Elizabeth Szabo, Emily Blackburn, Olivia N. Amodeo, Samantha Nadeau, Alexander A. Radecki, James P. Grady, Abhijit Rath, Christopher D. Heinen
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A Cell-Based Functional Assay Calibrated for Analysis of MSH6 and MSH2 Mismatch Repair Gene Variants
Variants of uncertain significance (VUS) in the DNA mismatch repair (MMR) genes can confound the diagnosis and treatment of suspected Lynch syndrome (LS) patients. To aid the reclassification of VUS, we developed the in cellulo analysis of MMR variants (inCAMA) and used known control variants to calibrate this assay such that results can be readily applied as functional evidence by expert classification panels. We used CRISPR gene engineering to introduce known pathogenic and benign variants into the MSH6 or MSH2 loci in human embryonic stem cells and assessed their effects on cellular MMR repair and damage response functions. Our functional assay successfully discerned known pathogenic and benign variants. Using these results and performing a linear regression analysis with available odds of pathogenicity scores for the known calibration variants, we created equations that can generate a functional odds of pathogenicity score for any future MSH6 or MSH2 variant tested. In summary, inCAMA represents a new, calibrated assay for testing the function of virtually any MSH6 or MSH2 variant. The conversion of assay results directly into odds of pathogenicity scores makes it possible to use any PS3 or BS3 evidence strength level toward the reclassification of VUS.
期刊介绍:
Human Mutation is a peer-reviewed journal that offers publication of original Research Articles, Methods, Mutation Updates, Reviews, Database Articles, Rapid Communications, and Letters on broad aspects of mutation research in humans. Reports of novel DNA variations and their phenotypic consequences, reports of SNPs demonstrated as valuable for genomic analysis, descriptions of new molecular detection methods, and novel approaches to clinical diagnosis are welcomed. Novel reports of gene organization at the genomic level, reported in the context of mutation investigation, may be considered. The journal provides a unique forum for the exchange of ideas, methods, and applications of interest to molecular, human, and medical geneticists in academic, industrial, and clinical research settings worldwide.