Functional analysis of vus (variant of uncertain significance) of human muts homolog 2/6 (hMSH2/6) proteins

Abstract

Lynch syndrome (LS) or hereditary non-polyposis colorectal cancer (HNPCC) is a genetic condition that raises the risk of colorectal cancer and related cancers. Germline genetic variants of the DNA mismatch repair genes MSH2, MSH6, MLH1, and PMS2 are primarily responsible for its occurrence. In eukaryotes, MSH2 and MSH6 combine to form the MutSα complex, which is responsible for recognizing mismatches and assembling the necessary proteins for mismatch repair. Defining the functional consequences of variants is crucial in enrolling LS patients in appropriate surveillance programs to reduce morbidity and mortality. Herein, the mutation profile of hereditary colorectal cancer in the Turkish population was determined by analyzing the variation spectrum of 26 cancer susceptibility genes in 371 patients with colorectal cancer using next-generation sequencing technology. The detected variants were interpreted based on The American College of Medical Genetics and Genomics (ACMG) recommendations. The MSH2 and MSH6 loci were screened for variants of unknown significance (VUS) to determine the effect of nucleotide substitution on protein function. Mismatch repair deficient cell line (LoVo) was transiently transfected with hMSH2 wild-type (MSH2-WT) and hMSH6 wild type (MSH6-WT) genes and selected mutated subclones (MSH6-R577C, MSH6- S1279N, and MSH2-A733T). Regulation of mRNA expression and protein expression of interacting proteins were investigated, which showed hMSH6 gene expression was decreased when LoVo cells were transfected with MSH2-A733T compared to MSH2- WT. Expression levels of the downstream targets and interaction partner proteins were not affected significantly due to mutated forms of proteins. In-vitro binding assays showed that mutations did not affect the interaction between MSH2 and MSH6. Purified MSH2-WT and MSH2-A733T proteins that were produced from HEK293T cells were not obtained in stable forms. Bacterial co-expression of genes exhibited soluble protein production, and purification method for wild type proteins was promoted. However, due to the low stability of high molecular weighted proteins, it was decided that this method could be used in domain-specific studies with improvements. In order to reclassify clinical importance of the selected VUS, functional studies are needed to be improved. Reclassification of VUS in MSH2/MSH6 has the potential to improve variant classification accuracy, increase risk assessment, facilitate recommended clinical decision making, and provide more accurate genetic counseling for affected individuals and their families. Consequently, implementing personalized management strategies can effectively reduce cancer mortality and morbidity in Lynch syndrome populations.