FUNCTIONAL-ANALYSIS OF HUMAN MLH1 MUTATIONS IN SACCHAROMYCES-CEVEVISIAE

Hereditary non-polyposis colorectal cancer (HNPCC; OMIM 120435-6) is a cancer-susceptibility syndrome(1) linked to inherited defects in huma n mismatch repair (MMR) genes(2). Germline missense human MLH1 (hMLH1) mutations are frequently detected in HNPCC (ref. 3), making functiona l characterization of mutations in hMLH1 critical to the development o f genetic testing for HNPCC. Here, we describe a new method for detect ing mutations in hMLHI using a dominant mutator effect of hMLH1 cDNA e xpressed in Saccharomyces cerevisiae. The majority of hMLH1 missense m utations identified in HNPCC patients abolish the dominant mutator eff ect. Furthermore, PCR amplification of hMLHI cDNA from mRNA from a HNP CC patient, followed by in vivo recombination into a gap expression ve ctor, allowed detection of a heterozygous loss-of-function missense mu tation in hMLH1 using this method. This functional assay offers a simp le method for detecting and evaluating pathogenic mutations in hMLH1.