Journal: Nature Communications

Article Title: Joint disease-specificity at the regulatory base-pair level

doi: 10.1038/s41467-021-24345-9

Figure Lengend Snippet: a Karyotype showing GWAS association of GDF5 with joint diseases. The strongest signal on chr20 is in the GDF5-UQCC1 locus (seven significant SNPs; see Supplemental section for more details). b Modified UCSC Genome Browser view (hg19) showing the intersection of DDH , and knee OA risk variants (top) with Human ATAC-seq regions (from acetabulum, proximal femur, distal femur, proximal tibia tissues) (middle), GDF5 regulatory elements ( R1-R5 , GROW1 )(bottom), followed by UCSC gene locations and peaks of phyloP100ways conservation. Two variants, rs4911178 in GROW1 , and rs6060369 in R4, overlap with functional regulatory sequences in embryonic human tissues and mouse (see also Supplementary Fig. ).

Article Snippet: The R4 rs6060369-T / rs6060369- + single allelic replacement mouse line contains a single “T” allelic base-pair replacement of the orthologous human rs6060369 variant in the R4 enhancer and was generated on the C57BL/6 J Mus musculus background by Applied StemCell and PI Capellini laboratory.

Techniques: Modification, Functional Assay

Journal: Nature Communications

Article Title: Joint disease-specificity at the regulatory base-pair level

doi: 10.1038/s41467-021-24345-9

Figure Lengend Snippet: a Occurrence of ‘T’ risk allele frequency at rs6060369 in subsampled OAI patients stratified by OA progression (see text for p -values). Points represent individual sub-samples ( n = 200) of individuals taken from patient groups presenting with no/moderate-OA (KL = 0/1) ( n = 1207), progressing to significant OA ( n = 208), and presenting with significant OA (KL ≥ 2) ( n = 1119). A two-sided Student’s T -test was used to compare risk-allele occurrence in subsamples of each group, with FDR correction for n = 3 tests. Central bar represents mean of distribution, lower/upper bars indicate first and third quartiles, respectively. b Significantly different anatomical features of the knee in OAI patients (WT n = 76, HET n = 161, HOMO n = 146). ANOVA with Dunnet post-hoc was used for pairwise comparisons to wild type. All p -values are two-sided and indicate significant differences compared to individual’s wild type for the non-risk allele. Bars indicate medians. c µCT measurements of significantly different anatomical features in rs6060369 single base-pair replacement mice at 1-year (WT n = 6, HET n = 15, HOMO n = 14). ANOVA with Dunnet post-hoc was used for pairwise comparisons to wild type. All p -values are two-sided and indicate significant differences compared to wild type. Bars indicate medians. d Histological analysis of rs6060369 single base-pair replacement mice at 1 year, showing coronal sections at mid-knee level. The sections represent knees with mild, moderate, and severe knee OA. Sections are color coded to their corresponding overall score listed in the graph in ( e ). The scale bars are 250 µm. e OARSI scores on wild type and homozygous rs6060369 single base-pair “T” replacement knees at 1 year (WT n = 5, HOMO n = 10). Mann–Whitney test with two-sided p values were used for comparison. f 3D comparative analysis indicating the anatomical locations of largest morphological differences between wild type R4 +/+ and homozygous R4 -/- hind limbs (top) as well as between wild type R4 rs6060369-A/rs6060369-A and homozygous R4 rs6060369-T/rs6060369-T hind limbs (bottom) at 1-year. All p -values are two-sided. See Supplementary Figs. – , Supplementary Tables – and the Supplementary Information for related analyses.

Article Snippet: The R4 rs6060369-T / rs6060369- + single allelic replacement mouse line contains a single “T” allelic base-pair replacement of the orthologous human rs6060369 variant in the R4 enhancer and was generated on the C57BL/6 J Mus musculus background by Applied StemCell and PI Capellini laboratory.

Techniques: MANN-WHITNEY, Comparison

Journal: Nature Communications

Article Title: Joint disease-specificity at the regulatory base-pair level

doi: 10.1038/s41467-021-24345-9

Figure Lengend Snippet: a Linear regression analysis showing a strong linear relationship between levels of Gdf5 expression and extent of bony shape changes in four distinct mouse lines, two of which harbor separate enhancer deletions (i.e., of R4 and GROW1 ) and two of which harbor separate, single-base-pair replacements in these enhancers (i.e., at rs6060369 and rs4911178). Dark circles = homozygous, open circles = heterozygous. See the Supplementary Information for related analyses. b Average fold change in anatomy between homozygous and heterozygous (enhancer null and single base-pair) mutations in R4 and GROW1 enhancers. Summary data are presented as mean and standard deviations. c Mechanistic model showing two separate non-coding regulatory variants on the same risk GDF5 haplotype, i.e., rs4911178 “A” variant in the GROW1 enhancer and rs6060369 “T” variant in the R4 enhancer control different pathologies (i.e., DDH and knee OA, respectively) in different joints, mediated by differential binding of PITX1 on each respective enhancer variant in each specific joint.

Article Snippet: The R4 rs6060369-T / rs6060369- + single allelic replacement mouse line contains a single “T” allelic base-pair replacement of the orthologous human rs6060369 variant in the R4 enhancer and was generated on the C57BL/6 J Mus musculus background by Applied StemCell and PI Capellini laboratory.

Techniques: Expressing, Variant Assay, Control, Binding Assay