Journal: PLoS Genetics

Article Title: Targeted Next-Generation Sequencing Identifies a Recurrent Mutation in MCPH1 Associating with Hereditary Breast Cancer Susceptibility

doi: 10.1371/journal.pgen.1005816

Figure Lengend Snippet: Frequency of the heterozygous MCPH1 c.904_916del mutation among familial, unselected and young breast cancer patients, and in population controls.

Article Snippet: The primary MCPH1 antibody (R&D systems, AF3998) was a goat polyclonal antibody raised against the amino acids 1–250 mapping to the N-terminus of MCPH1 of human origin.

Techniques: Mutagenesis

Journal: PLoS Genetics

Article Title: Targeted Next-Generation Sequencing Identifies a Recurrent Mutation in MCPH1 Associating with Hereditary Breast Cancer Susceptibility

doi: 10.1371/journal.pgen.1005816

Figure Lengend Snippet: Family history of cancers of MCPH1 c.904_916del positive index cases ^a .

Article Snippet: The primary MCPH1 antibody (R&D systems, AF3998) was a goat polyclonal antibody raised against the amino acids 1–250 mapping to the N-terminus of MCPH1 of human origin.

Techniques: Biomarker Discovery

Journal: PLoS Genetics

Article Title: Targeted Next-Generation Sequencing Identifies a Recurrent Mutation in MCPH1 Associating with Hereditary Breast Cancer Susceptibility

doi: 10.1371/journal.pgen.1005816

Figure Lengend Snippet: ( A ) Schematic presentation of the MCPH1 protein and the position of the observed truncating mutation. ( B ) Sequence chromatogram comparisons of genomic DNA and cDNA of heterozygous mutation carriers and a wild-type control. ( C ) Immunoblotting of 3 mutation carriers and 3 non-carriers with an antibody directed towards the amino-terminus of MCPH1. The representative image of altogether three independent experiments is shown.

Article Snippet: The primary MCPH1 antibody (R&D systems, AF3998) was a goat polyclonal antibody raised against the amino acids 1–250 mapping to the N-terminus of MCPH1 of human origin.

Techniques: Mutagenesis, Sequencing, Control, Western Blot

Journal: PLoS Genetics

Article Title: Targeted Next-Generation Sequencing Identifies a Recurrent Mutation in MCPH1 Associating with Hereditary Breast Cancer Susceptibility

doi: 10.1371/journal.pgen.1005816

Figure Lengend Snippet: Patients with breast cancer are marked with black half circles. Other cancer types are marked with grey squares. The age at diagnosis, when known, is marked below the cancer type. Individuals genotyped for MCPH1 c.904_916del are marked with either a plus (mutation positive) or a minus sign (mutation negative). A slashed pedigree symbol indicates a deceased individual. Triangle indicates the initially studied index patient (BR-0653, BR-0887 and BR-0154, respectively). Abbreviations: Bas: basalioma, Bt: brain tumor, Br: breast cancer, Col: colon cancer, Csu: cancer site unknown, Lar: laryngeal cancer, Ov: ovarian tumor, Pro: prostate cancer, Ut: uterine cancer, Vul: vulvar cancer.

Article Snippet: The primary MCPH1 antibody (R&D systems, AF3998) was a goat polyclonal antibody raised against the amino acids 1–250 mapping to the N-terminus of MCPH1 of human origin.

Techniques: Biomarker Discovery, Mutagenesis

Journal: PLoS Genetics

Article Title: Targeted Next-Generation Sequencing Identifies a Recurrent Mutation in MCPH1 Associating with Hereditary Breast Cancer Susceptibility

doi: 10.1371/journal.pgen.1005816

Figure Lengend Snippet: Occurrence of chromosomal aberrations in blood lymphocyte metaphases of heterozygous MCPH1 c.904_916del mutation carriers and healthy controls.

Article Snippet: The primary MCPH1 antibody (R&D systems, AF3998) was a goat polyclonal antibody raised against the amino acids 1–250 mapping to the N-terminus of MCPH1 of human origin.

Techniques: Mutagenesis

Journal: PloS one

Article Title: A novel MCPH1 isoform complements the defective chromosome condensation of human MCPH1-deficient cells.

doi: 10.1371/journal.pone.0040387

Figure Lengend Snippet: Figure 1. The human MCPH1 gene, its transcripts and predicted polypeptides. (A) Exon (filled boxes) and intron (open boxes) organization of the 241 906-bp encompassing MCPH1 gene locus. Red arrows indicate the positions of the regular and of the alternative (*) polyadenylation sites (polyA). (B) The full-length (FL) and the alternative transcripts De9–14, De1–3, and De8: numbered boxes indicate exons, black filled areas illustrate the entire coding regions (CDS), and colored areas show untranslated regions (UTR) as indicated. (C) Predicted polypeptides representing MCPH1 isoforms: blue boxes depict the positions of BRCT domains, while green boxes represent the site of the canonical nuclear localization signal sequence (NLS). Two additional amino acids, S and M, are included into MCPH1De9–14 prior to premature termination (#). (D) Expression of MCPH1 transcript variants. Columns represent the levels of MCPH1 transcripts in indicated adult and fetal tissues determined using quantitative real-time PCR. Data represent means 6 one S.D. of three independent experiments and are normalized to the geometric mean levels of UBC, GAPDH, B2M, and HPRT1 cDNA. doi:10.1371/journal.pone.0040387.g001

Article Snippet: Antibodies Rabbit antiserum (ab2612) and goat antiserum (AF3998) to MCPH1 were obtained from Abcam (Cambridge, UK) and R&D Systems (Minneapolis, MN, USA), respectively.

Techniques: Sequencing, Expressing, Real-time Polymerase Chain Reaction

Journal: PloS one

Article Title: A novel MCPH1 isoform complements the defective chromosome condensation of human MCPH1-deficient cells.

doi: 10.1371/journal.pone.0040387

Figure Lengend Snippet: Figure 2. Cell cycle-dependent regulation of MCPH1 transcripts. (A) HeLa cells were arrested in G1 phase by double thymidine block. Cultures harvested at various time points after release were analyzed using flow cytometry. (B) Plots represent numbers of cells as a function of their DNA content. A total of 90% of the cells synchronously progressed into S phase (0–4 h), entered G2 phase (4–6 h), started passing through mitosis after 7 h, and were completely in G1 phase after 12 h. (C) Levels of MCPH1-FL (diamonds, solid line), MCPH1De9–14 (squares, dotted line), and MCPH1De8 (circles, dashed line) mRNA. Data represent means 6 S.E.M. of three independent experiments and are normalized to the expression levels of GAPDH and B2M. doi:10.1371/journal.pone.0040387.g002

Article Snippet: Antibodies Rabbit antiserum (ab2612) and goat antiserum (AF3998) to MCPH1 were obtained from Abcam (Cambridge, UK) and R&D Systems (Minneapolis, MN, USA), respectively.

Techniques: Blocking Assay, Flow Cytometry, Expressing

Journal: PloS one

Article Title: A novel MCPH1 isoform complements the defective chromosome condensation of human MCPH1-deficient cells.

doi: 10.1371/journal.pone.0040387

Figure Lengend Snippet: Figure 3. Expression of GFP-tagged MCPH1 isoforms in MCPH1-deficient 562T fibroblasts. (A) Cells were transduced with GFP-tagged coding sequence of full-length MCPH1 cDNA in a conditional, doxycycline (DOX)-dependent construct with a second regulatory construct trKRAB. Cultures were exposed to increasing DOX concentrations as indicated. Whole-cell extracts were prepared 72 h later and analyzed for the expression of MCPH1 using immunoblotting with an antibody against GFP. (B) The graph shows MCPH1 band intensity relative to the loading control p84 plotted against DOX concentrations. Data represent means 6 one S.D. of three independent assays. (C) Immunoblot analysis of whole-cell extracts from non-transduced (NT) 562T cells (lane 1), 562T cells transduced with the regulatory construct only (lane 2), with GFP alone (30 kDa, lane 3) or with GFP fused to MCPH1-FL (120 kDa, lane 4), MCPH1De9–14 (94 kDa, lane 5), MCPH1De8 (78 kDa, lane 6), or MCPH1De1–7 (96 kDa, lane 7) with an antibody against GFP.. Nuclear matrix protein p84 served as the loading control. doi:10.1371/journal.pone.0040387.g003

Article Snippet: Antibodies Rabbit antiserum (ab2612) and goat antiserum (AF3998) to MCPH1 were obtained from Abcam (Cambridge, UK) and R&D Systems (Minneapolis, MN, USA), respectively.

Techniques: Expressing, Transduction, Sequencing, Construct, Western Blot, Control

Journal: PloS one

Article Title: A novel MCPH1 isoform complements the defective chromosome condensation of human MCPH1-deficient cells.

doi: 10.1371/journal.pone.0040387

Figure Lengend Snippet: Figure 4. Complementation of PCC in patient fibroblasts. Cells are derived from patient with homozygous truncating mutation c.427dupA (p.T143NfsX5) in MCPH1. Chromosome preparations from (A) non-transduced cells and (B) cells expressing GFP only, or GFP fusions with (C) full- length, (D) De9–14, (E) De8, or (F) De1–7 MCPH1. Arrows indicate nuclei of prophase-like cells (PLCs). (G) Mean rates of PLCs (filled columns) of slides from A-F. Open columns represent mean mitotic indices. Error bars denote the S.D. of counts of approximately 1000 cells each from three independent experiments. doi:10.1371/journal.pone.0040387.g004

Article Snippet: Antibodies Rabbit antiserum (ab2612) and goat antiserum (AF3998) to MCPH1 were obtained from Abcam (Cambridge, UK) and R&D Systems (Minneapolis, MN, USA), respectively.

Techniques: Derivative Assay, Mutagenesis, Expressing

Journal: PloS one

Article Title: A novel MCPH1 isoform complements the defective chromosome condensation of human MCPH1-deficient cells.

doi: 10.1371/journal.pone.0040387

Figure Lengend Snippet: Figure 5. Only simultaneous downregulation of endogenous full-length (FL) and De9–14 MCPH1 induces PCC. (A) Immunoblots with an anti-MCPH1 antibody after transfection of HeLa cells with different siRNAs demonstrate efficient downregulation of either MCPH1-FL (93 kDa) or MCPH1De9–14 (70 kDa) or both isoforms (exon 8 and FL+ De9–14) but no downregulation in response to non-targeting siRNA or mock-transfected controls. Nuclear matrix protein p84 served as a loading control. (B) Columns represent PLC rates in HeLa cells transfected with MCPH1 siRNA as indicated in A. Error bars denote the S.D. doi:10.1371/journal.pone.0040387.g005

Article Snippet: Antibodies Rabbit antiserum (ab2612) and goat antiserum (AF3998) to MCPH1 were obtained from Abcam (Cambridge, UK) and R&D Systems (Minneapolis, MN, USA), respectively.

Techniques: Western Blot, Transfection, Control

Journal: PloS one

Article Title: A novel MCPH1 isoform complements the defective chromosome condensation of human MCPH1-deficient cells.

doi: 10.1371/journal.pone.0040387

Figure Lengend Snippet: Figure 6. Intracellular distribution of MCPH1 isoforms. (A) MCPH1-deficient fibroblasts expressing GFP alone or the specified GFP-MCPH1 fusion proteins were fractionated and cytoplasmic (Cyt) and nuclear (Nuc) protein extracts were analyzed using immunoblotting with an antibody against GFP. The nuclear matrix protein p84 and GAPDH were used as index proteins and loading controls. (B) Cells indicated in A stained with an anti-GFP antibody (green), counterstained with DAPI (blue) and analyzed using fluorescence microscopy. Arrows indicate the prophase-like nuclei. Scale bar = 10 mm. All MCPH1 isoforms exhibit unambiguous nuclear localization. doi:10.1371/journal.pone.0040387.g006

Article Snippet: Antibodies Rabbit antiserum (ab2612) and goat antiserum (AF3998) to MCPH1 were obtained from Abcam (Cambridge, UK) and R&D Systems (Minneapolis, MN, USA), respectively.

Techniques: Expressing, Western Blot, Staining, Fluorescence, Microscopy

Journal: PloS one

Article Title: A novel MCPH1 isoform complements the defective chromosome condensation of human MCPH1-deficient cells.

doi: 10.1371/journal.pone.0040387

Figure Lengend Snippet: Figure 7. Nuclear localization signals (NLSs) in human MCPH1. (A) The positions of the putative NLS motifs and their amino acid sequences are highlighted in the diagram of the full-length MCPH1. (B) Subcellular distribution of GFP-tagged wild-type (wt) MCPH1 and mutants with deleted NLSs as indicated were transiently expressed in HeLa cells. Cytoplasmic (Cyt) and nuclear (Nuc) protein extracts were immunoblotted with an antibody against GFP (left panel). GAPDH (center panel) and the nuclear matrix protein p84 (right panel) served as index proteins and loading controls. (C) Ratios of relative GFP band intensity in the cytoplasmic (Cyt) vs. nuclear (Nuc) fractions. Absolute numbers were assessed using densitometry and normalized to the loading controls. Columns designate means, and error bars represent the S.D. from three different experiments. Significant differences to wt MCPH1 are indicated by asterisks denoting p,0.05 (Student’s t-test). Scale bar = 10 mm. doi:10.1371/journal.pone.0040387.g007

Article Snippet: Antibodies Rabbit antiserum (ab2612) and goat antiserum (AF3998) to MCPH1 were obtained from Abcam (Cambridge, UK) and R&D Systems (Minneapolis, MN, USA), respectively.

Techniques:

Journal: PloS one

Article Title: A novel MCPH1 isoform complements the defective chromosome condensation of human MCPH1-deficient cells.

doi: 10.1371/journal.pone.0040387

Figure Lengend Snippet: Figure 8. Colocalization of MCPH1 and cH2AX in ionizing irradiation-induced nuclear foci. (A) Non-transduced (NT) MCPH1-deficient 562T cells and 562T stably expressing GFP alone or the specified GFP-MCPH1 fusion proteins were fixed 2 h after irradiation with 10 Gy and co- stained with antibodies against cH2AX (red) and GFP (green). Nuclei were counterstained with DAPI (blue). Rectangles frame areas, which are shown enlarged in the bottom row. MCPH1 focus formation was observed for MCPH1 isoforms containing the C-terminal BRCT tandem. (B) Quantification of cells expressing foci containing cH2AX and/or (C) MCPH1. Error bars indicate the S.D. of three different measurements, counting approximately 300 nuclei. * p#0.05 vs. NT as calculated using the Student’s t-test. doi:10.1371/journal.pone.0040387.g008

Article Snippet: Antibodies Rabbit antiserum (ab2612) and goat antiserum (AF3998) to MCPH1 were obtained from Abcam (Cambridge, UK) and R&D Systems (Minneapolis, MN, USA), respectively.

Techniques: Irradiation, Stable Transfection, Expressing, Staining

Journal: PLoS ONE

Article Title: IGFBP3 Colocalizes with and Regulates Hypocretin (Orexin)

doi: 10.1371/journal.pone.0004254

Figure Lengend Snippet: Human narcolepsy susceptibility candidate genes analyzed by microarray, quantitative RT-PCR and distribution shown by in situ hybridization in mice.

Article Snippet: 236034_at , microcephaly, primary autosomal recessive 1 (MCPH1) , B , 83 , 2.6 , 0.030 , Hs00226253_m1 , , 0.8 , 0.361 , 6416651 , n.e..

Techniques: Microarray, Quantitative RT-PCR, In Situ Hybridization, Binding Assay

Journal: PLoS ONE

Article Title: Mcph1 -Deficient Mice Reveal a Role for MCPH1 in Otitis Media

doi: 10.1371/journal.pone.0058156

Figure Lengend Snippet: (A) Schematic of knockout strategy for Mcph1 gene based on knockout-first design. A promoterless cassette including LacZ and neo genes was inserted in the third intron of Mcph1 gene flanked by FRT sites. LoxP sites flank the critical exon (exon4 of Mcph1 gene in knockout-first design). See http://www.knockoutmouse.org/martsearch/project/41705 for more details. (B) Short range PCR for genotyping. Wild type allele produces one band of 366bp. Due to the insertion of the cassette, primers designed for the wild type allele do not have product for the mutant allele using the short range PCR (illustrated as the left panel, schematic illustration is not in scale). The homozygous allele produces only one band of 185 bp. The heterozygotes produce two bands of 185 bp and 366 bp. (C) Quantitative real-time PCR showed largely reduced transcript of Mcph1 in Mcph1 tm1a / tm1a (n = 3) mice compared to wild type mice (n = 3) and the residual levels vary in different organs.

Article Snippet: Real-time PCR was performed in quadruplicate for each sample using the probe (Applied Biosystem, Mm00557495_m1, covering exon 3–4 boundary) in an ABI Prism 7000 (Applied Biosystem).

Techniques: Knock-Out, Mutagenesis, Real-time Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: Mcph1 -Deficient Mice Reveal a Role for MCPH1 in Otitis Media

doi: 10.1371/journal.pone.0058156

Figure Lengend Snippet: (A) ABR measurement results of 14 week old Mcph1 tm1a/tm1a mice (n = 11) in MGP showed mild to moderate hearing impairment, or normal hearing compared to control mice. The green baseline area shows a reference range for the control wild type mice with the same genetic background, plotting the median and 2.5 to 97.5 percentile of the population (n = 440). (B) Input-output function (IOF) analysis. The peak-peak amplitude of wave 1 (P1-N1 amplitude) of click-evoked ABRs is plotted as a function of dB SL (Sensation Level, dB above threshold) for wild type (green) and Mcph1 tm1a/tm1a (red) mice. There was no significant difference of IOF slopes of Mcph1 tm1a/tm1a (n = 24, slope = 0.144+/−0.066; mean +/− SD) and wild type mice (n = 36, slope = 0.133+/−0.048) (t-test, p = 0.444).

Article Snippet: Real-time PCR was performed in quadruplicate for each sample using the probe (Applied Biosystem, Mm00557495_m1, covering exon 3–4 boundary) in an ABI Prism 7000 (Applied Biosystem).

Techniques: Control

Journal: PLoS ONE

Article Title: Mcph1 -Deficient Mice Reveal a Role for MCPH1 in Otitis Media

doi: 10.1371/journal.pone.0058156

Figure Lengend Snippet: (A) Results of recurrent ABR measurement (click thresholds) with age. Hearing impairment can be detected as early as 3 weeks old in Mcph1 tm1a/tm1a mice (n = 13). Hearing profile of the Mcph1 tm1a/tm1a mice showed either a stable, progressive, or fluctuating pattern with age (three of them marked dark). All the wild type (n = 13) and heterozygous (n = 17) mice displayed normal click thresholds with age. (B) Auditory chain (incus-stapes joint) and oval window sound transduction was severely impeded. Normal incus-stapes joint of auditory chain in a Mcph1 + / + mouse, and a clear oval window is necessary for sound vibration conduction. After removing some of the amorphous material in the middle ear cavity of a Mcph1 tm1a / tm1a mouse, the incus-stapes joint (arrow head) and the oval window (arrow) is present but embedded in the amorphous material. Scale bar, 1 mm. (C–F) Correlation between middle ear defects and hearing sensitivity change with time. (C) Normal ABR thresholds and middle ear structure in a wild type mouse: normal middle ear cavity is full of air, tympanic membrane is transparent and normal morphology of ossicles. (D) Progressively elevated ABR thresholds with age in a Mcph1 tm1a / tm1a mouse. Amorphous mass filled the middle ear cavity and outgrew into external ear canal. Ossicles were embedded in the amorphous mass and appeared to have thinner bony structure. (E) Fluctuating ABR thresholds in a Mcph1 tm1a / tm1a mouse. Watery effusion with bubbles was seen in the middle ear cavity and normal gross morphology of ossicles. (F) Stable and moderate hearing impairment in a Mcph1 tm1a / tm1a mouse. The middle cavity was filled with pus-like secretion. Normal gross morphology of ossicles but with rough surface. Scale bar, 1 mm.

Article Snippet: Real-time PCR was performed in quadruplicate for each sample using the probe (Applied Biosystem, Mm00557495_m1, covering exon 3–4 boundary) in an ABI Prism 7000 (Applied Biosystem).

Techniques: Transduction, Membrane

Journal: PLoS ONE

Article Title: Mcph1 -Deficient Mice Reveal a Role for MCPH1 in Otitis Media

doi: 10.1371/journal.pone.0058156

Figure Lengend Snippet: A & B illustrate click-evoked ABR waveforms recorded in a wildtype (green) and a Mcph1 tm1a / tm1a (red) mouse, respectively. The dB SPL of the click stimulus for each response is indicated on the abscissa. The scale bar indicates 5 µV amplitude of response. The heavy lines indicate the click-ABR threshold allocated to each mouse. C. (inset) Mean click-evoked ABR waveforms recorded at 21 dB sensation level for wildtype (n = 8, green) and Mcph1 tm1a / tm1a (n = 8, red).

Article Snippet: Real-time PCR was performed in quadruplicate for each sample using the probe (Applied Biosystem, Mm00557495_m1, covering exon 3–4 boundary) in an ABI Prism 7000 (Applied Biosystem).

Techniques:

Journal: PLoS ONE

Article Title: Mcph1 -Deficient Mice Reveal a Role for MCPH1 in Otitis Media

doi: 10.1371/journal.pone.0058156

Figure Lengend Snippet: Clear middle ear cavity (MEC) and thin mucoperiosteum in wild type mice (A,C). MECs of Mcph1 tm1a / tm1a mice (B,D) were filled with exudate and lined with thickened mucoperiosteum. High magnification for mucoperiosteum of MEC framed in A and B (C,D). Inflammatory cells (E,F) in MECs. Scale bar, 200 µm (A,B), 20 µm (C–F).

Article Snippet: Real-time PCR was performed in quadruplicate for each sample using the probe (Applied Biosystem, Mm00557495_m1, covering exon 3–4 boundary) in an ABI Prism 7000 (Applied Biosystem).

Techniques:

Journal: PLoS ONE

Article Title: Mcph1 -Deficient Mice Reveal a Role for MCPH1 in Otitis Media

doi: 10.1371/journal.pone.0058156

Figure Lengend Snippet: (A) Scanning electron microscope (SEM) showed normal development of hair cells at P4 in Mcph1 tm1a / tm1a mice ( Mcph1 + / + , n = 3; Mcph1 tm1a / tm1a , n = 3. scale bar, 10 µm). (B) HE slides displayed comparable structure of inner ears (basal turn) in Mcph1 + / + and Mcph1 tm1a / tm1a mice at 4–5 weeks old ( Mcph1 + / + , n = 3; Mcph1 tm1a / tm1a , n = 3. scale bar, 50 µm).

Article Snippet: Real-time PCR was performed in quadruplicate for each sample using the probe (Applied Biosystem, Mm00557495_m1, covering exon 3–4 boundary) in an ABI Prism 7000 (Applied Biosystem).

Techniques: Microscopy

Journal: PLoS ONE

Article Title: Mcph1 -Deficient Mice Reveal a Role for MCPH1 in Otitis Media

doi: 10.1371/journal.pone.0058156

Figure Lengend Snippet: Immunochemistry using an antibody shows that Mcph1 (brown labelling) is expressed in epithelial cells of the middle ear cavity at P7 (A,B) and P28 (C,D) wild type mice. Mcph1 is expressed in both ciliated (B,D) cells close to orifice of Eustachian tube and non-ciliated cells (A,C). Scale bar, 20 µm.

Article Snippet: Real-time PCR was performed in quadruplicate for each sample using the probe (Applied Biosystem, Mm00557495_m1, covering exon 3–4 boundary) in an ABI Prism 7000 (Applied Biosystem).

Techniques:

Journal: PLoS ONE

Article Title: Mcph1 -Deficient Mice Reveal a Role for MCPH1 in Otitis Media

doi: 10.1371/journal.pone.0058156

Figure Lengend Snippet: (A) X-ray assay showed comparable structure of craniofacial skeleton between wild type and Mcph1 tm1a / tm1a mice at 14 weeks old. (B) Measurement showed that skull width and length in female Mcph1 tm1a / tm1a mice (n = 6) are significantly smaller than those of wild type mice (n = 7) (Rank Sum test). The weight of the brain was measured at 16 weeks of age showed that female and male Mcph1 tm1a / tm1a mice (n = 3, each sex) had lighter brain than the local control mice (n = 3, each sex) and MGP wild type mouse baseline (female, n = 10; male n = 187).

Article Snippet: Real-time PCR was performed in quadruplicate for each sample using the probe (Applied Biosystem, Mm00557495_m1, covering exon 3–4 boundary) in an ABI Prism 7000 (Applied Biosystem).

Techniques: Control

Journal: PLoS ONE

Article Title: Mcph1 -Deficient Mice Reveal a Role for MCPH1 in Otitis Media

doi: 10.1371/journal.pone.0058156

Figure Lengend Snippet: Adult Mcph1 tm1a / tm1a (n = 7) mice showed increased genomic instability when compared to wild type mice (n = 34) as determined by an increased prevalence of micronucleated normochromatic erythrocytes (MN-NCE). Data is presented as the Mean ± SEM.

Article Snippet: Real-time PCR was performed in quadruplicate for each sample using the probe (Applied Biosystem, Mm00557495_m1, covering exon 3–4 boundary) in an ABI Prism 7000 (Applied Biosystem).

Techniques:

Journal: PLoS ONE

Article Title: Mcph1 -Deficient Mice Reveal a Role for MCPH1 in Otitis Media

doi: 10.1371/journal.pone.0058156

Figure Lengend Snippet: (A) No difference of weight change (Mean ± SEM) between Mcph1 tm1a/tm1a (n = 4) and Mchp1 +/+ (n = 8) mice infected by Salmonella Typhimurium and monitored by weight loss for 21 days. (B) Viable bacterial counts from the spleen (i) and the liver (ii) from the same mice infected by Salmonella Typhimurium at 21 days post infection. There was only one Mcph1 tm1a/tm1a mouse out of 4 tested with any bacteria present in the liver. Mann Whitney U tests were used, p (two-tailed) is indicated in the figure. (C) Viable bacterial counts (Mean ± SEM) being shed in the stool from Mcph1 tm1a/tm1a (n = 5) and Mchp1 +/+ (n = 5) mice were similar over a 28 day infection of Citrobacter rodentium .

Article Snippet: Real-time PCR was performed in quadruplicate for each sample using the probe (Applied Biosystem, Mm00557495_m1, covering exon 3–4 boundary) in an ABI Prism 7000 (Applied Biosystem).

Techniques: Infection, Bacteria, MANN-WHITNEY, Two Tailed Test

Journal: PLoS ONE

Article Title: Mcph1 -Deficient Mice Reveal a Role for MCPH1 in Otitis Media

doi: 10.1371/journal.pone.0058156

Figure Lengend Snippet: Mcph1 tm1a/tm1a and Mchp1 +/+ mice were immunised intranasally with fragment C of tetanus toxin on day 0, 7 and 21. Presence of antigen-specific Ig in serum isolated at day 28 was showed no difference in Mcph1 tm1a/tm1a (n = 6) and Mchp1 +/+ (n = 5). The solid bar represents the Mean ± SEM. Mann Whitney U tests were used, p (two-tailed) is indicated in the figure.

Article Snippet: Real-time PCR was performed in quadruplicate for each sample using the probe (Applied Biosystem, Mm00557495_m1, covering exon 3–4 boundary) in an ABI Prism 7000 (Applied Biosystem).

Techniques: Isolation, MANN-WHITNEY, Two Tailed Test

Journal: PLoS ONE

Article Title: Mcph1 -Deficient Mice Reveal a Role for MCPH1 in Otitis Media

doi: 10.1371/journal.pone.0058156

Figure Lengend Snippet: (A) Slit lamp images (12× magnification) revealed corneal (center) opacity and vascularisation (right) in Mcph1 tm1a / tm1a mice. The difference of ocular abnormality portion is significant between wild type (n = 23) and Mcph1 tm1/tm1a (n = 14) mice (Fischer's exact test: p = 0.002). (B) Wild type eye shows normal lens and retina. The anterior and posterior chamber spaces are well defined. ac = anterior chamber; pc = posterior chamber. (C) Mcph1 tm1a / tm1a eye shows cataractous lens and thin retina. The anterior and posterior chambers are collapsed. Scale bar, 500 µm (B,C). (D) Inset from (C) with solid line shows cataractous lens and loss of retinal cell layers. Scale bar, 100 µm.

Article Snippet: Real-time PCR was performed in quadruplicate for each sample using the probe (Applied Biosystem, Mm00557495_m1, covering exon 3–4 boundary) in an ABI Prism 7000 (Applied Biosystem).

Techniques:

Journal: Nature Communications

Article Title: Microcephalin 1/BRIT1-TRF2 interaction promotes telomere replication and repair, linking telomere dysfunction to primary microcephaly

doi: 10.1038/s41467-020-19674-0

Figure Lengend Snippet: Data collection and refinement statistics.

Article Snippet: MCPH1 TBM peptides, synthesized by GenScript China, at 1.0 mM concentration in the syringe were titrated into TRF2 TRFH at 0.1 mM concentration in the sample cell.

Techniques: Solvent

Journal: Nature Communications

Article Title: Microcephalin 1/BRIT1-TRF2 interaction promotes telomere replication and repair, linking telomere dysfunction to primary microcephaly

doi: 10.1038/s41467-020-19674-0

Figure Lengend Snippet: a Schematic representation of human TRF2 and MCPH1 domains, showing the interaction domains. b Comparison of MCPH1 TBM sequence with those of known TRF2-interacting protein. The conserved amino acid Y/H-X-L-X-P consensus sequence is highlighted. c Dimeric TRF2–MCPH1 structure is shown in a ribbon representation (TRF2, green/cyan; MCPH1, magenta/yellow). d TRF2 and MCPH1 are depicted in green and yellow, respectively, and the residues involved in their interaction are shown. Hydrogen bonding: magenta dashed lines. e MCPH1 TBM (in yellow) is buried inside a hydrophobic pocket formed by TRF2 helices α2 and α3 (in green). f ITC measurement of the interactions between TRF2 TRFH and different MCPH1 TBM mutant peptides. S333phos is a phosphorylated S333 peptide synthesized using a phosphorylated serine as starting material. Equilibrium dissociation constant ( K D ) values derived from ITC data are shown in Table .

Article Snippet: MCPH1 TBM peptides, synthesized by GenScript China, at 1.0 mM concentration in the syringe were titrated into TRF2 TRFH at 0.1 mM concentration in the sample cell.

Techniques: Comparison, Sequencing, Mutagenesis, Synthesized, Derivative Assay

Journal: Nature Communications

Article Title: Microcephalin 1/BRIT1-TRF2 interaction promotes telomere replication and repair, linking telomere dysfunction to primary microcephaly

doi: 10.1038/s41467-020-19674-0

Figure Lengend Snippet: Equilibrium dissociation constant ( K D ) values.

Article Snippet: MCPH1 TBM peptides, synthesized by GenScript China, at 1.0 mM concentration in the syringe were titrated into TRF2 TRFH at 0.1 mM concentration in the sample cell.

Techniques:

Journal: Nature Communications

Article Title: Microcephalin 1/BRIT1-TRF2 interaction promotes telomere replication and repair, linking telomere dysfunction to primary microcephaly

doi: 10.1038/s41467-020-19674-0

Figure Lengend Snippet: a Schematic representation of the human MCPH1 constructs generated. The TBM sequence for each construct is shown and amino acid substitutions are depicted in red. b Co-IP with anti-Myc antibody-conjugated agarose beads from lysates of 293T cells expressing Myc-tagged TRF2 and either FLAG-tagged WT MCPH1, FLAG-MCPH1 TBM mutants or FLAG-MCPH1 ΔBRCT . γ-tubulin was used as a loading control. The blot shown is representative of four independent experiments. c Immunostaining-PNA FISH in HeLa cells overexpressing either empty vector or one of the FLAG-tagged WT MCPH1, MCPH1 AA , MCPH1 S333A , MCPH1 S333D , MCPH1 ΔBRCT constructs and either empty vector or HA-TPP1 ΔRD . MCPH1 proteins were detected using an anti-FLAG antibody (green) while telomeres were detected with a Cy3-OO-(CCCTAA) 4 PNA probe (red). 4,6-diamidino-2-phenylindole (DAPI) was used to stain nuclei (blue). Representative images from three independent experiments are shown. White arrowheads indicate MCPH1 foci co-localizing with the telomere signals. Scale bar: 5 μm. d Quantification of the percentage of cells with >5 MCPH1-positive foci at telomeres from c . Data represent the mean ± standard deviation (SD) from three independent experiments. At least 200 cells were scored for each sample. Significance was determined with one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test. P values are shown. e Comparison of MCPH1 TBM amino acidic sequence across several mammalian species. The conserved residues are highlighted in yellow, while the residues in red differ from the canonical Y/H-X-L-X-P amino acid sequence. f Immunostaining-PNA FISH in MEFs overexpressing either Myc-WT MCPH1 or Myc-MCPH1 ΔBRCT together with either empty vector or FLAG-TIN2 A110R . Myc-MCPH1 proteins were detected with a Myc antibody (green), while telomeres were detected with either a telomeric PNA probe or a FLAG antibody that recognizes FLAG-TIN2 A110R (in red). Nuclei were stained with DAPI (blue). Representative images from three independent experiments. Scale bar: 5 μm. g Quantification of the percentage of cells with >5 MCPH1-positive foci at telomeres from f . Data are representative of the mean of three independent experiments ± SD. A minimum of 200 cells for each sample were scored. Statistical analysis: one-way ANOVA followed by Tukey’s multiple comparison test.

Article Snippet: MCPH1 TBM peptides, synthesized by GenScript China, at 1.0 mM concentration in the syringe were titrated into TRF2 TRFH at 0.1 mM concentration in the sample cell.

Techniques: Construct, Generated, Sequencing, Co-Immunoprecipitation Assay, Expressing, Control, Immunostaining, Plasmid Preparation, Staining, Standard Deviation, Comparison

Journal: Nature Communications

Article Title: Microcephalin 1/BRIT1-TRF2 interaction promotes telomere replication and repair, linking telomere dysfunction to primary microcephaly

doi: 10.1038/s41467-020-19674-0

Figure Lengend Snippet: a Immunostaining for MCPH1 telomeric localization in MCPH1 +/+ HCT116 and two CRISPR/Cas9 MCPH1 Δ/Δ HCT116 clones (B2 and A5) overexpressing the indicated constructs. MCPH1 localization at telomeres was assessed using an anti-MCPH1 antibody (green) and telomere were detected through PNA-FISH (red). Representative images from either three (MCPH1 +/+ and MCPH1 Δ/Δ B2 + vector, TRF2 ΔBΔM or TPP1 ΔRD ) or two (MCPH1 Δ/Δ A5 and samples with TRF2 ΔBΔM + TPP1 ΔRD ) independent experiments. b Percentage of cells with >5 MCPH1-positive foci at telomeres from ( a ). Data represent mean values ± SD. n = 3 for MCPH1 +/+ and MCPH1 Δ/Δ B2 + vector, TRF2 ΔBΔM and TPP1 ΔRD ; n = 2 for MCPH1 Δ/Δ A5 and samples with TRF2 ΔBΔM + TPP1 ΔRD . A minimum of 200 cells were scored for each sample. c BARD1 TIF analysis in WT and MCPH1 Δ/Δ cells reconstituted with either empty vector, WT MCPH1, MCPH1 S333A or MCPH1 S333D and overexpressing either empty vector or FLAG-TPP1 ΔRD . Representative images from two independent experiments. d Percentage of cells with >5 BARD1-positive TIFs from ( c ). The means from two independent experiments ± SD are shown. At least 200 cells were scored for each sample. e – g Quantification of the percentage of cells with >5 p-RPA32 (S33) ( e ) and CTIP ( f ) TIFs and with >3 EXOI ( g ) TIFs in WT and MCPH1 Δ/Δ cells reconstituted with the indicated constructs and overexpressing either empty vector or FLAG-TPP1 ΔRD (see also Supplementary Fig. ). Data represent the mean ± SD from two independent experiments. At least 200 cells were scored for each sample. h RAD51 TIF analysis in TPP1 ΔRD -expressing U2OS cells treated with either scrambled or MCPH1 shRNA and reconstituted with either empty vector, WT MCPH1, MCPH1 S333A or MCPH1 S333D . Representative images from three independent experiments. i Quantification of the percentage of cells with >5 RAD51-positive TIFs shown in ( h ). Data represent the mean values ± SD, n = 3. At least 200 cells were scored for each sample. The statistical analysis for b , d – g and i was performed using one-way ANOVA followed by Tukey’s multiple comparison test. Scale bars for a , c , h : 5 μm.

Article Snippet: MCPH1 TBM peptides, synthesized by GenScript China, at 1.0 mM concentration in the syringe were titrated into TRF2 TRFH at 0.1 mM concentration in the sample cell.

Techniques: Immunostaining, CRISPR, Clone Assay, Construct, Plasmid Preparation, Expressing, shRNA, Comparison

Journal: Nature Communications

Article Title: Microcephalin 1/BRIT1-TRF2 interaction promotes telomere replication and repair, linking telomere dysfunction to primary microcephaly

doi: 10.1038/s41467-020-19674-0

Figure Lengend Snippet: a Telomeric PNA-FISH staining of metaphase spreads from MCPH1 +/+ , MCPH1 Δ/Δ B2, and MCPH1 Δ/Δ A5 HCT116 cells overexpressing either empty vector, TRF2 ΔBΔM , TPP1 ΔRD , or both TRF2 ΔBΔM and TPP1 ΔRD . Telomeres were detected using a PNA probe (red) and DAPI was used to stain the chromosomes (blue). Representative images from either three (MCPH1 +/+ and MCPH1 Δ/Δ B2 + vector, TRF2 ΔBΔM , or TPP1 ΔRD ) or two (MCPH1 Δ/Δ A5 and samples with TRF2 ΔBΔM + TPP1 ΔRD ) independent experiments. White and green arrowheads indicate chromosome and chromatid fusions, respectively. Scale bar: 5 μm. b , c Quantification of the percentage of chromosome ( b ) and chromatid ( c ) fusions observed in metaphase spreads shown in ( a ). Data represent the mean values ± SD. n = 3 for MCPH1 +/+ and MCPH1 Δ/Δ B2 + vector, TRF2 ΔBΔM , and TPP1 ΔRD ; n = 2 for MCPH1 Δ/Δ A5 and samples with TRF2 ΔBΔM + TPP1 ΔRD . A minimum of 30 metaphases for each sample were examined per experiment. Significance was determined using one-way ANOVA followed by Tukey’s multiple comparison test. d Representative images of telomere sister chromatid exchanges (T-SCEs) (arrowheads) from three independent CO-FISH experiments on metaphase spreads of U2OS cells expressing TPP1 ΔRD and either scrambled or MCPH1 shRNA. Sister chromatid telomeres were labeled with a FAM-OO-(TTAGGG) 4 PNA probe (green) and with a Cy3-OO-(CCCTAA) 4 PNA probe (red) to detect telomeres generated by leading and lagging strand replication, respectively. Scale bar: 5 μm. e Quantification of the percentage of T-SCEs observed on metaphase spreads of U2OS cells expressing either empty vector or TPP1 ΔRD after MCPH1 depletion and reconstitution with the indicated constructs. Data are representative of the mean of three independent experiments ± SD. A minimum of 40 metaphases were analyzed per experiment. The indicated p values were calculated using one-way ANOVA followed by Tukey’s multiple comparison test.

Article Snippet: MCPH1 TBM peptides, synthesized by GenScript China, at 1.0 mM concentration in the syringe were titrated into TRF2 TRFH at 0.1 mM concentration in the sample cell.

Techniques: Staining, Plasmid Preparation, Comparison, Expressing, shRNA, Labeling, Generated, Construct

Journal: Nature Communications

Article Title: Microcephalin 1/BRIT1-TRF2 interaction promotes telomere replication and repair, linking telomere dysfunction to primary microcephaly

doi: 10.1038/s41467-020-19674-0

Figure Lengend Snippet: a Representative images from three independent experiments of telomeric PNA-FISH on chromosome spreads of WT and MCPH1 Δ/Δ HCT116 cells to detect multiple telomeric signals (MTS) (arrowheads). Scale bar: 5 μm. b Mean values ± SD of the percentage of MTS visualized by PNA-FISH in the indicated cell lines treated with either DMSO or 0.25 μM aphidicolin (APH). n = three independent experiments. At least 50 metaphases were scored. One-way ANOVA followed by Tukey’s multiple comparison test. c Co-IP with anti-TRF2 antibody from lysates of synchronized U2OS cells harvested at the indicated time points. Cyclin A was used as a control for cell cycle progression. Representative blots from two independent experiments. No Ab: no antibody control; *non-specific band. See also Supplementary Fig. . d Percentage of cells showing ≥4 MCPH1/FLAG-TRF1 co-localizations in IMR-90 and HeLa cells expressing CDT1 (G 1 ) and Geminin (S/G 2 ). See also Supplementary Fig. . Mean values ± SD from two independent experiments are shown. At least 200 nuclei were scored. Two-sided Student’s t test. e SMARD analysis of telomeric DNA fibers in U2OS treated with either Scrambled or MCPH1 shRNAs. Top: scheme of the CldU (green) and IdU (red) pulse label timing. Middle: representative images of telomeric fibers (telomeric DNA depicted in blue) from two independent experiments. Bottom: quantification of either CldU- or IdU-positive telomeric fibers. Scale bar: 10 μm. f Quantification of the length of CldU and IdU tracks from a representative experiment. Blue line: median. Two-sided Mann–Whitney test. g SMARD analysis of telomeric replication forks restart in U2OS cells treated with either Scrambled or MCPH1 shRNA and reconstituted with the indicated constructs. Top: pulse labeling timing scheme with hydroxyurea (HU)-induced replication block. Bottom: Representative images from two independent experiments. The dashed line separates the CldU-labeled portion (regular replication) and the IdU-labeled portion (replication restart). Scale bar: 10 μm. h Quantification of the IdU/CldU length ratio for the fibers labeled with both halogenated nucleotides in one representative experiment. Black line: median. Kruskal–Wallis test with Dunn’s multiple comparison test.

Article Snippet: MCPH1 TBM peptides, synthesized by GenScript China, at 1.0 mM concentration in the syringe were titrated into TRF2 TRFH at 0.1 mM concentration in the sample cell.

Techniques: Comparison, Co-Immunoprecipitation Assay, Control, Expressing, MANN-WHITNEY, shRNA, Construct, Labeling, Blocking Assay

Journal: Nature Communications

Article Title: Microcephalin 1/BRIT1-TRF2 interaction promotes telomere replication and repair, linking telomere dysfunction to primary microcephaly

doi: 10.1038/s41467-020-19674-0

Figure Lengend Snippet: a MCPH1 (red) and γ-H2AX (green) immunostaining in the indicated cells lines treated with either DMSO or 0.25 μM APH. Representative images from either three (MCPH1 +/+ and MCPH1 Δ/Δ + vector cells) or two (MCPH1 Δ/Δ + WT MCPH1 cells) independent experiments. b Mean values ± SD of the percentage of cells with >5 MCPH1/γ-H2AX co-localizations from ( a ). n = 3 for MCPH1 +/+ and MCPH1 Δ/Δ + vector cells, n = 2 for MCPH1 Δ/Δ + WT MCPH1 cells. At least 200 nuclei were scored. One-way ANOVA followed by Tukey’s multiple comparison test. c MCPH1 (red) and SMARCAL1 (green) immunostaining in U2OS cells treated with either DMSO or 0.25 μM APH. Representative images from three independent experiments. d Mean values ± SD of the percentage of cells with >5 co-localizing MCPH1/SMARCAL1 foci from ( c ). n = 3., at least 200 cells scored. Two-sided Student’s t test. e Percentage of cells with >5 BARD1, RAD51 or p-RPA32 (S4/S8) foci co-localizing with γ-H2AX in the indicated cell lines treated with either DMSO or 0.25 μM APH. Representative images are shown in Supplementary Fig. . Data represent mean values ± SD, n = 2. At least 200 nuclei were examined. One-way ANOVA followed by Tukey’s multiple comparison test. f Representative images from two independent time course experiments to assess γ-H2AX foci (green) resolution in the indicated cell lines after release of HU block. Cells were incubated with 2 mM HU for 24 h before replacing the media and then fixed at the indicated time points. Untreated cells were used as control. g Number of γ-H2AX foci observed in 50–100 cells from each sample shown in ( f ). Data from one representative experiment. Red line: median. Kruskal–Wallis test followed by Dunn’s multiple comparison test. h , i Cell viability assay of MCPH1 +/+ and MCPH1 Δ/Δ cells treated with increasing amount of HU ( h ) or Olaparib ( i ), with or without concomitant BRCA1 depletion. Data from one of three independent experiments. Statistical significance is shown in Supplementary Table (HU) and 2 (Olaparib). Scale bars for a , c , f : 5 μm.

Article Snippet: MCPH1 TBM peptides, synthesized by GenScript China, at 1.0 mM concentration in the syringe were titrated into TRF2 TRFH at 0.1 mM concentration in the sample cell.

Techniques: Immunostaining, Plasmid Preparation, Comparison, Blocking Assay, Incubation, Control, Viability Assay

Journal: Nature Communications

Article Title: Microcephalin 1/BRIT1-TRF2 interaction promotes telomere replication and repair, linking telomere dysfunction to primary microcephaly

doi: 10.1038/s41467-020-19674-0

Figure Lengend Snippet: MCPH1 interacts with TRF2 when S333 is de-phosphorylated, while this interaction is disrupted upon S333 phosphorylation. In unperturbed conditions, MCPH1 exists in equilibrium between the phosphorylated and the de-phosphorylated forms and its telomeric localization increases in S phase. Removal of POT1 through the overexpression of TPP1 ΔRD increases MCPH1 localization to telomeres that is dependent upon the interaction with both TRF2 and γ-H2AX. MCPH1 binding to TRF2 promotes the recruitment of HDR and end resection factors to initiate HDR at telomeres lacking POT1-TPP1. Cells lacking MCPH1 or expressing MCPH1 S333D display reduced recruitment of HDR factors and reduced T-SCEs, suggestive of HDR defects. Similarly, induction of replication stress at telomeres increases MCPH1 telomeric localization, promoted by its interaction with TRF2. In the absence of MCPH1 or upon overexpression of the phosphomimetic mutant MCPH1 S333D , replication forks stalling at telomeres increases and telomere replication is impaired, resulting in telomere fragility. These observations suggest that telomeric localization of MCPH1 is required for proper telomere replication by promoting stalled replication fork restart.

Article Snippet: MCPH1 TBM peptides, synthesized by GenScript China, at 1.0 mM concentration in the syringe were titrated into TRF2 TRFH at 0.1 mM concentration in the sample cell.

Techniques: Phospho-proteomics, Over Expression, Binding Assay, Expressing, Mutagenesis

Journal: Open Medicine

Article Title: The analyses of human MCPH1 DNA repair machinery and genetic variations

doi: 10.1515/med-2024-0917

Figure Lengend Snippet: Annotation of Exon Expression of MCPH1 derived from the GTEx Analysis V8. The functional annotations of the rare variants p.Val10SerfsTer5 andp.Ser571Ter to the exons and tissue specific expression values.

Article Snippet: More recently, mutations in the MCPH1 were associated with congenital hearing impairment (CHI) [ , ], and previously in otitis media in the mice model [ ].

Techniques: Expressing, Derivative Assay, Functional Assay

Journal: Open Medicine

Article Title: The analyses of human MCPH1 DNA repair machinery and genetic variations

doi: 10.1515/med-2024-0917

Figure Lengend Snippet: Overview of MCPH1 disease-associated variants identified in different domains of the canonical transcript. (ENST00000344683.9)

Article Snippet: More recently, mutations in the MCPH1 were associated with congenital hearing impairment (CHI) [ , ], and previously in otitis media in the mice model [ ].

Techniques:

Journal: Open Medicine

Article Title: The analyses of human MCPH1 DNA repair machinery and genetic variations

doi: 10.1515/med-2024-0917

Figure Lengend Snippet: Protein altering variants in identified in MCPH1 in the gnomAD data, and their VEP and ClinVar annotations considered pathogenic, likely pathogenic or uncertain significance

Article Snippet: More recently, mutations in the MCPH1 were associated with congenital hearing impairment (CHI) [ , ], and previously in otitis media in the mice model [ ].

Techniques:

Journal: Open Medicine

Article Title: The analyses of human MCPH1 DNA repair machinery and genetic variations

doi: 10.1515/med-2024-0917

Figure Lengend Snippet: The two highlighted regions in purple show where MCPH1 functions. The MCPH1 forms parts of the MRN complex, in conjunction with SET, condensin II, SMC2, SMC4, NCAPD3 , NCAPH2 , and NCAPG2 . The NHEJ pathway is initiated in response to the formation of DNA DSBs induced by DNA-damaging agents, such as ionizing radiation. The DNA DSBs are recognized by the MRN complex, leading to ATM activation and ATM-dependent recruitment of several DNA damage checkpoints and repair proteins to DNA DSB sites.

Article Snippet: More recently, mutations in the MCPH1 were associated with congenital hearing impairment (CHI) [ , ], and previously in otitis media in the mice model [ ].

Techniques: Activation Assay