title: Functional analysis of human MLH1 and MSH2 missense variants and hybrid human-yeast MLH1 proteins in Saccharomyces cerevisiae.
abstract: 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 majority of HNPCC. In addition to clear loss-of-function mutations conferred by nonsense or frameshift alterations in the coding sequence or by splice variants, genetic screening has revealed a large number of missense codons with less obvious functional consequences. The ability to discriminate between a loss-of-function mutation and a silent polymorphism is important for genetic testing for inherited diseases like HNPCC where the opportunity exists for early diagnosis and preventive intervention. In this study, quantitative in vivo DNA MMR assays in the yeast Saccharomyces cerevisiae were performed to determine the functional significance of amino acid replacements observed in the human population. Missense codons previously observed in human genes were introduced at the homologous residue in the yeast MLH1 or MSH2 genes. This study also demonstrated feasibility of constructing genes that encode functional hybrid human-yeast MLH1 proteins. Three classes of missense codons were found: (i) complete loss of function, i.e. mutations; (ii) variants indistinguishable from wild-type protein, i.e. silent polymorphisms; and (iii) functional variants which support MMR at reduced efficiency, i.e. efficiency polymorphisms. There was a good correlation between the functional results in yeast and available human clinical data regarding penetrance of the missense codon. The results reported here raise the intriguing possibility that differences in the efficiency of DNA MMR exist between individuals in the human population due to common polymorphisms.
authors.affiliation: BitTech, Inc., Westlake Village, CA 91361, USA.
authors.forename: A R
authors.initials: AR
authors.lastname: Ellison
authors.forename: J
authors.initials: J
authors.lastname: Lofing
authors.forename: G A
authors.initials: GA
authors.lastname: Bitter
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D020137:Base Pair Mismatch; D002352:Carrier Proteins; D004260:DNA Repair; D004268:DNA-Binding Proteins; D005787:Gene Frequency; D006801:Humans; D008969:Molecular Sequence Data; D000070957:MutL Protein Homolog 1; D051718:MutS Homolog 2 Protein; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D009363:Neoplasm Proteins / Q000235:genetics* / Q000502:physiology; D009687:Nuclear Proteins; D010957:Plasmids / Q000235:genetics; D011518:Proto-Oncogene Proteins / Q000235:genetics* / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D017386:Sequence Homology, Amino Acid
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D004268:DNA-Binding Proteins; C501486:MLH1 protein, human; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; C497172:MSH2 protein, human; D000070957:MutL Protein Homolog 1; D051718:MutS Homolog 2 Protein