Journal: American Journal of Human Genetics
Article Title: MCM9 Mutations Are Associated with Ovarian Failure, Short Stature, and Chromosomal Instability
Figure Lengend Snippet: Pathogenic Homozygous Recessive Variants in MCM9 Were Identified in Two Consanguineous Kurdish Families from Turkey (A and B) The MCM9 genotype is provided below each individual. WT denotes the wild-type allele. MT (for mutation) indicates either (A) MCM9 c.1732+2T > C or (B) MCM9 c. 394C > T. PCR amplification was completed with the KAPA HiFi HotStart PCR Kit with dNTPs (Kapa Biosystems) according to the manufacturer’s instructions (primers for A are 5′-TCTAGGAGGTCCCGAGATGG-3′ and 5′-CAAAGGCAGAGTGATTGCCG-3′; primers for B are 5′-GCCTGAGAGGCAAGTGAATTTAG-3′ and 5′-TACCTAAAACCAAGGATGTGGGA-3′). PCR products were sequenced at Beckman Coulter Genomics. Results were analyzed with Sequencher (Gene Codes Corporation). Sample chromatograms are shown (5′ to 3′ direction in A; 3′ to 5′ direction in B). Arrows point to the nucleotide peak of interest. Samples utilized for whole-exome sequencing (WES) are indicated by an asterisk. (C) MCM9 is encoded on chr6: 119,252,903–119,134,612 (UCSC Genome Browser hg19). The MCM9 c.1732+2T > C splice variant lies in the splice donor site of exon 9 (red arrow). The c.394C > T mutation lies near the 5′ end of exon 2 (blue arrow). Exons are indicated as full boxes (black). Alternative splice forms of MCM9 either include or exclude exon 11 (white). Introns are indicated as lines. (D) MCM9 consists of an N-terminal DNA binding domain and AAA + core domain. The c.1732+2T > C mutation is predicted to disrupt normal splicing after amino acid 577. The c.394C > T mutation causes insertion of a stop codon in the place of an arginine at position 132 (p.Arg132 ∗ ). This most likely results in a truncated protein if one is formed. The yellow box represents the arginine finger domain (RF), the pink box represents the Walker A motif (WA), and the deep green box represents the Walker B motif (WB).
Article Snippet: Using the KAPA HiFi HotStart PCR Kit with dNTPs (Kapa Biosystems) and primers to amplify exons 7–12 (c.1151_1996), we expected to observe two previously reported wild-type products: an 846 bp product and a 700 bp product resulting from exon 11 skipping ( A; B, S3C, and A).
Techniques: Mutagenesis, Polymerase Chain Reaction, Amplification, Sequencing, Variant Assay, Binding Assay, Western Blot