Classification of ambiguous mutations in DNA mismatch repair genes identified in a population‐based study of colorectal cancer

Identification of germline mutations in DNA mismatch repair genes in colorectal cancer probands without an extensive family history can be problematic when ascribing relevance to cancer causation. We undertook a structured assessment of the disease-causing potential of sequence variants identified in a prospective, populationbased study of 932 colorectal cancer patients, diagnosed at o55 years of age. Patient samples were screened for germline mutations in MLH1, MSH2 ,a ndMSH6. Of 110 carriers, 74 (67%) had one of 33 rare variants of uncertain pathogenicity (12 MLH1 ,1 1MSH2 ,a nd 10MSH6). Pathogenicity was assessed by determining segregation in families, allele frequency in large numbers of unaffected controls, effect on mRNA for putative splicesite mutations, effect on protein function by bioinformatic analysis and tumor microsatellite instability (MSI) status and DNA mismatch repair protein expression by immunohistochemistry. Because of the ambiguous nature of these variants and lack of concordance between functional assays and control allele frequency, we devised a scoring system to rank the degree of support for ap athogenic role.MLH1 c.200G4 Ap .G67E,MLH1 c.2041G4A p.A681T, and MSH2 c.263415G4C were categorized as pathogenic through assimilation of all available data, while 14 variants were categorized as benign (seven MLH1 ,t hreeMSH2, and four MSH6). Interestingly, there is tentative evidence suggesting a possible protective effect of three variants (MLH1 c.2066A4G pQ689R, c.2146G4A p.V716M, and MSH2 c.965G4A p.G322D). These findings support a causal link with colorectal cancer for several DNA mismatch repair gene variants. However, the majority of missense changes are likely to be inconsequential polymorphisms. Hum Mutat 29(3), 367–374, 2008. r 2007 Wiley-Liss, Inc.