Abstract 5236: Functional analysis of variants of uncertain significance of the MSH6 mismatch repair gene

Lynch syndrome (LS) is a hereditary condition that increases patients’ lifetime risk of cancer, primarily colorectal cancer. LS is caused by germline mutations in mismatch repair (MMR) genes, MLH1, MSH2, MSH6, and PMS2. Identification of these mutations is important in LS diagnosis in order to guide treatment plans and preventative care for patients’ and their families. However, the consequences of some variants in these genes are not immediately obvious, granting them classification as variants of uncertain significance (VUS). Half of known variants in the MMR gene MSH6 are VUS and, thus, the goal of my project is to provide evidence to help better determine their pathogenic significance. Laboratory functional analysis to determine whether these variants disrupt MMR function in human cells can provide such evidence toward this goal. We have previously used CRISPR gene editing to recreate variants in the endogenous loci for the MMR genes MSH2 and MLH1 in human embryonic stem cells (hESC) in order to test their effects on MMR function in a cellular environment. We will now use test whether this approach can be used to study MSH6 VUS. Using these edited hESC lines we are examining the impact of the variants on RNA and protein stability, repair of DNA microsatellites, which is a hallmark of normal MMR function, and induction of the MMR-dependent DNA damage response. Using a panel of known MSH6 pathogenic and benign controls, we will calibrate these functional assays in order to associate functional performance with likelihood of pathogenicity. We will next test a separate panel of known controls to validate these assays. Finally, we will examine up to 35 VUS. With this compilation of data, we will convert the results into a quantitative Odds of Pathogenicity value. This Odds of Pathogenicity score can be used by expert variant interpretation committees to help readily reclassify these variants, and provide a clearer diagnosis for these suspected Lynch syndrome patients. Citation Format: Elizabeth Szabo, Emily Blackburn, Patrick Pagano, Abhijit Rath, Christopher Heinen. Functional analysis of variants of uncertain significance of the MSH6 mismatch repair gene. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5236.