Assessing pathogenicity of mismatch repair variants of uncertain significance by molecular tumor analysis

Functional analyses are the main method to classify mismatch repair (MMR) gene variants of uncertain significance (VUSs). However, the pathogenicity remains unclear for many variants because of conflicting results between clinical, molecular, and functional data. In this study, we evaluated whether whole exome sequencing (WES) could add another layer of evidence to elucidate the pathogenicity of MMR variants with conflicting interpretations. WES was performed on formalin-fixed paraffin-embedded tumor tissue of eight patients with a constitutional MMR VUS (seven families), including eight colorectal and two endometrial carcinomas and one ovarian carcinoma. Cell-free CIMRA assays were performed to assign Odds of Pathogenicity to these VUSs. In four families, seven tumors showed MMR deficiency-associated mutational signatures, supporting the pathogenicity of the VUS. Moreover, somatic (second) MMR hits identified in the WES data were found to explain MMR staining patterns when the MMR staining was discordant with the reported germline MMR gene variant. In conclusion, WES did not significantly reclassify VUS in these cases but clarified some phenotypic aspects such as age of onset and explanations in case of discordant MMR stainings.