A Cell-Based Functional Assay Calibrated for Analysis of MSH6 and MSH2 Mismatch Repair Gene Variants

Area of Science:

• Genetics
• Molecular Biology
• Cancer Research

Background:

• Variants of uncertain significance (VUS) in DNA mismatch repair (MMR) genes complicate Lynch syndrome (LS) diagnosis.
• Accurate classification of these variants is crucial for patient management and genetic counseling.

Purpose of the Study:

• To develop and validate a novel in cellulo assay, inCAMA, for functional analysis of MMR gene variants.
• To enable the reclassification of VUS in MMR genes, specifically MSH6 and MSH2, to improve LS diagnosis.

Main Methods:

• Utilized CRISPR gene editing to introduce known pathogenic and benign variants into MSH6 or MSH2 genes in human embryonic stem cells.
• Assessed the impact of these variants on cellular MMR repair and DNA damage response functions.
• Calibrated the assay using known variants and linear regression analysis with pathogenicity scores.

Main Results:

• The inCAMA assay successfully differentiated between known pathogenic and benign variants in MSH6 and MSH2.
• Developed predictive equations to generate functional odds of pathogenicity scores for novel variants.
• Demonstrated that assay results can be directly converted into odds of pathogenicity for VUS reclassification.

Conclusions:

• inCAMA provides a reliable and calibrated functional assay for MSH6 and MSH2 variants.
• The assay facilitates the use of functional evidence in the reclassification of VUS, supporting diagnostic accuracy for Lynch syndrome.
• This tool aids expert panels in assigning evidence strength (PS3/BS3) for variant classification.