Functional analysis of human MLH1 and MSH2 missense variants and hybrid human-yeast MLH1 proteins in Saccharomyces cerevisiae

Hereditary non-polyposis colorectal cancer (HNPCC) is an autosomal dominant inherited disease caused by defects in the process of DNA mismatch repair (MMR), and mutations in the hMLH1 or hMSH2 genes are responsible for the ma jority of HNPCC. In addition to clear loss-of-function mutations conferred by nonsense or frameshift alterations in the coding sequence or by splice v ariants, genetic screening has revealed a large number of missense codons w ith less obvious functional consequences. The ability to discriminate betwe en a loss-of-function mutation and a silent polymorphism is important for g enetic testing for inherited diseases like HNPCC where the opportunity exis ts for early diagnosis and preventive intervention. In this study, quantita tive in vivo DNA MMR assays in the yeast Saccharomyces cerevisiae were perf ormed to determine the functional significance of amino acid replacements o bserved in the human population. Missense codons previously observed in hum an genes were introduced at the homologous residue in the yeast hMLH1 or MS H2 genes. This study also demonstrated feasibility of constructing genes th at encode functional hybrid human-yeast MLH1 proteins. Three classes of mis sense codons were found: (i) complete loss of function, i.e. mutations; (ii ) variants indistinguishable from wild-type protein, i.e. silent polymorphi sms; and (iii) functional variants which support MMR at reduced efficiency, i.e. efficiency polymorphisms. There was a good correlation between the fu nctional results in yeast and available human clinical data regarding penet rance of the missense codon. The results reported here raise the intriguing possibility that differences in the efficiency of DNA MMR exist between in dividuals in the human population due to common polymorphisms.