Functional analysis of MSH2 unclassified variants found in suspected Lynch syndrome patients reveals pathogenicity due to attenuated mismatch repair.

BACKGROUND: Lynch syndrome, an autosomal-dominant disorder characterised by high colorectal and endometrial cancer risks, is caused by inherited mutations in DNA mismatch repair (MMR) genes. Mutations fully abrogating gene function are unambiguously disease causing. However, missense mutations often have unknown functional implications, hampering genetic counselling. We have applied a novel approach to study three MSH2 unclassified variants (UVs) found in Dutch families with suspected Lynch syndrome. METHODS: The three mutations were recreated in the endogenous Msh2 gene in mouse embryonic stem cells by oligonucleotide-directed gene modification. The effect of the UVs on MMR activity was then tested using a set of functional assays interrogating the main MMR functions. RESULTS: We recreated and functionally tested three MSH2 UVs: MSH2-Y165D (c.493T>G), MSH2-Q690E (c.2068C>G) and MSH2-M813V (c.2437A>G). We observed reduced levels of MSH2-Y165D and MSH2-Q690E but not MSH2-M813V proteins. MSH2-M813V was able to support all MMR functions similar to wild-type MSH2, whereas MSH2-Y165D and MSH2-Q690E showed partial defects. CONCLUSIONS: Based on the results from our functional assays, we conclude that the MSH2-M813V variant is not disease causing. The MSH2-Y165D and MSH2-Q690E variants affect MMR function and are therefore likely the underlying cause of familial cancer predisposition. Since the MMR defect is partial, these variants may represent low penetrance alleles.

Functional analysis in mouse embryonic stem cells reveals wild-type activity for three MSH6 variants found in suspected Lynch syndrome patients.

Lynch syndrome confers an increased risk to various types of cancer, in particular early onset colorectal and endometrial cancer. Mutations in mismatch repair (MMR) genes underlie Lynch syndrome, with the majority of mutations found in MLH1 and MSH2. Mutations in MSH6 have also been found but these do not always cause a clear cancer predisposition phenotype and MSH6-defective tumors often do not show the standard characteristics of MMR deficiency, such as microsatellite instability. In particular, the consequences of MSH6 missense mutations are challenging to predict, which further complicates genetic counseling. We have previously developed a method for functional characterization of MSH2 missense mutations of unknown significance. This method is based on endogenous gene modification in mouse embryonic stem cells using oligonucleotide-directed gene targeting, followed by a series of functional assays addressing the MMR functions. Here we have adapted this method for the characterization of MSH6 missense mutations. We recreated three MSH6 variants found in suspected Lynch syndrome families, MSH6-P1087R, MSH6-R1095H and MSH6-L1354Q, and found all three to behave like wild type MSH6. Thus, despite suspicion for pathogenicity from clinical observations, our approach indicates these variants are not disease causing. This has important implications for counseling of mutation carriers.

Characterization of MSH2 variants by endogenous gene modification in mouse embryonic stem cells.

Mutations in the mismatch repair gene MSH2 underlie hereditary nonpolyposis colorectal cancer (Lynch syndrome). Whereas disruptive mutations are overtly pathogenic, the implications of missense mutations found in sporadic colorectal cancer patients or in suspected Lynch syndrome families are often unknown. Adequate genetic counseling of mutation carriers requires phenotypic characterization of the variant allele. We present a novel approach to functionally characterize MSH2 missense mutations. Our approach involves introduction of the mutation into the endogenous gene of murine embryonic stem cells (ESC) by oligonucleotide-directed gene modification, a technique we recently developed in our lab. Subsequently, the mismatch repair capacity of mutant ESC is determined using a set of validated functional assays. We have evaluated four clinically relevant MSH2 variants and found one to completely lack mismatch repair capacity while three behaved as wild-type MSH2 and can therefore be considered as polymorphisms. Our approach contributes to an adequate risk assessment of mismatch repair missense mutations. We have also shown that oligonucleotide-directed gene modification provides a straightforward approach to recreate allelic variants in the endogenous gene in murine ESC. This approach can be extended to other hereditary conditions.