive targets for further research in a larger group of patients and functional analyses. E. Strauss: None. M. Zmyslowski: None. B. Budny: None. L. Dzieciuchowicz: None. E. Wrotkowska: None. M. Ruchala: None. K. Ziemnicka: None. G. Oszkinis: None. Z. Krasinski: None.

P06 Metabolic and Mitochondrial Disorders
P06.01.C Identification of AADC patient through combined determination of 3-OMD biomarker and DDC gene sequencing and copy number variation analysis

C. Pereira 1 , C. Cozma 1 , L. Demuth 1 , G. Morales Torres 1 , M. Iurascu 1 , P. Bauer 1 , A. Rolfs 1 , C. Lourenco 2

1 Centogene AG, Rostock, Germany, 2 Universidade de São Paulo, Sao Paulo, Brazil
L-amino acid decarboxylase (AADC) deficiency is caused by bi-allelic variants in the DDC gene. This very rare neurometabolic disorder is clinically characterized by vegetative symptoms, oculogyric crises, dystonia, and severe neurologic dysfunction, usually beginning in infancy or childhood. Treatment has become available recently. We quantified 3-OMD (3-O-Methyldopa), a suggested AADC-specific biomarker, in dried blood spots by tandem mass spectrometry. Upon suggestive findings, we subsequently analyzed the DDC gene by amplicon-based next generation sequencing (NGS) and copy number variant (CNV) screening. This two-step approach identified several patients. The ones with at least one hitherto undescribed variant are described here in detail. All three patients had pathological levels of 3-OMD. In patient 1, we identified a novel whole DDC gene duplication in homozygosity. In patient 2, we identified the two missense variants p.(Ser147Ile) and p.(Val60Ala) in compound heterozygosity; interestingly, variant p.(Ser147Ile) was not only novel, but shown to have occurred de novo. In patient 3, p.(Trp121Arg) and p.(Arg347Gln) were identified in compound heterozygosity. The former is novel, while the latter has frequently been described in AADC patients previously. Using a combined biochemical, NGS and CNV approach, a positive identification and diagnosis of AADC deficiency patients is reliably reached. This allows for an early treatment of the patients, leading to an improvement of their quality of life. Moreover, the identification of novel DDC variants and the establishment of correlations with 3-OMD levels can be expected to increase our understanding of the disease. C. Pereira: None. C. Cozma: A. Employment (full or part-time); Significant; Centogene AG. L. Demuth: A. Employment (full or part-time); Significant; Centogene AG. G. Morales Torres: A. Employment (full or part-time); Significant; Centogene AG. M. Iurascu: A. Employment (full or part-time); Significant; Centogene AG. P. Bauer: A. Employment (full or part-time); Significant; Centogene AG. A. Rolfs: A. Employment (full or part-time); Significant; Centogene AG. C. Lourenco: None. P06.04.C Patients affected by autosomal recessive or dominant cutis laxa type 3 show diverse degrees of collagen alterations

N. El Choubassi 1,2 , L. Lugli 3 , C. Spada 4 , D. de Silva 5 , O. Klein 1 , D. Meierhofer 2 , D. Horn 1 , U. Kornak 1,2,6 , B. Fischer-Zirnsak 1,2

1 Charité Universitaetsmedizin Berlin, Berlin, Germany, 2 Max Planck Institute for Molecular Genetics, Berlin, Germany, 3 Division of Neonatal Intensive Care Unit, Department of Pediatrics, University Hospital, Modena, Italy, 4 Department of Medical and Surgical Sciences for Mothers, Children and Adults, Post Graduate School of Paediatrics, University of Modena & Reggio Emilia, Modena, Italy, 5 Department of Physiology, Faculty of Medicine, University of Kelaniya, Ragama, Sri Lanka, 6 Institute of Human Genetics, University Medical Center Göttingen, Göttingen, Germany
Introduction: Mutations in ALDH18A1 , encoding the mitochondrial enzyme pyrroline-5 carboxylate synthase (P5CS), lead to a broad spectrum of rare genetic conditions. Besides hereditary spastic paraplegia, ALDH18A1 mutations are causative for autosomal recessive (ARCL3) and dominant (ADCL3) cutis laxa with progeroid features. Materials and Methods: We performed whole exome (WES) and gene panel sequencing to identify pathogenic variants in our probands. These analyses were complemented by mRNA expression studies, immunofluorescence, immunoblot and mass-spectrometry approaches on patient derived fibroblasts. Results: We report on novel pathogenic variants in three affected individuals with ARCL3/ADCL3. These variants have variable impacts on the stability of the protein and its localization within the mitochondrial network. However, targeted metabolomics revealed substantial alterations of mitochondrial proline synthesis rates. Additionally, gene expression profiling, comparative proteomics and subsequent immunoblot analyses showed alterations in expression of different collagens. Conclusions: Our data expand the mutational spectrum of ALDH18A1 -related diseases and demonstrate the importance of correct ALDH18A1 localization within the mitochondria for proper functioning. Additionally, they suggest a potential connection of the mitochondrial proline synthesis pathways with the regulation of collagen synthesis. Funding: The study was supported by the Deutsche Forschungsgemeinschaft (FI 2240/1-1) to BF-Z N. El Choubassi: None. L. Lugli: None. C. Spada: None. D. de Silva: None. O. Klein: None. D. Meierhofer: None. D. Horn: None. U. Kornak: None. B. Fischer-Zirnsak: None. P06.06.B Clinical study of the relationship between histamie intolerance and variants in the AOC1/ABP1 Gene

J. Vinas-López 1 , P. Ojeda 2 , I. Ojeda 2 , A. Duelo 3 , B. García-Velasco 1 , C. Lenz 2 , T. Perucho 1 , M. Berbel 3 , A. Vásquez 3 , E. Ruiz-Casares 1,4

1 Genyca, Majadahonda, Spain, 2 Clínica Ojeda de Asma y Alergia Dres. Ojeda, Madrid, Spain, 3 Centro de Nutrición AD Dietistas, Barcelona, Spain, 4 Universidad San Pablo CEU, Madrid, Spain
Introduction: Histamine is a biogenic amine with many essential physiological activities. However, its deregulation and accumulation can cause a multitude of pathologies such as histamine intolerance. There are two enzymes involved in the metabolism of histamine: histamine N-methyltransferase (HNMT) is responsible for degrading histamine inside cells. Diamine-oxidase (DAO) is the other enzyme involved in the degradation of histamine, mainly exogenous histamine, ingested with the diet. The deficiency of DAO activity could be one of the main causes of histamine intolerance, an alteration in homeostasis that is caused by a reduced intestinal degradation of histamine, with the consequent increase in its plasma levels. Variants in AOC1/ABP1 gene, coding DAO enzyme, have been associated with a reduction in DAO activity and accumulation of histamine. Objective: The present study aims to demonstrate the negative effect that the variants rs10156191, rs1049742 and rs1049793 have on DAO plasma activity in Caucasian male patients with clinical manifestations associated with histamine intolerance. Methods: We analyzed 78 white male patients with a clinical manifestation associated with histamine intolerance. Variants analyzed were genotyped by multiplex SNPE (Single Nucleotide Primer Extension). Results: The variants rs10156191 and rs1049742 reduce significantly DAO plasma activity. Factors such as worsening symptoms after eating a diet rich in histamine or NSAID-type drugs (non-steroidal anti-inflammatory) are associated with a greater number of alternative alleles in the SNVs analyzed, and reduced DAO plasma activity. Conclusions: These results show the predictive value of this genetic study in patients who have a clinical manifestation associated with histamine intolerance. J. Vinas-López: C. Other Research Support (supplies, equipment, receipt of drugs or other in-kind support); Modest; Genyca. P. Ojeda: None. I. Ojeda: None. A. Duelo: None. B. García-Velasco: None. C. Lenz: None. T. Perucho: None. M. Berbel: None. A. Vásquez: None. E. Ruiz-Casares: None. P06.10.C Low prevalence of classic galactosemia in the Sverdlovsk Region (Russian Federation)

O. Lagutina 1 , S. Deryabina 1,2 , T. Belyaeva 1 , N. Nikitina 1 , E. Nikolaeva 1

1 Medical Center “Health Care of Mother and Child”, Yekaterinburg, Russian Federation, 2 Federal State Autonomous Educational Institution of Higher Education «Ural Federal University named after the first President of Russia B.N. Yeltsin, Yekaterinburg, Russian Federation
Introduction: Classic galactosemia is one of the five hereditary metabolic diseases, which has been included in the State Program of Neonatal Screening of the Russian Federation in 2006. Aim of the study: To estimate the incidence of galactosemia in the Sverdlovsk Region based on neonatal screening data. Patients and methods: 698 550 children born in the Sverdlovsk Region from 2006 to 2019 were examined. Initially, the level of total galactose and galactose-1-phosphate was determined in a dry blood spot, then an activity of the enzyme galactose phosphate-uridyl transferase (GALT) has been selected as a biochemical marker of galactosemia. The diagnosis of galactosemia should then be confirmed by genetic analysis. Results: During the 13-year study period 657 newborns were included in the risk group and assigned for molecular genetic testing. Eight children were identified as having classical galactosemia on the basis of the enzymatic and molecular genetic analysis. Moreover,142 babies had a galactosemia Duarte, another 134 had decreasing activity of the GALT enzyme due to non-genetic reasons. The spectrum of revealed mutations in the GALT gene: K285N (31.3%), Q188R (25.0%), M142K (12.5%), E352Q (12.5%), W316X (6.2%), R262W (6.2%) and one new variant P140L (6.2%), not described earlier. Conclusion. Based on the results of newborn screening programs, the prevalence of classic galactosemia in the Ural population is 1:87 318 live birth and unlikely that it will undergo significant changes with further increase in the screening period. O. Lagutina: None. S. Deryabina: None. T. Belyaeva: None. N. Nikitina: None. E. Nikolaeva: None. P06.12.B What is beyond the congenital lactic acidosis? From variants to personalized medicine

I. Bravo-Alonso , R. Navarrete, P. Ruiz-Sala, F. Leal, M. Ugarte, C. Pérez-Cerdá, B. Merinero, B. Pérez, P. Rodríguez-Pombo

Centro de Diagnóstico de Enfermedades Moleculares, Centro de Biología Molecular Severo Ochoa, UAM-CSIC, CIBERER, IDIPAZ, Madrid, Spain
Implementation of Next Generation Sequencing has dramatically increased the diagnosis success-rate of highly heterogeneous inborn-errors of metabolism such as those underlying congenital lactic acidosis. However, the lack of integrated phenotypic data, the scarce number of datasets of global metabolomics and the necessary expertise to analyze the functional effects of variants of unknown significance, are limiting the transfer of genetic data into clinical practice. Our aim in this study was to evaluate the powerful of an adequate clinical-biochemical and genetic analysis to drive a clinical response in a timely manner preventing the use of major invasive techniques. With a pipeline that combined specific datasets from metabolomics sub-sections and phenotypic information, with DNA and/or RNA analysis, we identified likely pathogenic variants in strong-candidate genes in 64% of a cohort of 39 patients with clinical suspicion of congenital lactic acidosis. These variants, 14 of which were new, were found in 19 different nuclear and two mitochondrial genes. Specific biochemical test and/or bioenergetics analysis in fibroblasts including measures of oxygen consumption rate, steady-state levels of OxPhos proteins using Blue-native geles, and changes in mitochondrial morphology, sustained the diagnosis for patients carrying novel genotypes. RNA analysis contributed to solve miss or undiagnosed patients, providing a direct insight into transcriptional perturbations caused by specific genetic changes. Finally, by rescuing normal splicing in a GFM1-patient cells carrying in heterozygous fashion the c.689+908G>A change with antisense oligonucleotide treatment, we corroborated the importance of molecular analysis to investigate personalized therapeutic options. LCF/PR/PR16/11110018; PI16/00573; CAM, B2017/BMD3721 I. Bravo-Alonso: None. R. Navarrete: None. P. Ruiz-Sala: None. F. Leal: None. M. Ugarte: None. C. Pérez-Cerdá: None. B. Merinero: None. B. Pérez: None. P. Rodríguez-Pombo: None. P06.13.C Hypoglycemia-some of its many reasons-a molecular update .

K. Brusgaard 1 , Y. Alhaidan 2 , M. Balwi 2 , H. Christesen 1

1 Odense University Hospital, Odense, Denmark, 2 King Abdullah International Medical Research Center, Riyadh, Saudi Arabia
Hypoglycemia can be caused by numerous somatic or hereditary causes. Three major reasons are; diabetes, hyperinsulinism and ketotic/non-ketotic hyperglycemia. The brain accounts for 60% of the glucose consumption in the infants’ resting state, leaving the brain vulnerable to hypoglycemia. Tight plasma glucose control is ensured by a number of complex mechanisms. In the majority of cases the underlying pathophysiology are unexplained. The goal in the presented study was to find new reasons underlying these disorders. 38 families were included for trio analyses performing exome sequencing for germline variation. Where available exome sequencing was performed on resected pancreatic tissue if no germline cause was elucidated. Data was analyzed using VarSeq and various filters considering recessive, X-linked, de-novo or autosomal dominant modes of inheritance. Candidate genes was analyzed by in vitro studies using RIN-m β-cell lines and KO of target genes using siRNA or Crispercas9. Measurement of RNA expression levels was performed by Q-RT-PCR or microarray analysis. Insulin measurements were performed. In 10 (26%) cases the hypoglycemia was explained. In three ketotic hypoglycemia patients the reason was found in a known GSD gene, four patients was explained by novel genes. In one diabetic and one hyperinsulinism patient a novel genetic cause was found. A single hyperinsulinism patient was explained by a previously undetected mutation in a known hyperinsulinism gene. 10 additional candidate genes are currently under in vitro cellular investigation for their role in manifestation of disease in individual families. As a result of the current study the treatment was changed in three individuals K. Brusgaard: None. Y. Alhaidan: None. M. Balwi: None. H. Christesen: None. P06.14.A Biochemical and molecular landscape of Fabry disease in Portugal

F. E. Laranjeira 1,2 , P. Rodrigues 1,3 , C. Caseiro 1 , I. Ribeiro 1,2 , H. Carmo 3 , D. Quelhas 1,2

1 Unidade de Bioquímica Genética, Centro de Genética Médica Doutor Jacinto Magalhães, Centro Hospitalar Universitário do Porto, Porto, Portugal, 2 Unidade Multidisciplinar de Investigação Biomédica, Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto, Porto, Portugal, 3 UCIBIO, REQUIMTE – Laboratório de Toxicologia, Departamento de Ciências Biológicas, Faculdade de Farmácia, Universidade do Porto, Porto, Portugal
Introduction: Fabry disease is an X-linked progressive inborn error of metabolism originated by mutations in the GLA gene causing a deficiency of the lysosomal α-galactosidase (α-Gal) hydrolase activity. The breakdown of glycolipids is impaired and the lysosomal substrate accumulation triggers the onset of this lysosomal storage disorder. Male patients usually present very low α-Gal activity and marked substrate accumulation, and at least some of the clinical hallmarks. In females, however, there is a wide range of phenotypes leading to great controversy regarding clinical diagnosis and therapeutic decisions. Patients and methods Data concerning leukocytes and plasma α-Gal activity together with plasma and urine substrate accumulation for 141 males and 210 females were obtained. Correlation among biochemical parameters and with the molecular defect was evaluated. Results: 37 genetic variants were found and the most prevalent genetic variant is c.337T>C p.(F113L), found in 50% of males and females. Males usually present a correlation of reduced α-Gal activity with increased substrate accumulation. In this group it was possible to identify some genetic variant-specific patterns. Heterozygous females show less pronounced effect on substrate accumulation and enzyme deficiency. The variability of patterns within carriers of the same genetic variant is also noticed. Conclusions: Some genetic variants whose pathogenicity is controversial are indeed associated with milder biochemical phenotypes. Plasma α-Gal activity is not recommended as only diagnosis testing because in the presence of specific variants it may give false negatives. Heterogeneity among heterozygous females should be further evaluated with relation to X chromosome inactivation profile. F.E. Laranjeira: None. P. Rodrigues: None. C. Caseiro: None. I. Ribeiro: None. H. Carmo: None. D. Quelhas: None. P06.15.B Mutation spectrum and corresponding clinical manifestation of patients with hypertriglyceridemia in a German outpatient clinic

F. Bardey 1 , L. Rieck 1 , D. Spira 1 , W. März 2 , S. Schwab 2 , P. Binner 2 , T. Bobbert 1 , J. Spranger 1 , E. Steinhagen-Thiessen 1 , I. Demuth 1 , U. Kassner 1

1 Charite Universitätsmedizin Berlin, Berlin, Germany, 2 SYNLAB MVZ Humangenetik GmbH, Mannheim, Germany
Introduction: A sizeable proportion of patients with severe hypertriglyceridemia (HTG) do have the monogenetic familial chylomicronemia syndrome (FCS). It remains difficult to clinically distinguish FCS from the multifactorial chylomicronemia syndrome (MCS) in the absence of genetic testing. Our aim was to characterize the genotype of our HTG patients with respect to MCS and FCS as well as to apply the clinical FCS-score proposed by Moulin and colleagues. Methods: We studied 90 genotyped patients with severe HTG in our clinic during the last seven years. We classified the identified genetic variants (SHERLOC-criteria), followed by categorization into MCS or FCS. Clinical data were gathered from the medical records and the FCS-score was calculated for each patient. Results: The gene sequencing revealed ten FCS patients. Four were compound heterozygous and six were homozygous for a pathogenic variant. Another 53 patients were categorized as MCS, of which seven were heterozygous carriers of a pathogenic variant. Altogether, we found 16 pathogenic variants of which six have not been described before. The most common was a missense variant (c. 644G>A, p.G215E) in the LPL gene. In 27 (30%) of our patients we were unable to detect any genetic alterations. In our cohort the FCS-score yielded a sensitivity of 80% and a specificity of 92.5%. Conclusions: At 11% the prevalence of FCS among our patients with severe HTG was higher than expected. The FCS score performed similar to the original study, however 20% of the FCS patients will not be detected correctly, relying on the score only. F. Bardey: None. L. Rieck: None. D. Spira: None. W. März: None. S. Schwab: None. P. Binner: None. T. Bobbert: None. J. Spranger: None. E. Steinhagen-Thiessen: None. I. Demuth: None. U. Kassner: None. P06.17.A Gene expression and pathway analysis of omental adipose tissue from Type 2 diabetic and non diabetic Asian obese patients

R. Dehghan 1,2 , R. Dorajoo 1 , M. H. Liu 3 , L. Wang 1 , Y. Y. Sia 1 , C. B. Lin 1 , T. H. Koh 1 , S. Huang 4 , E. Chan 4 , W. Liew 4 , Y. Z. Tan 5 , C. M. Khoo 6 , X. Sim 7 , J. Liu 1,6 , A. Shabbir 5 , E. S. Tai 4,6,8

1 Genome Institute of Singapore, Agency for Science, Technology and Research, Singapore, Singapore, 2 Department of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Science, Isfahan, Iran, Islamic Republic of, 3 Department of Food Science and Technology, National University of Singapore, Singapore, Singapore, 4 Division of Endocrinology, Department of Medicine, National University Health System, Singapore, Singapore, 5 Department of Surgery, National University Hospital, Singapore, Singapore, 6 Department of Medicine, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore, 7 Saw Swee Hock School of Public Health, National University of Singapore, Singapore, Singapore, 8 Duke-National University of Singapore Medical School, Singapore, Singapore
Introduction: Asians may have increased propensity to central obesity and related Type 2 diabetes mellitus (T2DM) risks, as compared to populations of European ancestry. Gene expression profiling of omental fat in Asians may identify additional T2DM relevant pathways. Methods: We performed RNA-seq on 37 (16 T2DM and 21 non-T2DM) omental fats from Asian obese patients, obtained during bariatric weight-loss surgery. DESeq2 was used for differential expression analysis. Results: 266 differentially expressed genes (DEGs) showed higher and 197 showed lower expression in T2DM vs non-T2DM samples ( P adj < 0.05). We identified GABRB2 as the top novel upregulated gene, highlighting a role for GABAergic system in omental fat with T2DM while ACVR1C expression was most significantly downregulated and loss of function variants in ACVR1C have been reported for reduced central obesity and T2DM risks. Upstream regulator analysis using our DEGs revealed OSM as the most significant regulator of up-regulated DEGs and PPARG for down-regulated DEGs. Pathway-based analyses using our DEGs connected with OSM revealed significant associations for immune-related pathways, validating recent associations of increased HMGB1 and TREM1 signaling for increased T2DM risks. Similar analyses on PPARG revealed pathways associated with adipogenesis, glucose and lipid metabolism and validated the protective association between increased Apelin signaling in omental fat and T2DM. Conclusions: Our study on omental fat identified novel DEGs associated with T2DM. Omental fats from T2DM subjects exhibit upregulation of immune pathways probably modulated by OSM, and downregulation in genes involved in adipogenesis, glucose and lipid metabolism probably modulated by PPARG. R. Dehghan: None. R. Dorajoo: None. M.H. Liu: None. L. Wang: None. Y.Y. Sia: None. C.B. Lin: None. T.H. Koh: None. S. Huang: None. E. Chan: None. W. Liew: None. Y.Z. Tan: None. C.M. Khoo: None. X. Sim: None. J. Liu: None. A. Shabbir: None. E.S. Tai: None. P06.18.B Lipodystrophy-associated PPARγ mutants define subclasses of target genes

M. F. Broekema 1 , M. Stahl Madsen 2 , M. Rønn Madsen 2 , A. Koppen 1 , M. E. Kranendonk 1 , M. Groot Koerkamp 1 , N. Hamers 1 , A. Borgmans 1 , A. M. Bonvin 3 , F. C. Holstege 1 , W. Spiering 1 , H. Monajemi 4 , D. Cassiman 5 , S. Mandrup 2 , E. Kalkhoven 1

1 University Medical Center Utrecht, Utrecht, Netherlands, 2 University of Southern Denmark, Odense, Denmark, 3 Utrecht University, Utrecht, Netherlands, 4 Rijnstate Hospital, Arnhem, Netherlands, 5 Leuven University Hospitals, Leuven, Belgium
Introduction: The nuclear receptor PPARγ, encoded by the PPARG gene, is pivotal for adipocyte differentiation and function. How the different domains of PPARγ exactly communicate on intra- and intermolecular level to generate the appropriate transcriptional output in the context of chromatin is unresolved. New insights can be obtained from natural PPARG mutations, as found in patients suffering from familial partial lipodystrophy subtype 3 (FPLD3), characterized by repartitioning of adipose tissue causing insulin resistance and type 2 diabetes. We identified two FPLD3 mutations (E379K and R212Q) - located in distinct PPARγ domains - that impair the adipogenic capacity. Methods and materials: We have used genome-wide sequencing-based strategies in PPARG knockout mouse embryonic fibroblasts transduced with PPARγ to determine how these PPARG mutations affect the ability of PPARγ to activate PPARG target genes in the context of native chromatin structure. Results: Both mutations impair the ability of PPARγ to induce an overlapping subset of target genes, including classical PPARγ target genes such as Lpl , while retaining the ability to activate other genes including Acox1 . Genome-wide DNA binding profiles indicated that regulatory regions that require PPARγ for chromatin remodeling are particularly sensitive to the mutations. In addition, the exact nucleotide sequence of the PPARγ binding sites can potentially contribute to target gene sensitivity. Conclusions: We propose a model in which recruitment of PPARγ to chromatin is determined by multiple protein-protein and protein-DNA interfaces. Our findings indicate that relatively subtle molecular defects in PPARγ are sufficient to cause lipodystrophy by dysregulating a subset of PPARγ target genes. M.F. Broekema: None. M. Stahl Madsen: None. M. Rønn Madsen: None. A. Koppen: None. M.E. Kranendonk: None. M. Groot Koerkamp: None. N. Hamers: None. A. Borgmans: None. A.M. Bonvin: None. F.C. Holstege: None. W. Spiering: None. H. Monajemi: None. D. Cassiman: None. S. Mandrup: None. E. Kalkhoven: None. P06.21.B ‘Congenital Nicotinamide adenine dinucleotide (NAD) Deficiency Disorder treated with niacin supplementation’

A. M. Cueto-González , S. Arevalo, M. Del Toro, G. Giralt, A. Creus, M. Torres, I. Valenzuela, A. Abuli, E. Tizzano

Hospital Vall d’Hebron, Barcelona, Spain
Congenital NAD Deficiency is an extremely ultrarare disorder (OMIM 617660, ORPHA 521438) with only two previous cases reported (Shi et al. 2017). We report a new case diagnosed during pregnancy presenting cardiac malformations (Shone syndrome with high risk of aortic coarctation) and progressive intrauterine growth retardation since 20w with preserved head circumference and abdominal perimeter, without renal malformations or vertebral segmentation. Prenatal exome revealed a homozygous frameshift mutation in exon 1 of the HAAO gene (p.R15Gfs*99). The parents were heterozygous carriers and referred non-consanguinity. After birth the length was -3.2SD, we performed an abdominal ultrasound (normal), skeletal X-rays (normal) and cranial MRI (normal). The patient had elevated levels of 3-hidroxyanthralinic acid (3HAA) and 3-OH-kynurenina and normal levels of niacin, similar to the two previously reported cases. Given that defects in Haao-null mice owing to NAD deficiency can be prevented with supplementation with nicotinic acid (Shi et al., 2017), compasive use of niacin supplementation was started at one month. Follow-up at 3 months shows a normal length (-1.63SD). There are only two reported patients from Australia with homozygous variants in HAAO predicting loss-of-function changes in HAAO (different mutations that our patient). Cardiac, vertebral and renal malformation and hearing loss were present in both and Mondini defect and intellectual disability in one of them. To our knowledge, this is the third patient reported with NAD Deficiency and the first treated with niacin. Our patient has only three months and long term follow-up is required to determine efficacy . A.M. Cueto-González: None. S. Arevalo: None. M. Del Toro: None. G. Giralt: None. A. Creus: None. M. Torres: None. I. Valenzuela: None. A. Abuli: None. E. Tizzano: None. P06.29.A Research Priorities for Primary Mitochondrial Disorders: Defined by Patients, their Carers and Clinicians .

J. Lowndes 1 , R. Thomas 2 , S. Rahman 3 , R. Wheeler 4 , J. Sharpe 5 , S. Holmes 6 , M. Votruba 7 , C. Feeney 2 , L. Weaver 8 , L. Curtis 9 , L. Butterworth 9 , P. Hossain 8 , K. Staley 10 , A. Hunter 10 , S. Upadhyaya 11

1 Oxford University Hospitals NHS Foundation Trust, Oxford, United Kingdom, 2 Wellcome Trust Centre for Mitochondrial Research, Newcastle, United Kingdom, 3 UCL Great Ormond Street Institute of Child Health, London, United Kingdom, 4 LHON Society, London, United Kingdom, 5 Muscular Dystrophy UK, London, United Kingdom, 6 National Hospital for Neurology and Neurosurgery, London, United Kingdom, 7 Cardiff University and The University Hospital of Wales, Cardiff, United Kingdom, 8 Metabolic Support UK, London, United Kingdom, 9 The Lily Foundation, London, United Kingdom, 10 Genetic Alliance UK, London, United Kingdom, 11 James Lind Alliance, London, United Kingdom
Introduction: Primary mitochondrial disorders are clinically and genetically heterogeneous, for which there is currently a lack of effective therapies. It is therefore vital to know that competitively funded research meets the needs of patients and their clinicians. The Priority Setting Partnerships (PSPs) are an established, collaborative methodology that brings patients, charity representatives and clinicians together to try to establish the most pressing and unanswered research priorities for a particular disease group. Methods: After a scoping exercise to identify relevant partners, we chose to include questions relevant to both children and adults with primary mitochondrial disorders. A web-based questionnaire was developed and widely disseminated; asking for patients, carers and clinicians to pose their important research questions. This yielded 709 questions from 50 patients, 47 carers and 50 clinicians. These were grouped in to themes and filtered to exclude statements that were personal stories. Extensive literature searching was performed to determine which questions are unanswered based on current evidence. This process resulted in a list of 42 discrete questions. In a second web-based questionnaire, patients, carers and clinicians were invited to rank these 42 questions. This identified 24 questions that were taken forward to a workshop attended by a diverse range of patients, carers, charity representatives and clinicians. The relative merits and strengths of each question were debated to create a definitive ‘top 10 of unanswered research questions’. Results/Conclusion: Here we present the ‘top 10 of unanswered research questions for primary mitochondrial disorders’, as defined by the patients, their carers and clinicians. J. Lowndes: None. R. Thomas: None. S. Rahman: None. R. Wheeler: None. J. Sharpe: None. S. Holmes: None. M. Votruba: None. C. Feeney: None. L. Weaver: None. L. Curtis: None. L. Butterworth: None. P. Hossain: None. K. Staley: None. A. Hunter: None. S. Upadhyaya: None. P06.31.C High coverage NGS of mtDNA proofs apparent de novo variant to be in fact not de novo

U. Ahting 1 , B. Lorenz-Depireux 2 , R. Berutti 2 , M. Radivojkov-Blagojevic 2 , F. Radelfahr 3 , C. Stendel 3 , J. U. Walther 1

1 Institut für Humangenetik. TUM, München, Germany, 2 Helmholtz-Zentrum München, Institut für Humangenetik, Neuherberg, Germany, 3 Friedrich-Baur-Institut, München, Germany
Introduction: Maternally inherited pathogenic mtDNA variants increase in heteroplasmy during passage from mother to affected offspring. A pathogenic variant, which is present in an affected offspring, is often not detectable in various tissues of the mother, and therefore classified as “de novo”. Here we present a case, where an apparent “de novo” variant is actually not de novo, as it is detectable by high coverage NGS in the mother and other maternal relatives. Case: The male index patient is affected by Leigh syndrome, a classical mitochondrial disease. Symptoms are psychomotor delay and generalized dystonia and MRI brain scans show bilateral symmetrical lesions in the basal ganglia and brainstem. Muscle biopsy showed ragged red fibers. Mother and grandfather are affected by Parkinson´s disease. Results: Whole exome sequencing identified pathogenic variant m.10158T>C in mtDNA isolated from blood. High coverage NGS revealed that the variant is present in this sample with heteroplasmy of 62% (2710 of 4371 reads). Testing of mother and healthy siblings showed that the variant is present in all investigated tissues with low heteroplasmy (~1%). Therefore the variant is inherited. Discussion: Apparent de novo mtDNA-variants may be inherited and maternal relatives may be carriers of such variants. High coverage NGS of mtDNA can detect such cases. Increase of heteroplasmy in cases like this must be due to a strong bottleneck effect. If the bottleneck works by unequal partitioning (segregation), at least 6 cell divisions without mtDNA replication must have happened in the reported affected case. U. Ahting: None. B. Lorenz-Depireux: None. R. Berutti: None. M. Radivojkov-Blagojevic: None. F. Radelfahr: None. C. Stendel: None. J.U. Walther: None. P06.32.A Molecular mitochondrial DNA analysis in patients with suspected mitochondrial disorder

K. Grigalionienė , B. Burnytė, D. Vansevičiūtė, G. Žukauskaitė, L. Ambrozaitytė, A. Utkus

Department of Human and Medical Genetics, Institute of Biomedical Sciences, Faculty of Medicine, Vilnius University, Vilnius, Lithuania
Introduction: Mitochondrial diseases represent important and common source of inherited metabolic disorders. Mitochondrial dysfunction is characterized by defects in oxidative phosphorylation and caused by pathogenic variants in either mitochondrial DNA or nuclear mitochondrial genes. A correct diagnosis is challenging due to the complexity of the clinical presentations, lack of classical diagnostic clues and genetic heterogeneity. Patients and methods: In this study, mtDNA was examined in a cohort of 71 affected individuals (69 unrelated families) with suspected mitochondrial disorder. Comprehensive analysis of mtDNA was performed using following molecular genetic methods: Sanger sequencing of whole-length mtDNA for detection of point variants, long PCR and MLPA for detection of mtDNA deletions/duplications and RT-PCR for mtDNA copy number analysis. Results: Six (8.70%) patients were identified with pathogenic mtDNA variants in the cohort of 69 unrelated patients. Common mtDNA pathogenic variant NC_012920.1:m.3243A>G with variable heteroplasmy levels was identified in three unrelated female patients with heterogeneous phenotype of MELAS. Homoplasmic mtDNA pathogenic variants NC_012920.1:m.8993T>C and NC_012920.1:m.9185T>C were identified in patients presenting with Leigh syndrome. Novel heteroplasmic 5888 bp mtDNA deletion NC_012920.1:m.6069_11956del causing Kearns-Sayre syndrome was identified using long PCR and Sanger sequencing while MLPA profile was normal. MtDNA haplogroup analysis revealed a 43.5% frequency of the most common H haplogroup in the patients group. Conclusions: Benefits of mtDNA analysis manifest in the identification of pathogenic variants that could direct patients care, but limitations of the methods should be taken into account while selecting the best investigation strategy. Supported by grant TAP LLT-02/2015 K. Grigalionienė: None. B. Burnytė: None. D. Vansevičiūtė: None. G. Žukauskaitė: None. L. Ambrozaitytė: None. A. Utkus: None. P06.34.C Mitochondrial disorder diagnosis; a combined genomic and transcriptomic approach

J. Vella 1 , S. Laurie 2 , L. Matalonga 3 , J. Borg 4 , D. Soler 5 , E. Said 6 , I. Borg 6 , N. Vella 7 , J. Aquilina 7 , A. Felice 8

1 Department of Physiology and Biochemistry and Centre for Molecular Medicine and Biobanking, University of Malta, Msida, Malta, 2 Centro Nacional de Analisis Genomico (CNAG-CRG); Centre for Genomic Regulation; Barcelona Institute of Science and Technology (BIST), Universitat Pompeu Fabra (UBF), Barcelona, Spain, 3 Centro Nacional de Analisis Genomico (CNAG-CRG); Centre for Genomic Regulation; Barcelona Institute of Science and Technology (BIST); Universitat Pompeu Fabra (UBF), Barcelona, Spain, 4 Department of Health Sciences and Centre for Molecular Medicine and Biobanking, University of Malta, Msida, Malta, 5 Department of Paediatrics, Mater Dei Hospital, Msida, Malta, 6 Department of Pathology, Mater Dei Hospital, Msida, Malta, 7 Department of Neurology, Mater Dei Hospital, Msida, Malta, 8 Department of Physiology and Biochemistry and Centre for Molecular Medicine and Biobanking, Msida, Malta
Mitochondrial disorders are rare, genetically heterogenous diseases which affect the oxidative phosphorylation (OXPHOS) system. A combined genomic and transcriptomic approach was adopted to diagnose mitochondrial disorders in a cohort of 14 Maltese patients. Phenotypic data was recorded in the RD-Connect PhenoTips instance. The nuclear and mitochondrial genome were evaluated by whole exome sequencing. Variant filtration and prioritisation was undertaken using the RD-Connect Genome Phenome Analysis Platform (GPAP). The full transcriptome was analysed by RNA sequencing. Differential expression of candidate variants was confirmed by q-PCR. A HEK-293 cell model was used to functionally characterise the effect of a rare homozygous missense variant on mitochondrial complex 1 activity in one of the patients. Pathogenic variants were characterised according to their functional roles in OXPHOS biogenesis (see Table). The most variants were identified in Complex 1 OXPHOS subunits (N=8) and Complex 1 assembly factor variants were also identified in 4 of these patients. Two patients were diagnosed with MELAS. Gene expression analysis also revealed the effects of these variants. This combined genomic and transcriptomic approach could aid in establishing a diagnosis for these rare disease patients. J. Vella: None. S. Laurie: None. L. Matalonga: None. J. Borg: None. D. Soler: None. E. Said: None. I. Borg: None. N. Vella: None. J. Aquilina: None. A. Felice: None. P06.35.A Mitochondriopathy in pediatric patients with unspecific neuropediatric disease

A. van der Ven 1 , J. Denecke 2 , K. Tsiakas 2 , S. Wortmann 3,4,5 , D. Lessel 1 , F. Kortüm 1 , T. Bierhals 1 , H. Prokisch 3,4 , R. Santer 2 , M. Hempel 1

1 Dept. Human Genetics, UKE Hamburg-Eppendorf, Hamburg, Germany, 2 Dept. Pediatrics, UKE Hamburg-Eppendorf, Hamburg, Germany, 3 Dept. Human Genetics, TU Munich, Munich, Germany, 4 Dept. Human Genetics, Helmholz Centre Munich, Neuherberg, Germany, 5 Dept. Pediatrics, Paracelsus Medical University, Salzburg, Austria
Introduction: Mitochondriopathies account for an important subgroup of neuropediatric conditions but often the clinical presentation may be too unspecific to hint to an a priori disease hypothesis. Materials and Methods: We report on a deep phenotyping and trio-WES study of 492 unrelated children affected by previously undiagnosed and diverse complex neuropediatric disorders. All children underwent a standardized and comprehensive clinical work-up and trio-WES. In a subset of 31 children we had an a priori clinical suspicion of a mitochondriopathy. Results: Within this group of patients with a clinical suspicion of an underlying mitochondriopathy, we confirmed the diagnosis by identifying a pathogenic variant in a known mitochondriopathy-associated gene in 9/31 children (29%). In 10/31 children (32%) we discovered a pathogenic variant in a disease gene not associated with a mitochondriopathy. For 3/31 children we detected a pathogenic mutation in a candidate gene for a mitochondriopathy. In the group of 461 children with no suspicion of a mitochondriopathy, we identified pathogenic mutations in known mitochondriopathy-associated genes in 13 children (3%). In 226/461 children (49%) we detected pathogenic mutations in other known disease genes. For 222/461 (48%) patients no definite genetic diagnosis could be established. Conclusions: Overall, we molecularly established the diagnosis of a mitochondriopathy in 22 of 492 (4,5%) unselected neuropediatric patients. This study highlights the importance of WES for the identification of children with a clinically less characteristic presentation of their underlying mitochondriopathy. A. van der Ven: None. J. Denecke: None. K. Tsiakas: None. S. Wortmann: None. D. Lessel: None. F. Kortüm: None. T. Bierhals: None. H. Prokisch: None. R. Santer: None. M. Hempel: None. P06.36.B Mitochondrial DNA sequence context in the penetrance of all mt-tRNA mutations across many species: A study diagnostic implications

H. O’Keefe, R. A. Queen, J. S. Steyn, P. Lord, J. L. Elson

Newcatsle University, Newcastle, United Kingdom
Introduction: Mitochondrial disorders are heterogeneous, showing variable presentation and penetrance. Over the last three decades, our ability to recognize mitochondrial patients and diagnose these mutations, linking genotype to phenotype, has greatly improved. However, it has become increasingly clear that these strides in diagnostics have not benefited all population groups. It has been suggested that haplogroup (or lineage) context might influence the presentation and penetrance of mtDNA disease; thus, the spectrum of mutations that are associated with disease in different populations. Methods and Materials: This paper conducted the most extensive investigation to date into the importance of haplogroup context on the pathogenicity of mtDNA mutations. We searched for proven human point mutations across 726 multiple sequence alignments derived from 33 non-human species absent of disease. Results: 58 pathogenic point mutations arise in the sequences of these species. We assessed the sequence context and found evidence of population variants that could modulate the phenotypic expression of these point mutations masking the pathogenic effects seen in humans. Conclusions: This supports the theory that sequence context is influential in the presentation of mtDNA disease and has implications for diagnostic practices. Our current understanding of the pathogenicity of mtDNA point mutations, primarily built on studies of individuals with haplogroups HVUKTJ, will not present a complete picture. This will impair our understanding of disease mechanisms and create a diagnostic inequality, whereby individuals who do not belonging to more commonly studied lineages will be less likely to receive a genetic diagnosis. H. O’Keefe: None. R.A. Queen: None. J.S. Steyn: None. P. Lord: None. J.L. Elson: None. P06.37.C Unraveling Atypical Phenotypes in Neuronal Ceroid Lipofucinosis type 2

A. M. Amaral 1 , F. Fola 1 , Z. A. Carneiro 1 , L. Vagnini 2 , C. Fischinger Moura De Souza 3 , A. Pessoa 4 , C. M. Lourenco 1

1 Faculdade de Medicina - Centro Universitario Estacio de Ribeirao Preto, Ribeirao Preto, Brazil, 2 Centro Paulista de Diagnostico e Pesquisa, Ribeirao Preto, Brazil, 3 Hospital de Clínicas de Porto Alegre, Porto Alegre, Brazil, 4 Hospital Infantil Albert Sabin, Fortaleza, Brazil
Introduction: Late-infantile neuronal ceroid lipofuscinosis (NCL), also called neuronal ceroid lipofuscinosis type 2 (CLN2), is caused by the deficiency of lysosomal enzyme tripeptidyl peptidase-1 (TPP1) due to mutations in the CLN2 gene. The classical phenotype is late infantile presenting with language developmental delay, with its clinical signs usually appearing between the ages of 2 to 4 years with seizures and a rapid decline of psychomotor functions. Most children are bedridden at 5 years and die around the age of 10 years. Nevertheless, atypical phenotypes have been reported in the literature, creating a clinical challenge to avoid diagnosis without significant delay. Methods: Clinical, biochemical and neuroradiological data retrospective analysis of five CLN2 patients showing atypical/protracted phenotypes Results: All four patients (3 males; 2 females) showed an atypical late onset phenotype. First symptons started around 5 - 10 years of age being cerebellar ataxia the most prominent feature. Abnormal findings in brain MRI were found in all patients (cerebellar atrophy and periventricular white matter changes). Seizures were present in three patients, cognitive decline was seen in 4 patients and mild learning disabilities in one. TPP1 enzyme was decreased in all patients, although not in the typical range found in “classical” CLN2 patients. Discussion: The protracted CLN2 phenotype was previously considered globally rare. In South America, the frequency is approximately 50% of affected individuals. Recently with the recent approval of intraventricular enzyme replacement therapy (ERT) with cerliponase alpha for CLN2 disease, early diagnosis is essential to start treatment before disease progression. A.M. Amaral: None. F. Fola: None. Z.A. Carneiro: None. L. Vagnini: None. C. Fischinger Moura De Souza: None. A. Pessoa: None. C.M. Lourenco: None. P06.38.A Comprehensive characterization of a large cohort of patients with Niemann Pick disease

P. Guatibonza , C. Beetz, J. Gutierrez, S. Zafari, V. Skrahina, C. Cozma, P. Bauer, A. Rolfs

Centogene AG, Rostock, Germany
The term Niemann Pick disease (NPD) denotes a genetically and clinically heterogeneous group of lysosomal storage disorders. A deeper insight into NPD would benefit from large and uniformly characterized cohorts of patients. We queried our database for patients that had received a genetic diagnosis of NPD between December 2015 and August 2019. Information was compiled on: affected gene, age, gender, and country of origin, along with available clinical and metabolic data. A genetic diagnosis of NPD was issued for 733 patients. With n=472 (64.4%), the majority had variants in NPC1 , while SMPD1 and NPC2 were identified as the affected genes in 255 (34.8%) and 6 cases (0.8%), respectively. Most patients were infants (median age at diagnosis 3.6 years; range 0 to 76 years). They had been referred from 52 distinct countries, with the top 4 countries (Iran, Germany, Brazil and Egypt) collectively accounting for more than 50% of referrals. Translation of the available clinical information into human phenotype ontology (HPO) terms revealed that hepato- and/or splenomegaly was present in 76.5% of patients, intellectual disability in 27.9%, and jaundice in 19.0%. Further frequent HPO terms (i.e. present in >10%) included, abnormality of movement, ataxia, failure to thrive, and seizures. The NPD-specific biomarker LysoSM-509 was pathologically increased in 97.9% of the patients. Our cohort is amongst the largest described to date. By being well-characterized at numerous levels, it represents a valuable basis for discovering novel correlations and, thereby, for increasing our understanding of Niemann Pick disease. P. Guatibonza: A. Employment (full or part-time); Significant; Centogene AG. C. Beetz: A. Employment (full or part-time); Significant; Centogene AG. J. Gutierrez: A. Employment (full or part-time); Significant; Centogene AG. S. Zafari: A. Employment (full or part-time); Significant; Centogene AG. V. Skrahina: A. Employment (full or part-time); Significant; Centogene AG. C. Cozma: A. Employment (full or part-time); Significant; Centogene AG. P. Bauer: A. Employment (full or part-time); Significant; Centogene AG. A. Rolfs: A. Employment (full or part-time); Significant; Centogene AG. P06.40.C A novel 160Kb deletion identified in PCCA gene using whole exome sequencing in a patient with propionic acidemia

S. Farajzadeh Valilou 1,2,3 , H. Heydari 1 , S. Shohani 1,4 , S. Samadpour 1 , H. Abdi 1 , J. Karimzad Hagh 5 , M. Salimi Asl 1

1 Division of Medical Genetics, TehranLab, Tehran, Iran, Islamic Republic of, 2 Department of Medical Genetics, Kasra Hospital, Tehran, Iran, Islamic Republic of, 3 Medical Genetics Network (MeGeNe), Universal Scientific Education and Research Network (USERN), Tehran, Iran, Islamic Republic of, 4 Department of Biotechnology, Faculty of Allied Medicine, Iran University of Medical Sciences, Tehran, Iran, Islamic Republic of, 5 IVF Center, Cuypers and Cuypers, Hochstr. 152, Heinsberg, Germany
Background: Propionic acidemia (PA) is a severe metabolic disorder characterized by various clinical features. Herein, we present an 18-month-old male patient from consanguineous parents with early-onset of symptoms such as anemia, respiratory problem, and developmental delay. Urine examination exhibited an increased level of 3-hydroxypropionic acid and methylcitric acid, led to the primary diagnosis of PA. Methods: To identify the causative variant, DNA was extracted from the peripheral blood of the child and exons were captured and enriched to perform whole exome sequencing (WES). Data were analyzed using bioinformatics techniques and in silico predictors. Conventional PCR and Sanger sequencing were used to confirm the findings in WES. Additionally, comparative genomic hybridization (CGH)-array was carried out to study the accurate size of the copy number variation (CNV). Results: Surprisingly, no pathogenic variants in propionic academia causing genes ( PCCA and PCCB ) or phenotypically related disorders’ genes. Assuming the probably low coverage, we analyzed the PCCA and PCCB regions in the BAM file. In the PCCA , unlike PCCB , exons 12 to 21 were identified with no coverage, probably suggesting deletion of the approximately 220kb-size region. Not only we confirmed the novel PCCA deletion via PCR but also performing CGH-array revealed the approximate deletion size of 160Kb. Conclusions: We identified a novel homozygous deletion in the PCCA in the patient with PA using WES. Our results showed that WES can be used for identifying CNVs instead of CGH-array. However, a lot of studies should be done to improve the accuracy of this approach. S. Farajzadeh Valilou: None. H. Heydari: None. S. Shohani: None. S. Samadpour: None. H. Abdi: None. J. Karimzad Hagh: None. M. Salimi Asl: None. P06.41.A A novel hemizygous loss-of-function variant in the PRPS1 gene found in a patient with features of Arts syndrome, but without hearing loss and normal levels of purine/pyrimidine metabolites

S. Puusepp 1,2 , K. Reinson 1,2 , S. Pajusalu 1,2,3 , A. B. P. van Kuilenburg 4 , D. Dobritzsch 5 , W. Stenzel 6,7 , K. Õunap 1,2

1 Department of Clinical Genetics, Institute of Clinical Medicine, Faculty of Medicine, University of Tartu, Tartu, Estonia, 2 Department of Clinical Genetics, United Laboratories, Tartu University Hospital, Tartu, Estonia, 3 Department of Genetics, Yale University School of Medicine, New Haven, CT, United States, 4 Department of Clinical Chemistry, Cancer Center Amsterdam, Amsterdam Gastroenterology & Metabolism, Amsterdam UMC, University of Amsterdam, Amsterdam, Netherlands, 5 Department of Chemistry – BMC, Uppsala University, Uppsala, Sweden, 6 Department of Neuropathology, Charité– - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin, Germany, 7 Leibniz Science Campus Chronic Inflammation, Berlin, Germany
Introduction: The PRPS1 gene, located on Xq22.3, encodes phosphoribosyl-pyrophosphate synthetase (PRPS), a key enzyme in purine de novo synthesis. Three clinical phenotypes are associated with loss-of-function PRPS1 variants and decreased PRPS activity: Arts syndrome, Charcot-Marie-Tooth disease type 5 (CMTX5) and X-linked non-syndromic deafness. Hearing loss is present in all of these patients. CMTX5 patients additionally present with peripheral neuropathy and optic atrophy. Arts syndrome includes developmental delay, intellectual disability, ataxia and susceptibility to infections. Case report: We report a boy who presented with marked generalized muscular hypotonia, progressive global developmental delay, lack of speech, trunk instability, exercise intolerance, hypomimic face with opened mouth, oropharyngeal dysphagia, dysarthria, and very frequent upper respiratory tract infections. Cardiac, audiological and ophthalmological investigations were normal. A novel hemizygous variant c.130A>G p.(Ile44Val) was found in the PRPS1 gene by panel sequencing. PRPS activity in erythrocytes was markedly reduced confirming the pathogenicity of the variant. In retrospect, serum uric acid levels and the first urine purine/pyrimidine analysis were normal, but analysis of the follow-up sample showed nearly absent hypoxanthine. Conclusions: We describe a patient with features of Arts syndrome and a novel PRPS1 loss-of-function variant. However, at the current age of five years, our patient does not present with hearing loss, which has so far been reported in all of the Arts syndrome patients as congenital/early-onset. We would also like to emphasize that the first purine/pyrimidine analysis was normal giving no clues towards a dysfunction in the purine-pyrimidine metabolism. Funding: Estonian Research Council grants PUT355, PRG471, PUTJD827. S. Puusepp: None. K. Reinson: None. S. Pajusalu: None. A.B.P. van Kuilenburg: None. D. Dobritzsch: None. W. Stenzel: None. K. Õunap: None. P06.43.C Infantile and early childhood onset mitochondrial myopathy due to mutations in the TK2 gene with a phenotype of spinal muscular atrophy 5q. The first cases in Russia .

P. Tsygankova 1 , S. Kurbatov 2,3 , I. Bychkov 1 , Y. Itkis 1 , A. Galushkin 1 , K. Molewa 4 , V. Zabnenkova 1 , E. Zakharova 1

1 Research centre for medical genetics, Moscow, Russian Federation, 2 Voronezh Regional Clinical Consulting and Diagnostic Center, Voronezh, Russian Federation, 3 Association of Neuromuscular Disorders Specialists, Medical Center “Practical Neurology”, Moscow, Russian Federation, 4 Department of Neurology, "Dagestan State Medical University", Machachkala, Russian Federation
Mitochondrial myopathy (MM) due to thymidine kinase 2 deficiency and spinal muscular atrophy (5q) (SMA-5q) are two potentially curable hereditary diseases with different levels of damage to the neuromuscular system and etiology. Early childhood forms have a similar phenotype and are difficult for differential diagnosis. We present the clinical characteristics and molecular defect of MM with TK2 deficiency and informing health care specialists about the possibility of optimizing differential diagnosis. Materials and methods. This study involved 9 patients with excluded SMA-5q diagnosis. TK2 mutations were detected by NGS-gene panel in 3 of them. In addition, 96 DNA samples of patients with SMA-phenotype were screened for TK2 mutations by Sanger sequencing. Results. Nine patients with MM with TK2 from 6 unrelated families have been diagnosed. Three of them have been found retrospectively by analysis of 96 SMA-5q negative samples. Screening for mutations in the TK2 gene can be recommended for all children with a manifestation of the disease up to 2 years old, with rapidly progressing muscle weakness, increased CPK and/or lactate in the blood and/or detection of damage to the leg muscles on MRI. The study revealed four previously undescribed mutations in the TK2 gene: c.169G>A (p.Gly57Ser), c.310C>T (p.Arg104Cys), c.338T>A (p.Val113Glu), c.655T>C (p.Trp219Arg). Early molecular diagnostic is important for the choice of experimental nucleoside bypass treatment for this group of mitochondrial diseases. P. Tsygankova: None. S. Kurbatov: None. I. Bychkov: None. Y. Itkis: None. A. Galushkin: None. K. Molewa: None. V. Zabnenkova: None. E. Zakharova: None. P06.44.A TYPE 2 DIABETES AND CANCER CO-MORBIDITY: IS THERE SHARED GENETIC SUSCEPTIBILITY?

L. Zudina 1 , A. Demirkan 1 , I. Pupko 1 , M. J. Gunter 2 , M. Kaakinen 1 , I. Prokopenko 1

1 Department of Clinical & Experimental Medicine, University of Surrey, Guildford, United Kingdom, 2 Nutrition and Metabolism Section, International Agency for Research on Cancer, World Health Organization, Lyon, France
A plethora of epidemiological studies showed relationships between Type 2 Diabetes (T2D) and common cancers, reporting increased risks for postmenopausal breast (RR=1.20), colorectal (RR=1.30) and pancreatic (RR=1.94) cancers, and decreased risk for prostate cancer (RR=0.84) in T2D patients. We aimed to dissect the genetic factors shared between T2D, pancreatic and three common cancers using genome-wide genetic variability in a large case-control and population-based context. We evaluated the role of signals established for T2D on the susceptibility to postmenopausal breast, prostate, colorectal and pancreatic cancers in EPIC (European Prospective Investigation into Cancer and Nutrition) a combined nested case-control study of 14,553 individuals, including 4,345/2,120/426/499 with postmenopausal breast/colorectal/prostate/pancreatic cancer diagnosis and 476 prevalent cases of T2D. We generated polygenic risk scores (PRS) for breast/colorectal/prostate/pancreatic cancers and T2D using 171/90/154/21 and 403 established DNA variants for each disease, respectively. To perform reciprocal evaluation of PRS effects on diabetes and four cancer risks, we used the variants from original phenotype and the estimates of per-allele genetic effects from the other outcome. For all individual PRSs, we observed significant (P < 5x10 −5 ) increase in the risk of original outcomes. T2D PRS was significantly associated with risk of pancreatic cancer (OR=3.82[1.65-8.84], P=1.71x10 −3 ). This effect was stronger compared to the T2D PRS effect on T2D risk (OR=1.53[1.35-1.75], P=9.87x10 −11 ). The T2D and pancreatic cancer share signals at GRP , HNF1B BCAR1 and LINC-PINT/KLF14 loci only. Using the pan-European EPIC study data, we provide evidence for shared genetic factors between T2D and pancreatic cancer. Funding: WCRF-2017/1641 L. Zudina: None. A. Demirkan: None. I. Pupko: None. M.J. Gunter: None. M. Kaakinen: None. I. Prokopenko: None. P06.45.B Milder form of Vici syndrome due to novel missense variant affecting splicing

M. Skopkova 1 , K. Brennerova 2 , M. Ostrozlikova 3 , S. Borecka 1 , D. Gasperikova 1

1 Institute of Experimental Endocrinology, Biomedical Research Center, Slovak Academy of Sciences, Bratislava, Slovakia, 2 Department of Pediatrics, Medical Faculty of Commenius University and National Institute of Children’s Diseases, Bratislava, Slovakia, 3 Department of Laboratory Medicine, National Institute of Children’s Diseases, Bratislava, Slovakia
Introduction: Vici syndrome is a rare autosomal recessive syndrome caused by mutations in the gene EPG5 encoding ectopic P granules protein involved in autophagy. It is a neurodevelopmental disorder with multisystem involvement including corpus callosum agenesis, cataracts, hypopigmentation, cardiomyopathy, and immunodeficiency. It has been hypothesized that the severity of phenotype is related to residual level of functional EPG5. Patient: A 4 years old boy of Roma ethnicity with preterm birth, hypotonia, severe hypotrophy and growth retardation, developmental delay, mild brain atrophy, and urine organic acid profile suggesting mitochondrial disease was referred for whole-exome sequencing. Results: We have found a novel variant c.4205G>A (p.Arg1402Lys) in the EPG5 gene. As it is the last base of exon 23, we performed a mini-gene assay to estimate its effect on splicing. This has shown decreased inclusion of exon 23 compared to wild-type (40% vs. 72%, respectively). RNA sequencing confirmed only presence of the correctly spliced transcript. The patient was further examined for symptoms common in Vici syndrome and hypopigmented skin areas and immunodeficiency were confirmed. Interestingly, the patient has present corpus callosum, although thin, no seizures, no cataracts and no cardiomyopathy. Additionally, he has hemodynamically important stenosis of left pulmonary artery. The typical sign - pale skin and hair were also not present. Conclusions: Novel variant c.4205G>A causes aberrant splicing only in a proportion of transcripts, therefore, milder presentation of Vici syndrome in our patient is probably due to the residual presence of the EPG5 protein. Supported by VEGA 2/083/17 and APVV-17-0296. M. Skopkova: None. K. Brennerova: None. M. Ostrozlikova: None. S. Borecka: None. D. Gasperikova: None.

P07 Immunology and Hematopoietic System
P07.05.A A rare pair in one patient - Bernard-Soulier syndrome unraveled by 22q11.2 microdeletion syndrome

G. Taurina 1,2 , D. Murmane 1,2 , I. Voitovica 1 , S. Nikulsins 1 , I. Micule 1,3

1 Childrens Clinical University Hospital, Riga, Latvia, 2 Riga Stradins University, Riga, Latvia, 3 Biomedical Research and Study Centre, Riga, Latvia
Introduction: The 22q11.2 deletion syndrome is one of the most common microdeletion syndromes. Bernard-Soulier syndrome, in turn, is a rare autosomal recessive bleeding disorder (prevalence <1:1 000 000) with dysfunctional glycoprotein Ib-V-IX complex, responsible for the adhesion activity of platelets. We report a patient having both diseases. Case report: A boy who was admitted to hospital on the third day of life due to epistaxis, petechias and umbilical cord bleeding. Brain MRI showed subdural hematoma among cerebellar lobes. Blood count revealed severe thrombocytopenia (8 x 10 3 /uL), that required several platelet infusions and did not improve with immunoglobulin treatment. An NGS analysis for Bleeding disorder gene panel was ordered, with CNV analysis. On the second month of life flow cytometry revealed reduced CD42b expressivity. Results: The NGS gene panel results revealed a heterozygous pathogenic 22q11.2 microdeletion, encompassing 73 genes with GP1BB gene included. The analysis also showed a heterozygous likely pathogenic variant in GP1BB gene c.395T>A, p.(Leu132Gln). The combination of the microdeletion on one and the mutation on the other GP1BB allele explains the bleeding disorder of our patient and confirms in him Bernard-Soulier syndrome. 22q11.2 microdeletion syndrome was not suspected before the DNA analysis. Afterwards at a repeated consultation the patient demonstrated signs of velopharyngeal insufficiency but as yet no other signs of the Velocardiofacial syndrome. Conclusions: In patients with early onset bleeding disorder genetic testing with CNV analysis must be performed in order to not miss an autosomal recessive disease unmasked by a seemingly presymptomatic frequent microdeletion syndrome. G. Taurina: None. D. Murmane: None. I. Voitovica: None. S. Nikulsins: None. I. Micule: None. P07.08.A High intrafamilial variability in a C15orf41 associated Congenital Dyserythropoietic Anemia family indicates involvement of C2orf69 in infantile epilepsy

A. Gurel 1 , S. Unal 2 , N. Yarali 3 , P. Simsek Kiper 4 , S. Ceylaner 5 , O. A. Bilir 3 , F. Gumruk 2 , N. A. Akarsu 1 , A. Cetinkaya 1

1 Department of Medical Genetics, Faculty of Medicine, Hacettepe University, Ankara, Turkey, 2 Pediatric Hematology Unit, Department of Pediatrics, Faculty of Medicine, Hacettepe University, Ankara, Turkey, 3 Division of Pediatric Hematology, Ankara Child Health and Diseases Hematology Oncology Training and Research Hospital, Ankara, Turkey, 4 Pediatric Genetics Unit, Department of Pediatrics, Faculty of Medicine, Hacettepe University, Ankara, Turkey, 5 Intergen Genetic Diagnosis Center, Ankara, Turkey
Congenital dyserythropoietic anemias (CDAs) are a heterogenous group of disorders characterized by dysplastic erythroblasts, ineffective erythropoiesis, iron overload, splenomegaly and occasionally congenital limb abnormalities. Here, we report a CDA family with a novel homozygous mutation in C15orf41 , one of six CDA associated genes, and unusually high intrafamilial clinical variability. The proband, 3-month-old male born to consangineous parents, presented with transfusion-dependent anemia, malnutrition, recurrent pulmonary infections, hypotonia, refractory seizures and cerebral atrophy. His affected sibling had a milder neonatal anemia with no additional findings. Whole exome sequencing in proband revealed a homozygous missense mutation in C15orf41 (c.58C>A,p.Pro20Thr), which is not present in GnomAD and also found in the affected sibling. Another mutation in this codon (c.58C>T,p.Pro20Ser) had already been associated with CDA. In order to discover a second mutation that may explain the neurological findings in the proband, we focused on twelve >4Mbp runs of homozygosity and detected a nonsense mutation in C2orf69 (c.929G>A,p.Trp310Ter), which is absent in GnomAD and in-house databases. No other family member was homozygous for this variant. C2orf69 is highly uncharacterized, but we predict that absence of any homozygous truncating mutations in GnomAD indicates selective pressure on C2orf69 . A high expression in brain is apparent in GTEx dataset. In conclusion, we demonstrate that a novel mutation in C15orf41 affects a hotspot codon leading to CDA. In addition, we associate biallelic mutations in C2orf69 with intractable infantile epilepsy. Multiple molecular diagnoses not only underlie the apparent phenotypic variability but also is useful in uncovering previously unidentified genes. A. Gurel: None. S. Unal: None. N. Yarali: None. P. Simsek Kiper: None. S. Ceylaner: None. O.A. Bilir: None. F. Gumruk: None. N.A. Akarsu: None. A. Cetinkaya: None. P07.09.B A novel disorder involving dyshematopoiesis, inflammation and HLH due to aberrant CDC42 function

S. Coppola 1 , M. Lam 2,3,4 , O. H. Krumbach 5 , G. Prencipe 6 , A. Insalaco 6 , C. Cifaldi 7,8 , I. Brigida 9 , E. Zara 1 , S. Scala 9 , S. Martinelli 10 , M. Di Rocco 10,11 , A. Pascarella 6 , M. Niceta 12 , F. Pantaleoni 12 , A. Ciolfi 12 , F. Carisey 2,4 , M. Akbarzadeh 5 , L. Farina 1,6 , L. Pannone 10,12 , H. C. Erichsen 13 , A. C. Horne 14,15 , Y. Bryceson 16,17 , L. Torralba-Raga 16 , K. Ramme 18 , A. Finocchi 19,8 , F. Locatelli 20 , C. Cancrini 19,8 , A. Aiuti 9 , M. R. Ahmadian 5 , J. S. Orange 2 , F. De Benedetti 6 , M. Tartaglia 12

1 National center for rare diseases, Istituto Superiore di Sanità, Rome, Italy, 2 Department of Pediatrics, Columbia University, Irving Medical Center, New York, NY, United States, 3 Translational Biology and Molecular Medicine Graduate Program and Medical ScientistTraining Program, Baylor College of Medicine, Houston, TX, United States, 4 Department of Pediatrics, Baylor College of Medicine, Houston, TX, United States, 5 Institute of Biochemistry and Molecular Biology II, Medical Faculty of the Heinrich-Heine University, Düsseldorf, Germany, 6 Division of Rheumatology, Ospedale Pediatrico Bambino Gesù, IRCCS, Rome, Italy, 7 Department of Pediatrics, Ospedale Pediatricon Bambino Gesù, IRCCS, Rome, Italy, 8 Department of Systems Medicine, University of Rome Tor Vergata, Rome, Italy, 9 San Raffaele Telethon Institute for Gene Therapy (SR-TIGET), IRCCS San Raffaele Scientific Institute, Milan, Italy, 10 Department of Oncology and Molecular Medicine, Istituto Superiore di Sanità, Rome, Italy, 11 Department of Cellular Biotechnology and Haematology, Sapienza University of Rome, Rome, Italy, 12 Genetics and Rare Diseases Research Division, Ospedale Pediatrico Bambino Gesù, IRCCS, Rome, Italy, 13 Department of Pediatric and Adolescent Medicine, Oslo University Hospital, Oslo, Norway, 14 Pediatric Rheumatology, Astrid Lindgren Childrens Hospital, Karolinska University Hospital, Stockholm, Sweden, 15 Department of Women’s and Children’s Health, Karolinska Institutet, Karolinska University Hospital Solna, Stockholm, Sweden, 16 Department of Medicine, Karolinska Institutet, Karolinska University Hospital Huddinge, Stockholm, Sweden, 17 Broegelmann Research Laboratory, Institute of Clinical Sciences, University of Bergen, Bergen, Norway, 18 Pediatric Hematology, Immunology and HCT Section, Astrid Lindgren Children’s Hospital, Karolinska University Hospital, Stockholm, Sweden, 19 Department of Pediatrics, Ospedale PediatricoBambino Gesù, IRCCS, Rome, Italy, 20 Department of Pediatric Hematology and Oncology, Ospedale Pediatrico Bambino Gesù, IRCCS, Rome, Italy
Introduction: The diagnosis and delineation of novel genetic syndromes is often difficult given the limited availability of patients, genetic heterogeneity, and clinical variability. Hemophagocytic lymphohistiocytosis (HLH) is characterized by hyper-inflammation driven by excessive activation and expansion of macrophages and defective Natural Killer (NK) cytotoxicity; it can be fatal if untreated. Here, we identify a previously unrecognized hematological and autoinflammatory condition in four unrelated patients with superimposable features, including neonatal-onset trilinear cytopenia with dyshematopoiesis, autoinflammation, rash, and HLH. Methods: To delineate this new syndrome, we performed phenotype and whole-genome/exome analyses. In silico, in vitro and in vivo ( C. elegans ) assays allowed to characterize the impact of mutation on localization and function. Results: All patients shared the same de novo CDC42 mutation (Chr1:22417990C>T, p.R186C) and altered hematopoietic compartment, immune dysregulation, and inflammation. We documented unique effects of the p.R186C on CDC42 localization and function. Indeed, CDC42^R186C showed impaired interaction with known CDC42 regulators and effectors, including RHOGDI, IQGAP1 and WASP, leading to aberrant Golgi accumulation, cytoskeleton rearrangement and reduced growth and migration, which likely underlie the distinctiveness of the trait of this syndrome. Aberrant CDC42 function caused defective NK cytotoxicity, which likely contributes to the HLH phenotype. Timely therapeutic neutralization of IL-1β and IFNγ was critical to the survival of one patient, who underwent successful bone marrow transplantation. Conclusions: The presented data emphasize the crucial role played by CDC42 in hematopoiesis and inflammation. Early recognition of the disorder and establishment of treatment followed by bone marrow transplant are important to survival. S. Coppola: None. M. Lam: None. O.H. Krumbach: None. G. Prencipe: None. A. Insalaco: None. C. Cifaldi: None. I. Brigida: None. E. Zara: None. S. Scala: None. S. Martinelli: None. M. Di Rocco: None. A. Pascarella: None. M. Niceta: None. F. Pantaleoni: None. A. Ciolfi: None. F. Carisey: None. M. Akbarzadeh: None. L. Farina: None. L. Pannone: None. H.C. Erichsen: None. A.C. Horne: None. Y. Bryceson: None. L. Torralba-Raga: None. K. Ramme: None. A. Finocchi: None. F. Locatelli: None. C. Cancrini: None. A. Aiuti: None. M.R. Ahmadian: None. J.S. Orange: None. F. De Benedetti: None. M. Tartaglia: None. P07.11.A Studying known autoimmune risk loci in patients with CFS-ME

R. Hajdarevic , B. Lie, M. Viken

University of Oslo, Oslo, Norway
Introduction: CFS/ME is a chronic debilitating disease of unknown origin and cause, but a heritable component is implicated, likely harbouring a multifactorial aetiology Evidence from recent studies suggest an involvement of the immune system as well as a high degree of comorbidity with other autoimmune disorders. This has led us to explore the genetic architecture of CFS/ME to further explore the immune component of this pathology. Previous genetic studies of CFS/ME were limited by heterogeneous diagnostic criteria as well as sample size (<100 patients), thereby lacking statistical substance. Materials and Methods: We have genotyped 426 CFS/ME patient (diagnosed according to the strict Canadian criteria) and 973 controls with the Illumnia immunochip. The Immunochip is designed to screen for more than 250,000 common genetic variants covering genetic regions, previously associated with autoimmune and inflammatory diseases like rheumatoid arthritis and multiple sclerosis. Results: Our analysis has shown 16 significant and close to significant (P values ranging from 6.35E-14 to 9.90E-05) genetic associations with CFS/ME scattered over several different chromosomes. The most robust finding is a block of 4 genetic variants located on chromosome 2. Conclusions: To the best of our knowledge, our study is the first of this magnitude in CFS/ME. The results from our discovery cohort are promising and replication studies in 2 different cohorts are in progress at the time of writing this abstract (independent Danish cohort with >700 samples and a UK Biobank cohort with >1500 samples). Grant provided by the Research Council of Norway Project number: 27268 R. Hajdarevic: None. B. Lie: None. M. Viken: None. P07.14.A Clonal relationship and mutational landscape of diffuse large B-cell lymphoma recurrences

M. R. Berendsen , M. van den Brand, J. Rijntjes, G. Astuti, C. J. Hess, K. M. Hebeda, W. B. C. Stevens, P. J. T. A. Groenen, J. H. van Krieken, B. Scheijen

Radboudumc, Nijmegen, Netherlands
Introduction: Diffuse large B-cell lymphoma (DLBCL) is an aggressive disease, with a 30-40% relapse rate after first-line chemoimmunotherapy treatment. Some patients develop multiple recurrences, but the clonal relationship between the initial diagnosis and subsequent lymphomas is not routinely assessed. The molecular mechanisms underlying therapy response or failure are still poorly understood. To support diagnostics, guide therapy choices and eventually improve outcome of patients with DLBCL, we aim to define the clonal relationship and mutational landscape of DLBCL recurrences. Materials and Methods: In a cohort of 37 matched DLBCL diagnosis and one to three relapse biopsies, clonality analyses were performed by next-generation sequencing (NGS) of immunoglobulin (IG) gene rearrangements. The clonotypes for the different IG gene rearrangements were identified by ARReST/Interrogate bioinformatics. Mutation analysis by whole exome sequencing is currently being performed on a subset of the paired diagnosis-relapse samples. Results: IG-NGS clonality analyses on matched diagnosis-relapse(s) samples revealed identical dominant clonotype(s) in 33/37 of the recurrences, even in cases with an interval period of 14 years. However, in 4/37 DLBCL patients, at least one of the recurrences was clonally unrelated. Conclusions: DLBCL recurrences are clonally unrelated lymphomas in ~10% of the cases, suggesting the occurrence of second primary lymphoma, possibly linked to a genetic predisposition or immunodeficiency. Our cohort of clonally related cases will be subjected to mutational analyses to define the patterns of clonal evolution and identify genes related to therapy resistance. These studies will help to define the genetic landscape of relapsed DLBCL. M.R. Berendsen: None. M. van den Brand: None. J. Rijntjes: None. G. Astuti: None. C.J. Hess: None. K.M. Hebeda: None. W.B.C. Stevens: None. P.J.T.A. Groenen: None. J.H. van Krieken: None. B. Scheijen: None. P07.17.A Clinical and cytogenetic assessment in a cohort of Egyptian patients with Fanconi anemia: A 10 year experience

M. Farid 1 , M. M. Thomas 2 , G. Y. Kamah 2 , M. M. Eid 1

1 Human Cytogenetics Department, Human Genetics and Genome Division, National Research Centre, Cairo, Egypt, 2 Clinical Genetics Department, Human Genetics and Genome Division, National Research centre, Cairo, Egypt
Introduction: Fanconi anemia (FA) is a rare autosomal recessive disorder. Common clinical presentations include progressive bone marrow failure, abnormal skin pigmentations, skeletal malformations and high risk of malignancy. Due to its rarity and heterogeneous presentation, it is rather challenging to establish a definite registry on a national level. Therefore, this study describes the FA cases in the NRC 10 year registry, focusing on family history, clinical, hematological and cytogenetic findings. Methods: This study included 225 patients. Their age ranged from 4 months to 26 years. All patients were subjected to thorough clinical examination, pedigree construction, hematological and radiological investigations. Cytogenetic assessment using diepoxybutane(DEB)-induced chromosomal breakage was done to differentiate Fanconi anemia from acquired aplastic anemia. Results: 131 patients (58%) showed positive chromosomal breakage and were diagnosed with FA. 93% of them were of consanguineous parents and 21% showed positive family history as an affected sibling. Clinical examination showed; limb anomalies (20%), abnormal skin pigmentation (31%), bleeding tendency (22%) and kidney anomalies (10%) of the cases. Hematological investigations showed: cytopenia in one or more of blood cell types (52%), bone marrow failure (42%) and 34% had received blood /blood products at least once at the time of diagnosis. Cytogenetic study showed mosaicism in 10% of the FA cases which is significant for treatment assessment. Conclusions: This study is the first to present the clinical and cytogenetic characterizations of a cohort of Egyptian FA patients. Further large scale studies are needed to identify the prevalence of FA in the Egyptian society. M. Farid: None. M.M. Thomas: None. G.Y. Kamah: None. M.M. Eid: None. P07.19.C Myeloperoxidase modulates inflammation in generalized pustular psoriasis and further rare pustular skin diseases

S. Haskamp

Institute of Human Genetics, University Hospital Erlangen, Erlangen, Germany
Generalized pustular psoriasis (GPP) is a severe multi-systemic inflammatory disease characterized by neutrophilic pustulosis and triggered by pro-inflammatory IL-36 cytokines in skin. While 19-41% of patients carry bi-allelic mutations in IL36RN , the majority of patients do not carry mutations in known genes. In order to identify and characterize new pathways involved in the pathogenesis of GPP we performed whole exome sequencing in 31 GPP patients and demonstrated effects of mutations in MPO encoding the neutrophilic enzyme myeloperoxidase (MPO) in myeloid cells. We discovered eight MPO mutations resulting in MPO-deficiency in neutrophils and monocytes. Mutations were associated with GPP, primarily those resulting in complete MPO-deficiency (p=3.49E-10; OR=8.63). Dosage of mutant MPO alleles showed significant differences between 82 patients and >3,600 controls (p=2.67E-10); this effect was stronger when including IL36RN mutations (p < 2.20E-16) and correlated with a younger age of onset (p=0.0018). Activity of four proteases, previously implicated as activating enzymes of IL-36 precursors, correlated with MPO-deficiency. Formation of neutrophil extracellular traps (NETs) was reduced following phorbol myristate acetate induction (p=0.015). Phagocytosis assays in MPO-deficient mice and human suggested that altered neutrophil function together with impaired clearance of neutrophils by monocytes (efferocytosis) allowed prolonged neutrophil persistence in patients’ skin. MPO mutations contribute significantly to GPP’s pathogenesis. Our study implicates MPO as an inflammatory modulator in humans by regulating protease activity and NET formation, and modifying efferocytosis. Our findings confine using MPO inhibitors in cardiovascular diseases. MPO and affected pathways represent attractive targets for inducing resolution of inflammation in neutrophil-mediated skin diseases. S. Haskamp: None. P07.21.B A rare cause of Acute Myeloid Leukemia and a delayed diagnosis of Fanconi Syndrome: Mutations in FANCE

B. Cavdarli 1 , G. Yigit 2 , Y. Li 2 , V. Topcu 1 , B. Wollnik 2

1 Ankara Bilkent City Hospital, Ankara, Turkey, 2 Institute of Human Genetics, University Medical Center Göttingen, Göttingen, Germany
Introduction: Genomic instability syndromes are a group of inherited disorders that include a high variety of symptoms such as congenital anomalies, bone marrow failure, and cancer susceptibility. Fanconi Syndrome(FS) is a rare syndrome belonging to this family characterized by neurodevelopmental delay, radial-ray defects, microcephaly and progressive deficiency of bone marrow production. Material and Methods: We present a 33-year-old female who was monitored for Acute Myeloid Leukemia. Clinical findings of the patient included short stature, microcephaly, neurodevelopmental delay, patchy hypopigmented dark-colored skin, signs of premature aging and chronic liver disease. Results: Chromosomal breakage rate was detected high after mitomycin-C(MMC) treatment of cells suggesting chromosomal instability syndrome. We performed whole-exome-sequencing on the DNA of the index patient and her affected brother who had parental consanguinity. We identified an intronic variant, c.1114-8G>A in FANCE that was homozygous in the index patient as well as her affected brother, and it is predicted to generate a new acceptor splice-site resulting in alternative-splicing and premature protein truncation (p.Ile372Leufs*2). So far, this variant has only been described in a single case also of Turkish origin with a similar phenotype. Besides this patient, there are currently only four additional patients carrying biallelic mutations in FANCE. All of these patients were diagnosed in the first decade of their lives. Conclusions: We expand the clinical spectrum of patients with FANCE mutations and show that the absence of renal, cardiac and radial ray anomalies might be the cause of late diagnosis of FS caused by FANCE mutations. B. Cavdarli: None. G. Yigit: None. Y. Li: None. V. Topcu: None. B. Wollnik: None. P07.25.C Cell-free DNA in plasma—an essential immune system regulator

M. Korabecna 1 , A. Zinkova 1 , I. Brynychova 1 , B. Chylikova 1 , P. Prikryl 1 , L. Sedova 1 , P. Neuzil 2,3 , O. Seda 1

1 First Faculty of Medicine, Charles University, Prague, Czech Republic, 2 Northwestern Polytechnical University, Xi’an, China, 3 Brno University of Technology, Brno, Czech Republic
Cell-free DNA (cfDNA) is always present in plasma, and it is utilized in prenatal diagnostics as well as in oncology and transplantology for therapy efficiency monitoring. But does the cfDNA have a physiological role? Here we show that cfDNA presence in plasma of healthy individuals plays an indispensable role in immune system regulation. We exposed THP1 cells to healthy individuals’ plasma with (NP) and without (TP) cfDNA. In cells treated with NP, we found elevated expression of genes whose products maintain immune system homeostasis. Exposure of cells to TP triggered an innate immune response (IIR), documented particularly by elevated expression of pro-inflammatory interleukin 8. The results of mass spectrometry showed a higher abundance of proteins associated with IIR activation due to the regulation of complement cascade in cells cultivated with TP. Our results provide evidence that the presence of cfDNA in plasma of healthy individuals blocks fundamental mechanisms of the inflammation process. The expression profiles show that this process could play an important role in the regulation of tissue homeostasis. This finding epitomizes one of many intriguing properties and features of the human immune system. Its detailed understanding is of crucial interest for future medical applications. Financial support: grants LTACH19005 and Progres Q25/LF1 of the Ministry of Education, Youth and Sport of the Czech Republic, grant RVO-VFN 64165 of the Ministry of Health of the Czech Republic. P.N. would like to acknowledge financial support from State Administration of Foreign Experts Affairs number W099109 and CEITEC Nano+ project (CZ.02.1.01/0.0/0.0/16_13/0001728). M. Korabecna: None. A. Zinkova: None. I. Brynychova: None. B. Chylikova: None. P. Prikryl: None. L. Sedova: None. P. Neuzil: None. O. Seda: None. P07.27.B The impact of rare and common genetic variation in the Interleukin-1 pathway for human cytokine responses

R. C. van Deuren 1,2,3 , P. Arts 2,4 , G. Cavalli 1,5,6 , M. Jaeger 1,3 , M. Steehouwer 2 , M. van der Vorst 2 , C. Gilissen 2,3 , L. A. B. Joosten 1,3,7 , C. A. Dinarello 1,6 , M. M. Mhlanga 8,9 , V. Kumar 1,10 , M. G. Netea 1,3,11 , F. L. van de Veerdonk 1,3 , A. Hoischen 1,2,3

1 Department of Internal Medicine, Radboud University Medical Center, Nijmegen, Netherlands, 2 Department of Human Genetics, Radboud University Medical Center, Nijmegen, Netherlands, 3 Radboud Institute of Molecular Life Sciences (RIMLS), Radboud University Medical Center, Nijmegen, Netherlands, 4 Department of Genetics and Molecular Pathology, Centre for Cancer Biology, SA Pathology and the University of South Australia, Adelaide, Australia, 5 Unit of Immunology, Rheumatology, Allergy and Rare Diseases, IRCCS San Raffaele Hospital and Vita-Salute San Raffaele University, Milan, Italy, 6 Department of Medicine, University of Colorado, Aurora, CO, United States, 7 Department of Medical Genetics, Iuliu Hatieganu University of Medicine and Pharmacy, Cluj-Napoca, Romania, 8 Division of Chemical Systems & Synthetic Biology, Institute for Infectious Disease & Molecular Medicine (IDM), Department of Integrative Biological & Medical Sciences, University of Cape Town, Cape Town, South Africa, 9 Faculty of Health Sciences, Department of Integrative Biomedical Sciences, University of Cape Town, Cape Town, South Africa, 10 University of Groningen, University Medical Center Groningen, Department of Genetics, Groningen, Netherlands, 11 Department for Genomics & Immunoregulation, Life and Medical Sciences Institute (LIMES), University of Bonn, Bonn, Germany
Interleukin(IL)-1 signaling is of major importance in human innate cytokine responses. Common genetic variants in underlying genes have been linked to various inflammation-mediated diseases and pathogen induced cytokine responses, but the role of rare variants remains to be elucidated. In this study, we characterize the role of rare and common genetic variation in 48 genes related to the IL-1 pathway, on in vitro stimulation-specific human cytokine responses from 463 healthy individuals. We identified strong NCF4 and CASP1 rare variant associations with IL-6 cytokine production induced by phytohaemagglutinin (P=7.2E −05 ) and lipopolysacharide (LPS) (P=3.0E −05 ). Additionally, common variants in IL36A and IL38 were associated to both C.albicans induced IL-1β and IL-6 ( IL36A P=0.0442 and 0.0037; IL38 P=0.0092 and 0.0082), an effect that was magnified on the respective IL-30 subpathway level (IL-1β P=0.0017; IL-6 P=1.8E −04 ). The common variant signature for the immunological response to C.albicans was confirmed by an association with the anti-inflammatory phenotype group (IL-1β P=1.87E −03 ; IL-6 P=5.75E −04 ), and we validated this for non-coding common variants. Lastly, we detected an association between rare variants in pro-inflammatory genes and LPS-induced IL-6 cytokine (P=2.42E −04 ), and discovered a role for rare variants in anti-inflammatory genes on S.aureus induced IL-6 production (P=6.71E −03 ). In conclusion, we show that both common and rare genetic variation in genes of the IL-1 pathway differentially influence in vitro cytokine responses to various stimuli in healthy individuals. This study therefore provides insight into potential mechanisms relevant for the characterization of genetic variant involvement in variability of immune responses and possibly inflammatory-mediated diseases. R.C. van Deuren: None. P. Arts: None. G. Cavalli: None. M. Jaeger: None. M. Steehouwer: None. M. van der Vorst: None. C. Gilissen: None. L.A.B. Joosten: None. C.A. Dinarello: None. M.M. Mhlanga: None. V. Kumar: None. M.G. Netea: None. F.L. van de Veerdonk: None. A. Hoischen: None. P07.28.C Correcting clinical diagnosis with targeted immundeficiency gene panel sequencing: the critical role of genetic counselling

K. Arvai 1 , A. Illés 1 , B. Kocsis-Deák 1 , V. Klujber 1 , B. Tóbiás 1 , B. Balla 1 , H. Pikó 1 , P. Lakatos 2 , J. Kósa 1,2

1 PentacoreLab Ltd., Budapest, Hungary, 2 Semmelweis University 1st Department of Internal Medicine, Budapest, Hungary
Immunodeficiency results from a failure or absence of elements of the immune system including lymphocytes, phagocytes and complement system. These immunodeficiencies can be either primary such as Bruton’s disease or secondary as the one caused by HIV infectionIn our case, a 35 years old healthy female patient was referred to our lab, who lost two of her brothers with severe immunodeficiency. She is about to have her first child and wanted to be tested before the pregnancy. The suspected familiar diagnosis, based only clinical symptoms, was X-linked agammaglobulinemia which is a X-linked monogenic disorder. After sequencing of the BTK gene, MLPA were carried out, both returned with negative results. After second genetic counselling it was revealed by the counsellor that the suspected phenotype is not fit entirely and targeted gene panel test was ordered, included 200 genes which are linked to immunodeficiencies. A pathogenic heterozygous deletion in the CD40LG gene (c.67dupA) was identified, which is a known cause of X-linked immunodeficiency, with hyper-IgM. HIGM is a rare immunodeficiency characterized by normal or elevated serum IgM levels associated with markedly decreased IgG, IgA, and IgE, resulting in a profound susceptibility to bacterial infections and an increased susceptibility to opportunistic infections. Patients also tend to have neutropenia, as well as a high rate of gastrointestinal and central nervous system infections. Based on estimation only 20% of patients will reach the third decade of life. Our results emphasize the importance of multiple genetic counselling and highlights the difficulties to interpret clinical results decades ago. K. Arvai: None. A. Illés: None. B. Kocsis-Deák: None. V. Klujber: None. B. Tóbiás: None. B. Balla: None. H. Pikó: None. P. Lakatos: None. J. Kósa: None. P07.31.C Hyperinflammation and secondary HLH in a patient with Interferon alpha/beta receptor 2 (IFNAR2) deficiency

C. Passarelli 1 , A. Civino 2 , M. N. Rossi 3 , V. Lanari 1 , G. M. Moneta 3 , A. Pascarella 3 , C. Bracaglia 3 , R. Montinaro 4 , F. De Benedetti 3 , A. Novelli 1 , G. Prencipe 3

1 U.O.C. Laboratory of Medical Genetics, IRCCS Paediatric Hospital “Bambino Gesu”, Rome, Italy, 2 Rheumatology and Paediatric Immunology, PO "Vito Fazzi", Lecce, Italy, 3 Division of Rheumatology, IRCCS Paediatric Hospital “Bambino Gesu”, Rome, Italy, 4 Division of Pediatry, PO "Santa Caterina Novella", Galatina, Italy
Introduction: Interferons α/β (Type I IFNs) play an important role in defense against viral infections. Here we report the case of a 22-months-old Caucasian boy presenting with high fever and lethargy five days after live-attenuated MMR (measles-mumps-rubella) vaccine. Laboratory parameters were suggestive for secondary Hemophagocityc lymphohistiocytosis (HLH), with progressive decrease in cell blood count, hyperferritinemia, elevation of liver enzymes and lactate dehydrogenase and hypofibrinogenemia. Materials and Methods: Clinical Exome, using a custom panel including 6920 genes known as associated to genetic diseases, was sequenced on Illumina NovaSeq6000® platform. In silico analysis was performed on the basis of the patient’s clinical phenotype. Peripheral blood mononuclear cells (PBMCs) were isolated from the patient and his family and stimulated with IFNα or IFNγ; phosphorylation of STAT1 and Type I and II IFN signatures were analyzed by FACS analysis and RT-PCR. Results: We identified the two novel frameshift mutations c.234delT and c.555_559delAAAAG, in a compound heterozygous status, in IFNAR2 gene (OMIM# 602376), resulting respectively in p.Leu79Ter and p.Ile185MetfsTer12 variants. Ex vivo studies on patient’s PBMCs confirmed the defect in the activation of the type I IFN signaling, but not in type II IFN response. Treatment with high dose of intravenous methylprednisolone allowed a progressive improvement of patient condition and of clinical and laboratory features. Conclusions: Our data demonstrate that HLH episodes following administration of live-attenuated MMR vaccine could suggest the presence of a defect in the type I IFN response. C. Passarelli: None. A. Civino: None. M.N. Rossi: None. V. Lanari: None. G.M. Moneta: None. A. Pascarella: None. C. Bracaglia: None. R. Montinaro: None. F. De Benedetti: None. A. Novelli: None. G. Prencipe: None. P07.32.A Genome wide association study of circulating interleukin 6 levels identifies novel loci

T. S. Ahluwalia 1 , N. J. Armstrong 2 , S. Aslibekyan 3 , M. Beekman 4 , Y. Cheng 5 , E. deGeus 6 , G. E. Delgado 7 , D. Marek 8 , S. Kanoni 9 , I. M. Nolte 10 , E. Porcu 11 , I. Seppälä 12 , M. Standl 13 , A. Teumer 14 , A. Thalamuthu 15 , S. Trompet 16 , E. J. Benjamin 17 , M. F. Feitosa 18 , G. Homuth 19 , J. Lahti 20 , Y. Liu 21 , N. J. Timpson 17 , S. Visvikis-Siest 22 , U. Völker 19 , B. T. Baune 23 , D. Boomsma 24 , I. J. Deary 25 , D. M. Evans 17 , M. A. Ferreira 26 , T. Gaunt 17 , V. Gudnason 27 , A. Hamsten 28 , S. E. Humphries 29 , W. Koeing 30 , M. Kumari 31 , D. A. Lawlor 17 , M. Nauck 32 , J. F. Price 33 , T. I. A. Sørensen 34 , D. Stacey 35 , M. G. Stathopoulou 22 , T. Tanaka 36 , S. G. Wannamethee 37 , J. I. Rotter 38 , A. Dehghan 39 , E. Boerwinkle 40 , H. Sneider 10 , B. M. Psaty 41 , B. P. Prins 10 , B. Z. Alizadeh 10

1 Steno Diabetes Center Copenhagen, Gentofte, Denmark, 2 Murdoch university, Perth, Australia, 3 University of alabama at birmingham school of public health, Birmingham, AL, United States, 4 Leiden university medical center, Leiden, Netherlands, 5 University of Maryland school of medicine, Baltimore, MD, United States, 6 Vrije universiteit amsterdam, Amsterdam, Netherlands, 7 Vth department of medicine university of heidelberg, Mannheim, Germany, 8 SIB swiss institute of bioinformatics, Lausanne, Switzerland, 9 William harvey research institute, queen mary university of london, London, United Kingdom, 10 university of groningen, university medical center groningen,, Groningen, Netherlands, 11 Istituto di ricerca genetica e biomedica, consiglio nazionale delle ricerche, Cagliari, Italy, 12 Department of clinical chemistry, tampere university, Tampere, Finland, 13 Institute of epidemiology, helmholtz zentrum münchen - german research center for environmental health, Neuherberg, Germany, 14 Institute for community medicine, university medicine greifswald, Greifswald, Germany, 15 Neuropsychiatric institute, prince of wales hospital, Sydney, Australia, 16 Department of cardiology, leiden university medical center,, Leiden, Netherlands, 17 MRC integrative epidemiology unit, university of bristol, Bristol, United Kingdom, 18 Department of genetics, washington university school of medicine, St. Louis, MO, United States, 19 Interfaculty institute for genetics and functional genomics, university medicine Greifswald, Greifswald, Germany, 20 Turku institute for advanced studies, university of turku, Turku, Finland, 21 Department of epidemiology and prevention, wake forest school of medicine, winston-Salem, NC, United States, 22 Université de lorraine, INSERM, Nancy, France, 23 Department of psychiatry and the Florey institute of neuroscience and mental health the university of Melbourne, Melbourne, Australia, 24 Department of biological psychology, behaviour and movement sciences, vrije universiteit Amsterdam, Amsterdam, Netherlands, 25 Centre for cognitive ageing and epidemiology, university of edinburgh, Edinburgh, United Kingdom, 26 QIMR berghofer medical research institute, Brisbane, Australia, 27 Faculty of medicine, university of iceland, Reykjavik, Iceland, 28 Department of medicine solna, center for molecular medicine, karolinska institutet, Stockholm, Sweden, 29 Institute of cardiovascular science, university college london, London, United Kingdom, 30 German Heart Centre Munich, Technical University Munich, Munich, Germany, 31 Department of epidemiology and public health, university college london, London, United Kingdom, 32 Institute of clinical chemistry and laboratory medicine, university medicine greifswald, Greifswald, Germany, 33 Usher institute of population health sciences and informatics, university of edinburgh, Edinburgh, United Kingdom, 34 Department of public health, university of copenhagen, Copenhagen, Denmark, 35 MRC cardiovascular epidemiology unit, university of cambridge, Cambridge, United Kingdom, 36 Translational gerontology branch, national institute on aging, Baltimore, MD, United States, 37 Institute of epidemiology & health care, UCL, London, United Kingdom, 38 Departments of pediatrics and medicine, harbor-ucla medical center, Los Angeles, CA, United States, 39 Department of epidemiology, erasmus mc, Rotterdam, Netherlands, 40 Human genetics center, university of texas, Houston, TX, United States, 41 Cardiovascular health research unit, department of medicine, university of washington, Seattle, WA, United States
Interleukin-6 (IL-6) is a multifunctional cytokine with both pro and anti-inflammatory properties, synthesized by a wide range of tissues and cell types. Increased levels of circulating IL-6 in blood is associated with the pathophysiology of complex disorders like type 2 diabetes, cardiovascular and autoimmune diseases. Albeit, IL-6 levels are heritable with estimates up to 61%, only a few common genetic loci associated with circulating IL-6 levels have been identified. We therefore conducted a two stage (discovery and replication) meta genome-wide association study (GWAS) of circulating serum IL-6 concentrations comprising up to 67,428 individuals of european ancestry. About 2.5 million single nucleotide polymorphisms (SNPs) were available for testing after imputation to Hap Map 2 reference panel. We conducted an inverse variance based fixed effects meta-analysis. We identified three IL-6 associated, independent signals on chromosomes (chr) 2q14, 6p21 and 1q21, reaching genome-wide significance ( p < 5.0 × 10 −8 ) in the combined meta-analyses. Among the identified loci IL1F10/IL1RN (chr 2q14, p = 1.8 × 10 −11 ), and HLA-DRB1 / DRB5 (chr 6p21, p =1.5 × 10 −10 ) were novel while IL6R (chr 1q21, p = 1.2 × 10 −122 ) was a known locus. Our study identifies 2 novel loci for circulating IL-6 levels uncovering new immunological and inflammatory pathways that may influence IL-6 pathobiology. T.S. Ahluwalia: None. N.J. Armstrong: None. S. Aslibekyan: None. M. Beekman: None. Y. Cheng: None. E. deGeus: None. G.E. Delgado: None. D. Marek: None. S. Kanoni: None. I.M. Nolte: None. E. Porcu: None. I. Seppälä: None. M. Standl: None. A. Teumer: None. A. Thalamuthu: None. S. Trompet: None. E.J. Benjamin: None. M.F. Feitosa: None. G. Homuth: None. J. Lahti: None. Y. Liu: None. N.J. Timpson: None. S. Visvikis-Siest: None. U. Völker: None. B.T. Baune: None. D. Boomsma: None. I.J. Deary: None. D.M. Evans: None. M.A. Ferreira: None. T. Gaunt: None. V. Gudnason: None. A. Hamsten: None. S.E. Humphries: None. W. Koeing: None. M. Kumari: None. D.A. Lawlor: None. M. Nauck: None. J.F. Price: None. T.I.A. Sørensen: None. D. Stacey: None. M.G. Stathopoulou: None. T. Tanaka: None. S.G. Wannamethee: None. J.I. Rotter: None. A. Dehghan: None. E. Boerwinkle: None. H. Sneider: None. B.M. Psaty: None. B.P. Prins: None. B.Z. Alizadeh: None. P07.33.B KIR-HLA class I ligand combinations among cystic fibrosis patients

K. Sosnina 1 , D. Zastavna 1,2 , O. Terpyliak 1 , H. Makukh 1 , L. Bober 3

1 SI “Institute of Hereditary Pathology Ukrainian National Academy of Medical Sciences”, Lviv, Ukraine, 2 Department of Biotechnology and Bioinformatics, Faculty of Chemistry, Rzeszow University of Technology, Al. Powstańców Warszawy 6, Rzeszow, Poland, 3 Lviv Regional Children’s Specialized Clinical Hospital, Lviv, Ukraine
KIR and HLA loci play a critical role in NK cell regulation. Different compound KIR-HLA genotypes can impart different thresholds of activation to the NK repertoire and such genotypic variation has been found to confer altered risk in a number of human diseases including infectious diseases. The aim of this study was to establish and analyze the spectrum of KIR-HLA genes in people with a confirmed diagnosis of CF, homozygote of F508del mutation of the СFTR gene for understanding the genetic predisposition of congenital immunity key part functioning during CF. Results: KIR and HLA class I ligands (C1,C2) genes distribution was established in 48 CF patients and 104 practically healthy individuals of control group. HLAC genotypes frequency did not differ in the CF group and in the control. The lower frequency of KIR2DL3 , KIR2DS1 and KIR2DS4 genes (37.5%, 6.25% and 0%) was found among CF patients (p < 0.05) compared to control group (67.3%, 20.2% and 10.5%) indicating a strong inhibitory effect on NK cells. Respectively, the frequency of KIRBB haplotype 8.34% in CF patients was significantly higher compared to the control 0.96 % (p < 0.05). Analysis of combined frequencies for iKIR+HLA (> = <) aKIR+HLA showed no significant difference between the study groups. iKIR+ HLA⁄aKIR analysis showed significantly lower frequency iKIR+HLA

K. Varvagiannis 1 , D. Gerasoudis 1 , A. Filia 1 , N. Panousis 2 , E. Dermitzakis 1,3,4,5 , G. Bertsias 6,7 , D. Boumpas 1,8,7,9 , A. Banos 1 , P. Makrythanasis 1,3

1 Biomedical Research Foundation Academy Of Athens, Athens, Greece, 2 Wellcome Trust Sanger Institute, Hinxton, Cambridge, United Kingdom, 3 Department of Genetic Medicine and Development, University of Geneva Medical School, Geneva, Switzerland, 4 Institute of Genetics and Genomics in Geneva (iGE3), University of Geneva, Geneva, Switzerland, 5 Swiss Institute of Bioinformatics, Geneva, Switzerland, 6 Department of Rheumatology, Clinical Immunology and Allergy, University of Crete, Medical School, Heraklion, Greece, 7 Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology - Hellas (FORTH), Heraklion, Greece, 8 Joint Academic Rheumatology Program, National and Kapodistrian University of Athens, Medical School, Athens, Greece, 9 Medical school, University of Cyprus, Nicosia, Cyprus
Background: Systemic lupus erythematosus (SLE) is a chronic autoimmune disease that can affect virtually any organ/system. Clinical features in individual patients can be highly variable, ranging from mild arthritis, skin manifestations and antinuclear antibody production to life-threatening organ disease. SLE exhibits a striking female predominance, with ratios exceeding 9:1. The disease often displays a more aggressive clinical course in men, resulting in poorer prognosis. Several hypotheses have been put forward to explain this gender bias, including X-chromosome inactivation, sex hormones and TLR/miRNAs altered expression. Methods: By employing data from mRNA sequencing in whole blood samples from 142 SLE patients and 58 healthy volunteers we explore gender/disease specific patterns of gene expression and their putative functional gene networks as suggested by DAVID analysis. Results: Genes displaying a stepwise up-/down-regulation upon comparison of their expression in the order of healthy males (HM), healthy females (HF), affected females (AF) and affected males (AM) groups, were the most over-represented among all possible combinations. These genes appeared to belong to pathways highly relevant to SLE pathogenesis, including NOD-/Toll-like receptor signaling, ubiquitin mediated proteolysis, nucleotide excision repair and spliceosome ( p -values < 0.05). Conclusions: Although preliminary, our results suggest that gender bias may - at least partially - be reflected by patterns of altered expression of genes/pathways relevant to SLE. Future analysis in lupus mouse model will reveal genetic impact on gender-specific gene expression. K. Varvagiannis: None. D. Gerasoudis: None. A. Filia: None. N. Panousis: None. E. Dermitzakis: None. G. Bertsias: None. D. Boumpas: None. A. Banos: None. P. Makrythanasis: None. P07.35.A MicroRNAs expression may affect the lymphocyte concentration in Down syndrome

M. F. Mattos 1 , O. B. Nascimento 1 , G. A. S. Passos 2 , J. M. Biselli 3 , E. M. Goloni-Bertollo 1 , É. C. Pavarino 1

1 Unidade de Pesquisa em Genética e Biologia Molecular da Faculdade de Medicina de São José do Rio Preto, São José do Rio Preto, Brazil, 2 2 Faculdade de Medicina de Ribeirão Preto da Universidade de São Paulo – USP, Ribeirão Preto, Brazil, 3 3Universidade Estadual Paulista Júlio de Mesquita Filho- UNESP, São José do Rio Preto, Brazil
Introduction: Studies have showed the importance of microRNAs in Down syndrome(DS) pathology and impaired T cell proliferation may result of altered microRNAexpression. Therefore, in this study we investigated the relationship between expressionpattern of hsa-miR-378a-3p, hsa-miR-668-3p and hsa-miR 942-5p and lymphocytesconcentration in DS. Material and Methods: Peripheral blood mononuclear cells were obtained from 52healthy individuals with full trisomy 21 (DS) (age 1 to 20) and 53 healthy control (HC)(age 1 to 12). MicroRNAs expression was investigated by real time polymerase chainreaction using Taqman assays. Statistical analysis was performed using One-SampleWilcoxon Signed Rank Test, in the GraphPad Prism 6 software. Bioinformatic analysiswas carried out to identify genes regulated by expressed differentially microRNAs andthe interaction between proteins using MiRTarbase v9 and STRING t-v11.0 tool. Results: DS individuals presented over-expression of hsa-miR-378a-3p (RQ 1.22, P=0.036) and down-expression of hsa-miR-668-3p (RQ 0.42, P=0.018). Moreover, thisindividuals presented lower lymphocytes concentration than HC (mean 36.70, 48.68; respectively, P=0.007). Network analysis showed that genes regulated by hsa-miR-378a-3p and hsa-miR-668-3p are significantly involved in cell differentiation, celldivision, cell cycle (P=<0.001) and regulation of cell growth (P=0.02). Conclusions: The lower lymphocytes concentration in DS may in part explain the highfrequency of immune alterations. Genes that regulates the lymphocyte concentrationsare probably affected by impaired microRNA expression. MicroRNA target genes areinvolved in biological process relevant to immune response as cell proliferation. Grants: FAPESP (2018/09126-0 and 2018/24825-2), CNPQ (310806/2018-6), CAPES(001), FAMERP/FUNFARME. M.F. Mattos: None. O.B. Nascimento: None. G.A.S. Passos: None. J.M. Biselli: None. E.M. Goloni-Bertollo: None. É.C. Pavarino: None. P07.36.B Surprising genetic results in a case of neurofibromatosis-like syndrome

M. T. Bataneant 1,2 , M. Puiu 1,2 , M. Baica 3 , E. Boeriu 1,2 , P. Urtila 1,2 , C. Zaica 3 , A. Beloia 4 , A. Chirita-Emandi 1

1 University of Medicine and Pharmacy "Victor Babes", Romania, Romania, 2 Clinical Emergency Hospital for Children „Louis Turcanu”, Timisoara, Romania, 3 Louis Turcanu Emergency Children Hospital, Timisoara, Romania, 4 Louis Turcanu Emergency Children Hospital, Romania, Romania
Introduction: Neurofibromatosis is an autosomal dominant disorder consisting in cutaneous, neurologic, ophthalmologic and tumoral signs. But always other disorders that can mimic neurofibromatosis should be excluded. Case presentation: A 10-year-old girl, from consanguineous gipsy parents, was admitted for anemia. Family history showed a sister who died due to cerebral tumors at the age of 3. The personal medical history was insignificant. The clinical examination revealed more than 6, cafe au lait” spots larger than 0,5cm and multiple axillary freckling, signs which support the diagnosis of neurofibromatosis. The clinical exam of the parents was normal. Laboratory explorations showed a normochromic, normocytic anemia, normal ferritin level, liver and kidney function, folate deficiency, but also immunological abnormalities: IgA, IgG2 and IgG4 deficiency, poor response to vaccination, low memory B cells and a normal number of T cell subpopulations. Ophthalmologic examination was normal. The cerebral MRI-scan highlighted multiple 0,5 and 4 cm in diameter tumors. The cerebral biopsy revealed a diffuse astrocytoma IDH mutant and she started chemotherapy. Taking in account the consanguinity, family history and the negative clinical exam of the parents we performed genetic exam - WES that revealed a pathogenic homozygous c.2653A>T mutation in MSH6 gene, parents being carriers for the same mutation. Conclusions: Homozygous mutation in MSH6 gene is a very rare type of immunodeficiency from DNA repair syndromes and it must be suspected in atypical cases of neurofibromatosis associating immunological abnormalities. A correct diagnosis allows genetic counseling and monitoring for the cancer development in other members of the family. M.T. Bataneant: None. M. Puiu: None. M. Baica: None. E. Boeriu: None. P. Urtila: None. C. Zaica: None. A. Beloia: None. A. Chirita-Emandi: None. P07.37.C Novel candidate genes and sequence variants for susceptibility to mycobacterial infections revealed by exome sequencing

A. M. Varzari 1,2 , M. Dembic 3,4,5 , G. Hoffmann Bruun 3 , I. V. Deyneko 6 , W. Hofmann 7 , V. M. Cebotari 8 , E. G. Privalova 9 , B. S. Andresen 3 , T. Illig 2,7

1 Laboratory of Human Genetics, Chiril Draganiuc Institute of Phthisiopneumology, Kishinev, Moldova, Republic of, 2 Hannover Unified Biobank, Hannover Medical School, Hannover, Germany, 3 Department of Biochemistry and Molecular Biology and The Villum Center for Bioanalytical Sciences, University of Southern Denmark, Odense M, Denmark, 4 Department of Clinical Genetics, Odense University Hospital, Odense, Denmark, 5 Department of Mathematics and Computer Science, University of Southern Denmark, Odense, Denmark, 6 Group of Functional Genomics, К.А. Timiryazev Institute of Plant Physiology, Moscow, Russian Federation, 7 Department of Human Genetics, Hannover Medical School, Hannover, Germany, 8 Department of Paediatrics, Municipal Hospital of Phthisiopneumology, Kishinev, Moldova, Republic of, 9 Laboratory of Immunology, Chiril Draganiuc Institute of Phthisiopneumology, Kishinev, Moldova, Republic of
Introduction: Mendelian Susceptibility to Mycobacterial Disease (MSMD) is a rare genetic disorder with impaired immunity against mycobacterial infections; however only half of patients with MSMD have an identified genetic etiology. Here, we attempt to detect causal mutations in three paediatric patients with possible presentations for MSMD. Methods: Whole exome sequencing was performed on two patients with severe localized BCG infection (#1 and #2) and their parents, and another individual patient with disseminated TB (#3). The functional effects of the identified variants were characterized using RT-PCR analysis of endogenous and minigene transcripts, and SPRi analysis. Results: Six rare heterozygous variants were detected in the established MSMD genes: IL12B (case #1, compound heterozygote), TYK2 (cases #1 and #2), IL12RB1 and STAT1 (case #3). In addition, four disease-associated variants in three presumed causative genes were identified by trio-based analysis: compound missense and intronic variants in GBP2 (case #2), a de novo heterozygous frameshift mutation in HEATR3 (case #1), and a homozygous missense mutation in IFNW (case #1). cDNA analysis for splice-site mutation c.373-2A>C in STAT1 demonstrated abnormalities in STAT1 pre-mRNA splicing. Characterization of the c.1149+14T>C variant in GBP2 via minigene assay revealed some exon-skipping event from the mutant construct. Additionally, SPRi analysis showed a greater affinity of silencer-bound hnRNP A1 protein to RNA oligonucleotides harboring c.1149+14T>C compared to the wild type oligonucleotides, providing support to the minigene assay. Conclusions: Our findings expand the spectrum of genetic variation possibly predisposing to mycobacterial infections in children. Funding: Alexander von Humboldt Foundation and Hanover Unified Biobank. A.M. Varzari: None. M. Dembic: None. G. Hoffmann Bruun: None. I.V. Deyneko: None. W. Hofmann: None. V.M. Cebotari: None. E.G. Privalova: None. B.S. Andresen: None. T. Illig: None. P07.38.A An indel in the promoter region of NFKB1 confers protection against infection but increases risk of allergy

A. Y. Chong 1 , N. Brenner 2 , A. Jimenez Kaufmann 3 , A. Cortes 4 , R. Almond 5 , M. Hill 6 , T. J. Littlejohns 7 , J. J. Gilchrist 1,8 , B. P. Fairfax 9 , F. Hodel 10,11 , J. Fellay 10,11,12 , G. McVean 4 , T. Waterboer 2 , A. Moreno Estrada 3 , A. V. S. Hill 1,13 , A. J. Mentzer 1,4

1 Wellcome Centre for Human Genetics, University of Oxford, Oxford, United Kingdom, 2 Infections and Cancer Epidemiology, German Cancer Research Center, Heidelberg, Germany, 3 Human Population Genomics Lab, Laboratorio Nacional de Genómica para la Biodiversidad, Irapuato, Mexico, 4 Big Data Institute, Li Ka Shing Centre for Health Information and Discovery, University of Oxford, Oxford, United Kingdom, 5 UK Biobank, Stockport, United Kingdom, 6 MRC-Population Health Research Unit, University of Oxford, Oxford, United Kingdom, 7 Nuffield Department of Population Health, University of Oxford, Oxford, United Kingdom, 8 Department of Paediatrics, University of Oxford, Oxford, United Kingdom, 9 Department of Oncology, University of Oxford, Oxford, United Kingdom, 10 Global Health Institute, School of Life Sciences, EPFL, Lausanne, Switzerland, 11 Swiss Institute of Bioinformatics, Lausanne, Switzerland, 12 Precision Medicine Unit, Lausanne University Hospital and University of Lausanne, Lausanne, Switzerland, 13 Jenner Institute, University of Oxford, Oxford, United Kingdom
Introduction: Infectious agents contribute significantly to the global burden of diseases, through both acute infection and increasing risk of diverse chronic conditions. We leveraged the rich phenotypic data in the UK Biobank to identify genetic loci that influence response to multiple infections. Methods: We undertook 45 genome-wide association studies of antibody responses against common pathogens, in 9,611 participants from UK Biobank, identifying NFKB1 as a common locus. Using incidence of disease in 487,409 UK Biobank participants, we investigated the association between NFKB1 variation and 91 infection and inflammation phenotypes. To better understand the impact of variation on underlying immune mechanisms, we also explored the effect of these variants on eight blood cell traits and cellular expression data. Results: We identified an insertion-deletion variant (rs28362491), known to affect NFKB1 promoter binding, as a likely causal variant. The insertion allele has a protective effect against infection, but increases the risk of allergic disease. The same variant was associated with shifts in haematopoietic pathways, and likely impacts cell survival, antibody production and inflammation. Interestingly, this variant is relatively common in all human populations, suggesting that variation within this locus is largely maintained through balancing selection. Conclusions: We find that disruptions to the tightly regulated immune responses and inflammatory processes can tip the balance between exacerbated immune responses and allergy, or increased risks of infection and impaired resolution of inflammation. This subtle yet demonstrable effect across the population is likely driven by modulation of NFKB1 gene expression. A.Y. Chong: None. N. Brenner: None. A. Jimenez Kaufmann: None. A. Cortes: None. R. Almond: None. M. Hill: None. T.J. Littlejohns: None. J.J. Gilchrist: None. B.P. Fairfax: None. F. Hodel: None. J. Fellay: None. G. McVean: None. T. Waterboer: None. A. Moreno Estrada: None. A.V.S. Hill: None. A.J. Mentzer: None. P07.39.B CN-LOH in PIGV causes PNH, in a case of ET and nephrotic syndrome

A. Maurel-Ribes 1 , A. Knaus 2 , C. Garcia 1 , H. Engels 3 , S. Sivalingam 2 , H. Hundertmark 3 , D. Ribes 4 , S. Tavitian 5 , B. Payrastre 6 , S. Faguer 4 , P. Krawitz 2 , F. Vergez 7

1 Laboratoire d’Hématologie, Centre Hospitalier Universitaire de Toulouse, Toulouse, France, 2 Institute for Genomic Statistics and Bioinformatics, University of Bonn, School of Medicine & University Hospital Bonn, Bonn, Germany, 3 Institute of Human Genetics, University of Bonn, School of Medicine & University Hospital Bonn, Bonn, Germany, 4 Department of Nephrology and Organ Transplantation, University Hospital of Rangueil, Toulouse, France, 5 Service D’Hématologie, Centre Hospitalier Universitaire de Toulouse, Institut Universitaire du Cancer de Toulouse, Toulouse, France, 6 Inserm U1048 and Paul Sabatier University, Institute of Cardiovascular and Metabolic Diseases, Toulouse, France, 7 Department of Biology Hematology, Toulouse-Oncopole University Cancer Institute (IUCT-O), Toulouse, Germany
Classical paroxysmal nocturnal hemoglobinura (PNH) is an acquired glycosylphosphatidylinositol (GPI) anchor deficiency in a hematopoietic stem cell (HSC) due to mutations in PIGA . However, the reasons for clonal expansion in an acquired loss of function of GPI biosynthesis, is still a matter of debate. Here we describe, how PNH also manifested in a patient with essential thrombocythemia (ET) after a copy number neutral loss-of-heterozygosity (CN-LOH) in PIGV , another gene of the GPI-anchor deficiency. Similar to other proliferative myeloid malignancies, the clonal expansion of the HSC with ET resulted in our case from a de novo mutation in MPL (p.W515L) on chromosome 1 (1p34.2). 16.7 MB upstream of the acquired MPL mutation in cis, the patient carried a heterozygous pathogenic mutation in PIGV (p.R469X, 1p36.11), congenitally. A somatic CN-LOH in the HSC comprised almost the entire p-arm of chromosome 1. Hence, the MPL mutation p.W5151L gained dominance and deteriorated ET and while homozygosity of p.R469X in PIGV resulted in a GPI-anchor deficiency and manifestation of PNH. In addition to ET and PNH, the patient presented with nephrotic syndrome that ceased after treatment with eculizumab and hydroxycarbamide. We determined the origin of kidney injury as a result of podocyte instability due to an increase of non-GPI linked soluble uPAR. In this report we identified PIGV as a novel gene causing PNH and provide insights into the pathomechanism of acquired GPI anchor deficiency and expansion of PNH cells. Furthermore, we discuss the contribution of PNH to kidney injury. A. Maurel-Ribes: None. A. Knaus: None. C. Garcia: None. H. Engels: None. S. Sivalingam: None. H. Hundertmark: None. D. Ribes: None. S. Tavitian: None. B. Payrastre: None. S. Faguer: None. P. Krawitz: None. F. Vergez: None. P07.40.C Human genomics of the humoral response against polyomaviruses

F. Hodel 1,2 , A. Y. Chong 3 , P. Scepanovic 4 , C. W. Thorball 1,2 , Z. M. Xu 1,2 , P. Marques-Vidal 5 , M. Begemann 6 , H. Ehrenreich 6 , N. Brenner 7 , N. Bender 7 , T. Waterboer 7 , A. J. Mentzer 3,8 , A. V. S. Hill 3,9 , C. Hammer 10,11 , J. Fellay 1,2,12

1 Global Health Institute, School of Life Sciences, EPFL, Lausanne, Switzerland, 2 Swiss Institute of Bioinformatics, Lausanne, Switzerland, 3 The Wellcome Centre for Human Genetics, University of Oxford, Oxford, United Kingdom, 4 Roche Pharmaceutical Research and Early Development, F. Hoffmann-La Roche Ltd, Basel, Switzerland, 5 Service of Internal Medicine, Department of Medicine, Lausanne University Hospital and University of Lausanne, Lausanne, Switzerland, 6 Clinical Neuroscience, Max Planck Institute of Experimental Medicine, DFG Research Center for Nanoscale Microscopy and Molecular Physiology of the Brain, Göttingen, Germany, 7 Infections and Cancer Epidemiology, German Cancer Research Center, Heidelberg, Germany, 8 Big Data Institute, Li Ka Shing Centre for Health Information and Discovery, University of Oxford, Oxford, United Kingdom, 9 The Jenner Institute, Oxford, United Kingdom, 10 Department of Cancer Immunology, Genentech Inc., San Francisco, CA, United States, 11 Department of Human Genetics, Genentech Inc., San Francisco, CA, United States, 12 Precision Medicine Unit, Lausanne University Hospital and University of Lausanne, Lausanne, Switzerland
Introduction: Human polyomaviruses are widespread in human populations and some cause severe disease in immunocompromised individuals. There remains an incomplete understanding of the potential impact of human genetic variation on inter-individual responses to polyomaviruses. Material and methods: To identify human genetic determinants of the humoral immune response against polyomaviruses, we performed genome-wide association studies and meta-analyses of immunoglobulin G (IgG) responses against the major capsid protein VP1 of Human polyomavirus 6 (HPyV6), BK virus (BKV), JC virus (JCV) and WU polyomavirus (WUPyV), using data from three independent studies grouping 15’660 individuals of European ancestry. Results: We found evidence for an involvement of HLA class II variation in the determination of antibody responses to JCV (P=1.5e-78) and HPyV6 (P=3.2e-14). We also observed associations in ABO for JCV (P=6.2e-11), and FUT2 for BKV, JCV and WUPyV (P=2.3e-18; P=9.7e-26; P=9.0e-09, respectively), involved in blood group determination and secretor status. Lastly, we found a functional variant in MUC1 , previously associated with the risk of developing gastric cancer and Helicobacter pylori associated gastritis, to be associated with IgG levels against WUPyV (rs4072037, P=3.6e-33). Conclusions: These results provide insights into the human genetic control of a family of very prevalent viruses, highlighting polymorphisms that play a modulating role on the humoral immune response against specific human polyomaviruses. Functional follow-up studies will be needed to uncover the biological mechanisms responsible for the newly discovered associations. F. Hodel: None. A.Y. Chong: None. P. Scepanovic: None. C.W. Thorball: None. Z.M. Xu: None. P. Marques-Vidal: None. M. Begemann: None. H. Ehrenreich: None. N. Brenner: None. N. Bender: None. T. Waterboer: None. A.J. Mentzer: None. A.V.S. Hill: None. C. Hammer: None. J. Fellay: None. P07.42.B Increasing the diagnostic yield in patients suspected for primary immunodeficiency

A. Niskakoski , P. Kokkonen, I. Saarinen, J. Sistonen, H. Junnila, A. Kere, M. Andreevskaya, M. Muona, J. Djupsjöbacka, L. Koskinen, H. Duzkale, S. Myllykangas, J. Koskenvuo, T. Alastalo

Blueprint Genetics, Espoo, Finland
Background: Finding the genetic diagnosis for patients with suspicion of primary immunodeficiency (PID) is becoming increasingly important in the management of primary immunodeficiency and estimating the risk for family members. Simultaneous sequence and copy number variant analysis combined with customized analysis for difficult to sequence regions in specific genes such as NCF1 improves the diagnostic yield in patients suspected for PID. Methods: A retrospective review of nearly 1700 patients suspected for PID and referred for genetic testing at Blueprint Genetics between 2018 and 2019 was performed. Patients were tested with Blueprint Genetics next generation sequencing panels related to immunology. Results: Diagnostic yield including all immunology related panels was 14.9% (253/1698). The diagnostic yield was highly variable between the different panels; diagnostic yield for Primary Immunodeficiency Panel was 13.3% (138/1036), whereas for Severe Combined Immunodeficiency Panel it was 46.7% (7/15). The diagnostic yield for Chronic Granulomatous Disease Panel is high (10/21, 48%) although analysis of the NCF1 gene included in this panel is complicated by two highly homologous pseudogenes NCF1B and NCF1C . Out of the 296 reported diagnostic sequence variants, 234 (79%) were unique, and only 7 diagnostic variants were reported over 3 times. Copy number variants, including deletions and duplications, were reported for 19 patients from 17 different genes. Conclusions: We constantly increase the diagnostic yield for PIDs by improving the sequencing technology, updating the panels with new genes discovered related to PID, and finding diagnoses from difficult to sequence regions and regions with high homology. A. Niskakoski: A. Employment (full or part-time); Significant; Blueprint Genetics. P. Kokkonen: A. Employment (full or part-time); Significant; Blueprint Genetics. I. Saarinen: A. Employment (full or part-time); Significant; Blueprint Genetics. J. Sistonen: A. Employment (full or part-time); Significant; Blueprint Genetics. H. Junnila: A. Employment (full or part-time); Significant; Blueprint Genetics. A. Kere: A. Employment (full or part-time); Significant; Blueprint Genetics. M. Andreevskaya: A. Employment (full or part-time); Significant; Blueprint Genetics. M. Muona: A. Employment (full or part-time); Significant; Blueprint Genetics. J. Djupsjöbacka: A. Employment (full or part-time); Significant; Blueprint Genetics. L. Koskinen: A. Employment (full or part-time); Significant; Blueprint Genetics. H. Duzkale: A. Employment (full or part-time); Significant; Blueprint Genetics. S. Myllykangas: A. Employment (full or part-time); Significant; Blueprint Genetics. E. Ownership Interest (stock, stock options, patent or other intellectual property); Significant; Blueprint Genetics. J. Koskenvuo: A. Employment (full or part-time); Significant; Blueprint Genetics. E. Ownership Interest (stock, stock options, patent or other intellectual property); Significant; Blueprint Genetics. T. Alastalo: A. Employment (full or part-time); Significant; Blueprint Genetics. E. Ownership Interest (stock, stock options, patent or other intellectual property); Significant; Blueprint Genetics. P07.45.B Common RUNX3 missense variant contributes to psoriatic arthritis by affecting splicing and modifying signaling, activation and differentiation of T-cells

U. D. Hüffmeier 1 , S. Löhr 1 , S. Uebe 1 , B. Popp 1,2 , J. Bowes 3 , P. Kirchner 1 , E. Giardina 4 , E. Korendowych 5 , A. B. Ekici 1 , P. Ho 3 , F. Behrens 6 , M. Köhm 6 , G. Schett 7 , J. Rech 7 , G. Assmann 8 , A. Nimeh 9 , L. Padyukov 10 , G. Alenius 11 , N. J. McHugh 12 , H. Sticht 13 , H. Burkhardt 6 , A. Barton 3,14 , A. Reis 1

1 Human Genetics, University of Erlangen, Erlangen, Germany, 2 Institute of Human Genetics, University of Leipzig Hospitals and Clinics, Leipzig, Germany, 3 Arthritis Research UK Centre for Genetics and Genomics, University of Manchester, Manchester, United Kingdom, 4 University of Rome Tor Vergata and Fondazione Policlinico Tor Vergata, Rome, Italy, 5 Royal National Hospital for Rheumatic Diseases, NHS Foundation Trust, Bath, United Kingdom, 6 Division of Rheumatology and IME-Fraunhofer Project Group Translational Medicine and Pharmacology, Johann Wolfgang Goethe University, Frankfurt/ Main, Germany, 7 Department of Internal Medicine 3 - Rheumatology and Immunology, University of Erlangen, Erlangen, Germany, 8 Department of Internal Medicine I, José-Carreras Centrum for Immuno- and Gene Therapy, University of Saarland Medical School, Homburg/ Saar, Germany, 9 Department of Rheumatology, Fachklinik Bad Bentheim, Bad Bentheim, Germany, 10 Karolinska Institute, Stockholm, Sweden, 11 University Hospital of Umeå, Umeå, Sweden, 12 University of Bath, Bath, United Kingdom, 13 Bioinformatics, Institute of Biochemistry, University of Erlangen-Nürnberg, Erlangen, Germany, 14 NIHR Manchester Musculoskeletal Biomedical Research Unit, Central Manchester Foundation Trust and University of Manchester, Manchester Academy of Health Sciences, Manchester, United Kingdom
Psoriatic Arthritis (PsA) is a chronic inflammatory joint disease occurring in up to 30% of patients with the most common psoriatic skin manifestation, psoriasis vulgaris (PsV). PsA is considered a T-cell mediated disease with a complex genetic basis. To date, GWAS identified >60 largely overlapping susceptibility loci for PsA/ PsV including RUNX3 , while the respective disease-contributing mechanism remained mostly uncertain. RUNX3 encodes a transcription factor expressed e.g. in immune cells, including T-cells, and skin. As genotyping arrays had insufficient coverage at this locus, we selected 32 tagging SNPs for further fine-mapping. Association analysis in 3,049 PsA patients and 6,178 controls of the Psoriatic Arthritis Genetics in Europe consortium showed significant association to an LD-wise isolated intronic SNP (p=1.50E-11) and to 5 SNPs (5.43E-12≤p≤2.78E-07) within one intragenic LD block. Genomic annotations for 38 SNPs in high LD (r 2 ≥0.9) with the SNPs were inconclusive, including histone modifications and DNAse-sensitive sites in immune cells. Haplotype and conditional analyses, though, pointed to disease-contribution of the common, evolutionary conserved variant c.53T>A/p.Ile18Asn in alternative exon 1. Targeted deep-sequencing of cDNA from CD8+ T-cells unveiled alternative splicing in homozygous wildtype carriers, but a reduced number of splicing products and lack of alternative splicing in homozygous risk-allele carriers. Comparative transcriptome analysis in CD8+ T-cells of homozygous carriers provided evidence for differentially expressed genes involved in T-cell exhaustion signaling, Th1/ Th2 activation and T-helper-cell differentiation. Our study indicates that the RUNX3 risk-allele in PsA reduces splicing variability thereby contributing to altered signaling, activation and differentiation of T-cells. U.D. Hüffmeier: None. S. Löhr: None. S. Uebe: None. B. Popp: None. J. Bowes: None. P. Kirchner: None. E. Giardina: None. E. Korendowych: None. A.B. Ekici: None. P. Ho: None. F. Behrens: None. M. Köhm: None. G. Schett: None. J. Rech: None. G. Assmann: None. A. Nimeh: None. L. Padyukov: None. G. Alenius: None. N.J. McHugh: None. H. Sticht: None. H. Burkhardt: None. A. Barton: None. A. Reis: None. P07.46.C Functional study of peptidylarginine deiminases in collagen-induced arthritis mice

A. Suzuki , T. Shibuya, K. Yamamoto

RIKEN, Yokohama, Japan.
Previously, peptidylarginine deiminase type 4 (PADI4) was identified as a susceptibility gene for Rheumatoid arthritis (RA) by genome-wide association studies in Japanese population. PADI genes can provide peptidyl citrulline which is a target antigen of anti-citrullinated peptide antibodies (ACPAs) that is highly sensitive and specific antibodies in RA. After that, PADI2 was also identified as susceptibility gene with RA. But, it is not clear how these PADI genes are involved in the development of rheumatoid arthritis. The aim of this study was to investigate the relationship between PADI4 gene and PADI2 gene in the progression of RA. To clarify the physiological function of PADI4 and PADI2, we used collagen-induced arthritis (CIA), known as a RA model mouse. We examined that localization of PADI4 and PADI2 protein was indicated by immunohistochemistry in CIA mice. We also measured expression of PADI genes and various inflammatory cytokines in immune cells by real-time TaqMan assay and ELISA, respectively. We generated PADI4−/− and PADI2−/− mice, and performed experimental arthritis. We demonstrated that the clinical disease score was significantly decreased in PADI4−/− mice and PADI2−/− mice. PADI4 expression was induced by CII immunization. Interestingly, PADI2 expression was compensationally induced in CD11b+ cells of PADI4-/- mice. However, in PADI2-/- mice, conpensational induction of PADI4 was not obsered. It appears that PADI4 and PADI2 enhance collagen-initiated inflammatory responses. A. Suzuki: None. T. Shibuya: None. K. Yamamoto: None. P07.49.C Likely pathogenic variant within zinc finger domain 4 of BCL11B in a child with facial and dermal abnormalities and severe combined immunodeficiency

L. Olfe 1 , S. v. Hardenberg 1 , C. Klemann 2 , U. Baumann 2 , E. Tolosa 3 , D. Lessel 4 , B. Auber 1

1 Department of Human Genetics, Hannover Medical School, Hanover, Germany, 2 Department of Pediatric Pneumology, Allergy and Neonatology, Hannover Medical School, Hanover, Germany, 3 Department of Immunology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany, 4 Institute of Human Genetics, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
BCL11B encodes a Cys2His2 zinc finger transcription factor involved in the development of nervous and immune systems. BCL11B heterozygous loss-of-function variants have been associated with a neurodevelopmental disorder, alterations in the T-cell development and reduced number of type 2 innate lymphoid cells (ILC2s). Moreover, a single patient presenting with syndromic features and severe combined immunodeficiency (SCID) due to a pathogenic missense variant in BCL11B has been reported. Here we report a newborn with developmental SCID diagnosed through neonatal SCID screening based on T-cell receptor excision circles. The child presented with microcephaly, hirsutism, loose skin folds and dysmorphic facial features, which include thin eyebrows, hypertelorism, short palpebral fissures, broad nasal bridge, anteverted, small nares, thin upper and lower lip vermillion and long philtrum. Immunophenotyping showed absence of T-cells with remaining B- and NK-cells, and in strong contrast to loss-of-function BCL11B cases a normal amount of ILC2-cells. Whole-exome sequencing identified a likely pathogenic BCL11B missense variant c.2428A>C, p.(Asn810His), affecting one of the four specificity residues of the DNA-contacting alpha-helix within the fourth zinc finger domain. Bioinformatic prediction algorithms suggest this missense variant binds to identical alternative genomic sequences, resulting in acquisition of novel DNA-binding regions, as the previously reported one. Thereby, providing an explanation for the striking clinical similarities of the two missense cases. Our findings further suggest that only specific BCL11B missense variants result in a severe immunodeficiency. This case further highlights the importance of neonatal SCID screening for early diagnosis and consequently the therapeutic option of hematopoietic stem cell transplantation. L. Olfe: None. S. v. Hardenberg: None. C. Klemann: None. U. Baumann: None. E. Tolosa: None. D. Lessel: None. B. Auber: None. P07.50.A An atypical presentation of Immunodeficiency Centromeric Instability Facial anomalies syndrome

C. Mehawej 1 , H. Khalife 2 , G. Dbaibo 3 , R. Wakim 4 , C. Farra 1

1 Unité de Génétique Médicale, Faculté de Médecine, Saint-Joseph University, Beirut, Lebanon, 2 Al Zahraa Hospital University Medical Center, Beirut, Lebanon, 3 Pediatrics Department, American University of Beirut, Beirut, Lebanon, 4 American University of Beirut, Beirut, Lebanon
Severe Combined Immunodeficiency Diseases (SCID) are genetic disorders characterized by profound impairment of cellular and humoral immunity. They are defined by a significant deficiency in T lymphocytes. Patients with SCID are asymptomatic at birth but infections can lead to their early death unless appropriate treatment is applied. A 10 day old girl, born to consanguineous Lebanese parents was referred to our Medical Genetics Unit for genetic evaluation, because of a family history of SCID. Lymphocytic immunophenotyping and serum immunoglobulin testing revealed a low absolute lymphocyte count, T cell lymphopenia with a significant decrease in naïve T cells and normal circulating B cells count including mainly immature B cells. SCID/atypical SCID was suspected in the patient. Whole Exome Sequencing (WES) was conducted and did not reveal any mutation in the SCID genes. However, reanalysis of WES data showed the presence of a homozygous missense variant, likely pathogenic, in the DNMT3B gene (NM_ 006892; c.2477G>A, p.R826H). This gene is involved in the autosomal recessive ICF1 syndrome (Immunodeficiency- Centromeric instability- Facial anomalies syndrome). A reevaluation of the patient’s clinical presentation and laboratory findings revealed undetectable immunoglobulin levels and facial dysmorphism (hypertelorism, micrognathia and low set ears), both characteristic of ICF1. T cell lymphopenia presenting at an early age in our proband is an unusual feature in ICF1 patients. Altogether, this case describes a severe presentation of ICF1 and highlights the heterogeneity of primary immunodeficiencies. It also stresses on the importance of early management of patients born to families with a history of SCID. C. Mehawej: ; Research council of Saint-Joseph University (FM362). H. Khalife: None. G. Dbaibo: None. R. Wakim: None. C. Farra: None. P07.51.B Uncovering rare hematological entities: Shwachman Diamond syndrome in a pair of siblings with congenital neutropenia and recurrent infections

J. Trizuljak 1,2 , Z. Vrzalová 1,2 , I. Blaháková 1,2 , L. Radová 2 , Š. Pospíšilová 1,2 , M. Doubek 1,2

1 University Hospital, Brno, Czech Republic, 2 Central European Institute of Technology, Brno, Czech Republic
Shwachman-Diamond syndrome (SDS) is an autosomal recessive disorder with clinical features that include pancreatic exocrine insufficiency, hematologic dysfunction, and skeletal abnormalities, Haematological malignancies occur in one third of patients. SDS is caused by homozygous or compound heterozygous mutations in SBDS gene. We present a sibling pair with mild to moderate neutropenia, episodes of autoimmune hemolysis and recurrent infections of the oral cavity and respiratory airways. Prior investigations were inconclusive. Family history suggested AR mode of inheritance. We performed whole-exome sequencing of siblings and parents. Whole-exome libraries were prepared according to the Nimblegen SeqCap EZ Exome v3 protocol and sequencing was performed on NextSeq 500 for all of them. Furthermore, we performed in silico analysis of a virtual gene panel, focused on congenital neutropenias. Whole-exome sequencing identified a compound heterozygous genotype in SBDS gene in both siblings. We identified a likely pathogenic missense variant c.355T>C; p.(Cys119Arg) in exon 3 of SBDS gene, leading to substitution of strongly conserved cysteine for arginine. This variant has been previously reported in a French family with SDS (Donadieu et al., 2012). Furthermore, we identified a rare, previously undescribed missense variant, c.536C>T; p.(Pro179Leu), leading to prolin - leucine substitution. In-silico analysis (Align GVGD, SIFT, MutationTaster) predicts pathogenic or likely pathogenic effect. Molecular-genetic analysis of parents confirmed heterozygous carrier status. Compound heterozygous mutations in SBDS leads to disruption of SBDS gene and clinical manifestation of Shwachman-Diamond syndrome in the siblings. This study was supported by Czech Ministry of Health (grant AZV 16-29447A) and Masaryk University (grant MUNI/A/1395/2019). J. Trizuljak: None. Z. Vrzalová: None. I. Blaháková: None. L. Radová: None. Š. Pospíšilová: None. M. Doubek: None. P07.52.C Genome-to-genome analysis identifies an Asian-specific variant in the gene encoding NTCP that leads to specific HBV escape mutations during chronic infection

Z. Xu 1 , S. Rüeger 1 , P. Shea 2 , M. Buti 3 , H. L. Y. Chan 4 , P. Marcellin 5 , O. Podlaha 6 , Z. Jiang 6 , V. Suri 6 , A. Gaggar 6 , M. Subramanian 6 , E. Gane 7 , D. Goldstein 2 , J. Fellay 1,8

1 School of Life Sciences, École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland, 2 Institute for Genomic Medicine, Columbia University, New York, NY, United States, 3 Liver Unit, Hospital Universitario Vall d’Hebron and CIBEREHD del Instituto Carlos III, Barcelona, Spain, 4 The Chinese University of Hong Kong, Hong Kong, China, 5 Service d’Hépatologie, Hôpital Beaujon, Clichy, France, 6 Gilead Sciences Inc, Foster City, CA, United States, 7 Auckland Clinical Studies, Auckland, New Zealand, 8 Precision Medicine Unit, Lausanne University Hospital, Lausanne, Switzerland
Introduction: Hepatitis B virus (HBV) is a major cause of chronic liver diseases, including liver cirrhosis and hepatocellular carcinoma. In chronic infections, the interplay between human and viral genetics contributes to HBV evolutionary changes. Here, we use a genome-to-genome (G2G) approach to identify sites of host-pathogen genomic interactions. Materials and Methods: We obtained paired human exome and HBV genome data from 424 chronically infected individuals of Asian ancestry from three Phase 3 studies evaluating tenofovir-based regimens. For each variable HBV amino acid, a genome-wide association study was conducted using generalized linear mixed models and controlling for human and viral population structures. Results: We identified strong associations (p=1.9E-12) between an Asian-specific missense SNP in the SLC10A1 gene encoding the HBV receptor NTCP (rs2296651) and amino acids in HBV large surface (preS1 domain) and polymerase proteins. The minor allele is present in 7% of East Asians (1KG Project) and was previously reported to associate with decreased risk of cirrhosis and hepatocellular carcinoma in chronic HBV. We also identified significant associations (p=8.7E-13) between variants of HLA-A protein and amino acids in HBV polymerase and core proteins. Conclusions: Our joint analysis of human and viral genomes shows that variation in NTCP exerts selective pressure on HBV during chronic infection, and confirms the impact of HLA class I variation on HBV epitopes. These results demonstrate the power of the G2G approach to identify host restriction factors and identify specific HBV amino acids likely to be involved in the interaction between HBV and its cellular receptor. Z. Xu: None. S. Rüeger: None. P. Shea: None. M. Buti: None. H.L.Y. Chan: None. P. Marcellin: None. O. Podlaha: A. Employment (full or part-time); Significant; Gilead Sciences Inc. Z. Jiang: A. Employment (full or part-time); Significant; Gilead Sciences Inc. V. Suri: A. Employment (full or part-time); Significant; Gilead Sciences Inc. A. Gaggar: A. Employment (full or part-time); Significant; Gilead Sciences Inc. M. Subramanian: A. Employment (full or part-time); Significant; Gilead Sciences Inc. E. Gane: None. D. Goldstein: None. J. Fellay: None. P07.53.A Relationships between smoking methylation biomarkers and GPR15+ helper T cell levels in peripheral blood

W. Philibert 1 , S. R. H. Beach 2 , A. M. Andersen 1

1 University of Iowa, Iowa City, IA, United States, 2 University of Georgia, Athens, GA, United States
Introduction: Smoking causes epigenetic changes associated with inflammatory diseases and elevated mortality. Of particular interest are changes in the level of T cells expressing G-protein-coupled receptor 15 (GPR15), a chemokine receptor linked with inflammatory bowel disease, multiple sclerosis and psoriasis. Accordingly, a better understanding of the mechanisms by which smoking influences variation in GPR15 + T cell subpopulations is of potential interest. Materials and Methods: Flow cytometry and digital PCR assays were used to measure the GPR15 + CD3 + CD4 + helper T (T h ) populations in peripheral blood from n = 62 primarily African American young adults (aged 27-35 years) with a high rate of tobacco and cannabis use. Results: Tobacco and cannabis smoking predicted GPR15 + (T h ) cell levels using linear regression models. Methylation of two candidate CpGs, cg19859270, located in GPR15 , and cg05575921, located in Aryl Hydrocarbon Receptor Repressor ( AHRR ), were both significant predictors of GPR15 + T h cell levels, mediating the relationship between smoking and increases in GPR15 + T h cells. As hypothesized, the interaction between cg05575921 and cg19859270 was significant, indicating that low cg05575921 methylation was more strongly associated with GPR15 + T h cell levels for those with lower cg19859270 methylation. Conclusions: Smoking leads changes in two CpGs, cg05575921 and cg19859270, that mediate 38.5% of the relationship between tobacco and cannabis smoking and increased GPR15 + T h levels in this sample. The impact of cg19859270 in amplifying the association between cg05575921 and increased GPR15 + T h levels is of potential theoretical interest because it may reflect a permissive interaction between different parts of the immune system. W. Philibert: None. S.R.H. Beach: None. A.M. Andersen: None. P07.54.B Somatic mutations in HLA loci in patients with myeloid leukemia

J. Neupauerová 1 , P. Crivello 2 , Š. Půbalová 1 , C. Šálek 1 , S. Nazarová 1 , M. Vraná 1

1 Institute of hematology and blood transfusion, Prague 2, Czech Republic, 2 Institute of experimental cellular therapy, University Hospital Essen, Essen, Germany
Introduction: In the HLA department of Institute of hematology and blood transfusion in Prague we deal with HLA typing of hematooncological patients and their related/unrelated donors. The best donor compatibility is important for succesful hematopoetic stem cell transplantation (HSCT). Materials and methods: Using NGS (Omixon, Hungary), we investigated more than 2000 patients and their HSC donors. New alleles found were confirmed by Sanger sequencing (Inno-train, Germany; CareDx, USA). Results: . From our recent studies (2017 - 2019), in our cohort of patients, 15 new alleles were detected. Two of them were a somatic mutation in patients suffering from acute myeloid leukemia (AML); one was in the HLA-B and the other in the HLA-C locus. In both cases, the somatic mutation represents a single nucleotide polymorphism. Conclusions: Our findings are in agreement with the known genome instability in AML. In the HLA region, somatic mutations are not so common, however, they should be given more attention since they can affect disease progression and may impact also on HSCT. Our future studies will focus on the precise identification of the new allelles and their characterization in HLA typing. Supported by the Ministry of Health, Czech Republic-conceptual development of research organization (CZ00023736, UHKT). J. Neupauerová: None. P. Crivello: None. Š. Půbalová: None. C. Šálek: None. S. Nazarová: None. M. Vraná: None. P07.55.C High-throughput sequencing of T-cell receptor alpha chain clonal rearrangements in B-lineage acute lymphoblastic leukemia

A. Miroshnichenkova 1 , A. Komkov 1,2 , A. Popov 1 , E. Volchkov 1 , M. Maschan 1 , A. Karachunskiy 1 , Y. Olshanskaya 1 , I. Mamedov 2

1 Dmitry Rogachev National Research Center of Pediatric Hematology, Oncology and Immunology, Moscow, Russian Federation, 2 Shemyakin-Ovchinnikov Institute of bioorganic chemistry, Moscow, Russian Federation
Introduction: Rearrangements of T- and B-cell receptor (TCR and BCR) genes are useful markers for clonality assessment and minimal residual disease (MRD) monitoring during the treatment of hematological malignancies. Despite their estimated lineage, B-cell leukemias manifest a remarkably high incidence of TCR gene rearrangements. According to indirect estimation, TCR alpha (TRA) incidence in B-cell precursor ALL could reach 46%. Currently, rearrangements of all BCR and 3/4 TCR loci are used for this purpose with the exception of TRA due to the large number of multiplex primers needed to amplify all TRA segments. Materials and Methods: We developed the first targeted sequencing-based method for identification of TRA clonal rearrangements. gDNA was extracted from a bone marrow of children with B-ALL. Amplification of TRA rearrangements was performed in multiplex PCR and subsequently sequenced on Illumina platform. Results: TRA rearrangements were detected in 5/45 samples and associated with myeloid CD33+ co-expression ( p -value 0.0147, two-tailed Mann-Whitney U-test). Clonal rates of TRA varied from 31% to 98% and corresponded to main leukemic clones. All found TRA rearrangements were nonfunctional. TRA clones arose after leukemic transformation and have high clonal rate, which indicates these clones are most viable of all other leukemic clones. Conclusions: The innovative study provides pathbreaking analyses of TRA occurrence in B-ALL at the DNA level and suggests that particular TRA rearrangements may be of clinical relevance in childhood B-ALL by adding a significant marker to MRD detection panel based on TCR/BCR rearrangements analysis. The study is supported by PFBR grants 20-015-00462, 18-29-09132. A. Miroshnichenkova: None. A. Komkov: None. A. Popov: None. E. Volchkov: None. M. Maschan: None. A. Karachunskiy: None. Y. Olshanskaya: None. I. Mamedov: None. P07.56.A Gene mutation spectrum of thalassemia patients from Azerbaijan

A. AGHAYEV 1 , V. Huseynov 2 , E. Aliyev 2 , A. Najafli 2 , K. Jafarova 2 , A. Mammadova 2 , R. Bayramli 2 , Z. Uyguner 3

1 National Hematology and Transfusiology Center, Department of Genetic Diagnosis, Baku, Azerbaijan, 2 National Hematology and Transfusiology Center, Baku, Azerbaijan, 3 Department of Medical Genetics, Istanbul Medical Faculty, Istanbul, Turkey
Introduction: Thalassemias are defined by the absence or decrease of globin chain(s), which may lead imbalances of globin chains, ineffective erythropoiesis, hemolysis, and eventually to a variable degree of anemia. With the estimated carrier frequency of β-thalassemia is 4%-8.6% in country, the importance of premarital genetic testing, informed family counseling and preventive medical treatment cannot be overstated. Screening for traits by hemoglobin electrophoresis and biochemical analysis has become a routine application test prior to the issuance of a marriage license throughout Azerbaijan. Methods: We aimed to evaluate prevalent mutations spectrum, the effect and their co-inheritance and/or role of polymorphisms (BCL11A, HBS1L-MYB and Xmn1 SNP) on disease phenotype by using reverse dot-blot hybridization using customized strips, sequencing analysis of the complete genes and MLPA. Results: We report here a summary finding of HBB gene analysis for 265 patients and HBA1 - HBA2 genes analysis for 28 patients, along with their family members totaling 570 individuals. β; thalassemia -66 were found to have homozygous, 76 possessed compound heterozygous and 80 possessed heterozygous mutations. Overall, a total of 416 alleles were found to have 34 mutations. The first 20 frequented mutations covered 87% of the entirety of all mutations. The summary range is as following: c.25_26delAA(p.Lys9Valfs) 27.9%; c.93-21G>A(IVS1+110G>A) 11.3%; c.315+1G>A(IVS2+1G>A) 8.9%. α; thalassemia -Genotyping of 45 alleles revealed 9 mutations, the 3.7 kb deletion is the most frequent mutation (35.6%), followed by 20.5 kb deletion (24.4%) and α2 polyA2( HBA2: c.*92A > G, 13.3%). Conclusions: The results may help inform decisions in the design and implementation of prevention strategies and diagnostic approaches. A. Aghayev: None. V. Huseynov: None. E. Aliyev: None. A. Najafli: None. K. Jafarova: None. A. Mammadova: None. R. Bayramli: None. Z. Uyguner: None. P07.59.A Genome-wide association study of susceptibility to respiratory syncytial virus infection during infancy

D. Lawless 1 , C. Rosas-Salazar 2 , T. Gebretsadik 2 , K. Turi 2 , B. Snyder 2 , P. Wu 2 , J. Fellay 1 , T. Hartert 2

1 Global Health Institute, Lausanne, Switzerland, 2 Vanderbilt University School of Medicine, Nashville, TN, United States
Introduction: Human respiratory syncytial virus (RSV) infection is associated with asthma development. However, this association is confounded by shared heritability, and genetic studies have focused exclusively on severe infection. Longitudinal, population-based studies of RSV infection are thus required to assess the causal association between RSV and asthma. To determine whether there are genetic variants involved with susceptibility or resistance to infection, we performed the first GWAS on non-severe RSV infection. Methods: We performed GWAS analyses of healthy term infants in a population-based birth cohort (INSPIRE) specifically designed to determine the effect of infant RSV infection on later child health. We compared infants who were and were not infected with RSV in the first year of life as determined by biweekly surveillance for RSV infection and serology at age 1 year. Results: Among 1959 enrolled, 54% of infants were RSV infected and 46% were uninfected at age 1. There were significant differences in environmental factors associated with RSV infection, including daycare (p=0.001), siblings (p=0.002) and black race (p=0.002). GWAS analyses of a subset of 663 participants with available genotyping data, adjusted for birth month, sex, race, daycare and siblings revealed no significant associations. Conclusions: We found no genome wide associations with risk of infant RSV infection; environmental factors were significantly associated with infection risk. These findings support using RSV infection as a means of natural randomization free from genetic confounding to study the effects of RSV infection on long-term outcomes. Grants: NIH-U19AI095227, NIH-UL1RR024975, SNSF IZSEZ0_191968 D. Lawless: None. C. Rosas-Salazar: None. T. Gebretsadik: None. K. Turi: None. B. Snyder: None. P. Wu: None. J. Fellay: None. T. Hartert: None. P07.60.B The HLA and ABO genetic background impact on SARS-CoV-2 infection and COVID-19 severity

T. Vaisitti 1 , A. Amoroso 2 , P. Magistroni 3 , F. Vespasiano 4 , F. Puoti 4 , S. Alizzi 3 , P. Grossi 5 , S. Trapani 4 , L. Lombardini 4 , P. Pezzotti 6 , M. Cardillo 4

1 Department of Medical Sciences, University of Turin, Turin, Italy, 2 Department of Medical Sciences, University of Turin and Immunogenetics and Transplant Biology Service, Città della Salute e della Scienza University Hospital, Turin, Italy, 3 Immunogenetics and Transplant Biology Service, Città della Salute e della Scienza University Hospital, Turin, Italy, 4 National Transplant Center, Istituto Superiore di Sanità, Rome, Italy, 5 Department of Medicine & Surgery University of Insubria – ASST-Sette Laghi, Varese, Italy, 6 Epidemiology, Biostatistics and Mathematical models, Istituto Superiore di Sanità, Rome, Italy
At the beginning of 2020, the SARS-CoV-2 outbreak has been defined by the WHO as a global health emergency. A peculiar feature of this infection is the extreme variability in clinical presentation, ranging from very mild sub-clinical flu-like symptoms to acute respiratory distress syndrome with high mortality. This heterogenous presentation poses the question whether individual variability of immune response to the virus can be influenced by genetic factors. Specifically, it is unknown whether HLA and AB0 can explain the different prevalence in population of SARS-CoV-2 infection and its clinical evolution. To address this point, a retrospective analysis was performed on an Italian cohort composed by subjects who received organ transplantation and candidates in a waiting list in a 2002-March 2020 time frame. A total of 56,304 cases were studied with the aim of comparing HLA and AB0 frequencies according to the presence (COVID + ) or absence (COVID − ) of SARS-CoV-2 infection. The prevalence of COVID was 0.112% in the Italian population and 0.462% in waitlisted and transplanted patients. HLA-B*51 and HLA-DRB1*08 were more frequent in COVID + compared to COVID − , while HLA-DRB1*07 showed a protective effect. In transplant recipients, HLA-DRB1*08 was an indicator of mortality (1.9% in living vs 15.2% in the deceased, OR=9.4 95%CI[1.9-47.3]; P=.006). Moreover, blood group A was more frequent in COVID + (45.5%) than in COVID − (39.0%). Overall, this study shows for the first time that some HLA antigens influence SARS-CoV-2 infection and its clinical evolution and confirms that blood group A subjects are at greater risk of infection. T. Vaisitti: None. A. Amoroso: None. P. Magistroni: None. F. Vespasiano: None. F. Puoti: None. S. Alizzi: None. P. Grossi: None. S. Trapani: None. L. Lombardini: None. P. Pezzotti: None. M. Cardillo: None.

P08 Intellectual Disability
P08.003.C Angelman syndrome: a novel UBE3A splice variant

M. G. Williams 1,2 , B. Hanna 3 , M. Freckmann 4 , S. Bommireddipalli 5 , A. Bournazos 5 , S. Cooper 5 , J. Harraway 6 , P. Dawson 1 , H. Heussler 1,2,7

1 Mater Research Institute – The University of Queensland, Woolloongabba, QLD, Australia, 2 Faculty of Medicine, University of Queensland, St Lucia, QLD, Australia, 3 Clinical Genetics, Westmead Hospital, Westmead, NSW, Australia, 4 Clinical Genetics, Royal North Shore Hospital, St Leonards, NSW, Australia, 5 Kids Neuroscience Centre, The Children’s Hospital at Westmead, Westmead, NSW, Australia, 6 Mater Pathology, South Brisbane, QLD, Australia, 7 Child Development, The Queensland Children’s Hospital, South Brisbane, QLD, Australia
Introduction: Angelman syndrome (AS) is a neurodevelopmental disorder characterised by moderate to severe developmental delay, absent or near absent speech, gait ataxia, microcephaly and seizures. Deficient expression or function of the maternally inherited UBE3A allele results in AS. Pathogenic UBE3A sequence variants (predominantly loss-of-function) account for ~11% of Angelman syndrome cases. A maternally inher