Analysis of structure and conservation for supporting functional evaluation of PMS2 missense variants

Germline defects in mismatch repair (MMR) genes are known to significantly increase the risk of developing certain types of cancers, notably colorectal and endometrial cancers. These conditions are characterized under Lynch syndrome. Accurate diagnosis of this predisposition, along with meaningful predictive testing for family members, necessitates the identification of pathogenic variants. However, classifying small coding genetic variants identified in cancer patients is very challenging, specifically in the case of PMS2 variants, since PMS2 pathogenic variants display a lower penetrance and less severe phenotype and therefore a lower tumor burden in affected families. We have assembled clinical data on four PMS2 missense variants of uncertain significance (VUS) identified in 23 patients (p.(Asp286Gly), p.(Asn335Ser), p.(Ile679Thr) and p.(Arg799Trp)). For these variants, functional testing was performed (RNA splicing, protein stability and catalytic activity). Since many protein ortholog sequences and accurate predictive models from AlphaFold2 are available, we also included a systematic analysis of residue conservation and structural role (ConStruct assessment). Overall, our findings indicate that p.(Asp286Gly) and p.(Arg799Trp) behave similarly to wild-type PMS2 and are thus probably neutral. In contrast, p.(Asn335Ser) and p.(Ile679Thr) conferred defects in protein expression or MMR activity. These could be explained by the relevant roles of these amino acids in MLH1-PMS2-N-terminal dimerization (p.Asn335) and C-terminal dimerization (p.Ile679). Our data thus suggest that p.(Asp286Gly) and p.(Arg799Trp) are benign, while the tumor risk in the other two variants remains to be established. Taken together, we suggest roadmaps for the individualized evaluation of difficult uncertain variants by comprising information from all available sources.