Detailed characterization of MLH1 p.D41H and p.N710D variants coexisting in a Lynch syndrome family with conserved MLH1 expression tumors.

Lynch syndrome (LS) is an autosomal dominant cancer-susceptibility disease caused by inactivating germline mutations in mismatch repair (MMR) genes. Variants of unknown significance (VUS) are often detected in mutational analysis of MMR genes. Here we describe a large family fulfilling Amsterdam I criteria carrying two rare VUS in the MLH1 gene: c.121G > C (p.D41H) and c.2128A > G (p.N710D). Collection of clinico-pathological data, multifactorial analysis, in silico predictions, and functional analyses were used to elucidate the clinical significance of the identified MLH1 VUS. Only the c.121G > C variant cosegregated with LS-associated tumors in the family. Diagnosed colorectal tumors were microsatellite unstable although immunohistochemical staining revealed no loss of MMR proteins expression. Multifactorial likelihood analysis classified c.2128A > G as a non-pathogenic variant and c.121G > C as pathogenic. In vitro functional tests revealed impaired MMR activity and diminished expression of c.121G > C. Accordingly, the N710 residue is located in the unconserved MLH1 C-terminal domain, whereas D41 is highly conserved and located in the ATPase domain. The obtained results will enable adequate genetic counseling of c.121G > C and c.2128A > G variant carriers and their families. Furthermore, they exemplify how cumulative data and comprehensive analyses are mandatory to refine the classification of MMR variants.