Functional significance and clinical phenotype of nontruncating mismatch repair variants of MLH1

Background & Aims: Germline mutations in mismatch repair genes are associated with hereditary nonpolyposis colorectal cancer. A significant proportion of mutations are nontruncating and associated with a variability of clinical phenotype and microsatellite instability and with occasional presence of residual protein in tumor tissue that suggests impaired functional activity but not total lack of mismatch repair. To address pathogenic significance and mechanism of pathogenicity, we studied the functionality of 31 nontruncating MLH1 mutations found in clinically characterized colorectal cancer families and 3 other variations listed in a mutation database. Methods: Mutations constructed by site-directed mutagenesis were studied for protein expression/stability, subcellular localization, protein-protein interaction, and repair efficiency. The genetic and biochemical data were correlated with clinical data. Finally, comparative sequence analysis was performed to assess the value of sequence homology as a tool for predicting functional results. Results: Altogether, 22 mutations were pathogenic in more than one assay, 2 variants were impaired in one assay, and 10 variants acted like wild-type protein. Twenty of 34 mutations affected the quantity of MLH1 protein, whereas only 15 mainly amino-terminal mutations were defective in an in vitro repair assay. Comparative sequence analysis correctly predicted functional studies for 82% of variants. Conclusions: Pathogenic nontruncating alterations in MLH1 may interfere with different biochemical mechanisms but generally more than one. The severe biochemical defects are mirrored by phenotypic characteristics such as early age at onset and high microsatellite instability, whereas variants with no or mild defects in functionality are associated with variable clinical phenotypes.