Functional analysis of MMR gene VUS from potential Lynch syndrome patients

Lynch syndrome is caused by inactivating variants in DNA mismatch repair genes, namely MLH1, MSH2, MSH6 and PMS2. We have investigated five MLH1 and one MSH2 variants that we have identified in Turkish and Tunisian colorectal cancer patients. These variants comprised two small deletions causing frameshifts resulting in premature stops which could be classified pathogenic (MLH1 p. (His727Profs*57) and MSH2 p. (Thr788Asnfs*11) ), but also two missense variants (MLH1 p. (Asn338Ser) and p. (Gly181Ser) ) and two small, in-frame deletion variants (p. (Val647-Leu650del) and p. (Lys678Cys680del) ). For such small coding genetic variants, it is unclear if they are inactivating or not. We here provide clinical description of the variant carriers and their families, and we performed biochemical laboratory testing on the variant proteins to test if their stability or their MMR activity are compromised. Subsequently, we compared the results to in-silico predictions on structure and conservation. We demonstrate that neither missense alteration affected function, while both deletion variants caused a dramatic instability of the MLH1 protein, resulting in MMR deficiency. These results were consistent with the structural analyses that were performed. The study shows that knowledge of protein function may provide molecular explanations of results obtained with functional biochemical testing and can thereby, in conjunction with clinical information, elevate the evidential value and facilitate clinical management in affected families.