Abstract 5286: A pipeline for developing a novel, predictive tool to classify variants of uncertain significance (VUS) in Lynch Syndrome

Lynch syndrome (LS) highly predisposes individuals and their families to an increased risk of colorectal cancers and to cancers of the endometrium, ovary, gastrointestinal tract, pancreas, upper urinary tract, and other tissues. The occurrence of LS has been attributed to germline, heterozygous mutations in four genes in the DNA mismatch repair (MMR) pathway: MLH1, MSH2, MSH6 and PMS2. Mutations in the MLH1 and MSH2 genes account for the majority of detectable mutations in LS, with more infrequent mutations in MSH6 and PMS2. Clinical diagnosis of LS helps direct the management of the disease and risk assessment for future cancers in the family. A major challenge is the determination of variant pathogenicity in MMR genes, particularly those that code for missense mutations. According the InSiGHT database, there are >5000 unclassified variants of unknown significance (VUS), and this number continues to grow every day due to the increased prevalence of genomic testing. Such VUS present an enormous challenge to effective genetic counseling of LS families. There are numerous predictive methods, such as SIFT and Polyphen-2, for predicting the effects of missense mutations in any gene including LS genes. Further, there is a great deal of promise for gene-specific predictors, such as PON-MMR and CoDP, that are trained on the physical and evolutionary features of known deleterious and benign mutations in the target genes of interest. Since the number of available mutations for the LS genes with experimental phenotypes is limited, we have developed and applied a pipeline to perform experimental validation of VUS in MMR genes. MMR genes with introduced VUS were assessed in vitro in comparison to wild type genes for the effect of the VUS on steady state protein expression, localization, and functionality in inducing DNA damage checkpoints and influencing cell survival following treatment with a panel of DNA-damaging agents. We then assessed a panel of VUS in MSH2, MLH1 and PMS2 in HEK293 kidney cells and colorectal cancer (CRC) cell lines bearing deletions in the MMR genes being tested. This mid-throughput pipeline allowed effective validation of the functional consequences of each VUS, generating additional data to enhance the efficacy of gene-specific predictors for the LS genes that we are developing. Such tools may greatly benefit the assessment of MMR gene variants identified through genetic testing and for genetic counseling of LS families. Citation Format: Sanjeevani Arora, Peter J. Huwe, Rahmat Sikder, Manali Shah, Sanat Deshpande, Michael J. Hall, Roland L. Dunbrack, Erica A. Golemis. A pipeline for developing a novel, predictive tool to classify variants of uncertain significance (VUS) in Lynch Syndrome. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5286.