Functional analysis of human MLH1 mutations in Saccharomyces cerevisiae. Nat Genet

Department of Clinical Oncology, Institute of Development, Aging and Cancer, Tohoku University, Sendai, Japan.
Nature Genetics (Impact Factor: 29.35). 09/1998; 19(4):384-9. DOI: 10.1038/1277
Source: PubMed


Hereditary non-polyposis colorectal cancer (HNPCC; OMIM 120435-6) is a cancer-susceptibility syndrome linked to inherited defects in human mismatch repair (MMR) genes. Germline missense human MLH1 (hMLH1) mutations are frequently detected in HNPCC (ref. 3), making functional characterization of mutations in hMLH1 critical to the development of genetic testing for HNPCC. Here, we describe a new method for detecting mutations in hMLH1 using a dominant mutator effect of hMLH1 cDNA expressed in Saccharomyces cerevisiae. The majority of hMLH1 missense mutations identified in HNPCC patients abolish the dominant mutator effect. Furthermore, PCR amplification of hMLH1 cDNA from mRNA from a HNPCC patient, followed by in vivo recombination into a gap expression vector, allowed detection of a heterozygous loss-of-function missense mutation in hMLH1 using this method. This functional assay offers a simple method for detecting and evaluating pathogenic mutations in hMLH1.

Download full-text


Available from: Hideki Shimodaira, Jun 02, 2014
16 Reads
  • Source
    • "Several methods have been applied to evaluate the spontaneous mutation rates in various yeast strains expressing mutant MMR proteins, all of which make use of reporter genes that are either located in the yeast chromosome or expressed extra-chromosomally. These reporter genes serve as selection markers; when mutated, they lose or regain their function and can be used to determine the MMR capacity of the introduced mutant MMR protein [Polaczek et al., 1998; Shimodaira et al., 1998; Takahashi et al., 2007; Tran et al., 1997; Wanat et al., 2007]. The reporter genes vary in the length of repeat sequences located in the gene. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Lynch syndrome (LS) is caused by germline mutations in DNA mismatch repair (MMR) genes and is the most prevalent hereditary colorectal cancer syndrome. A significant proportion of variants identified in MMR and other common cancer susceptibility genes are missense or noncoding changes whose consequences for pathogenicity cannot be easily interpreted. Such variants are designated as "variants of uncertain significance" (VUS). Management of LS can be significantly improved by identifying individuals who carry a pathogenic variant and thus benefit from screening, preventive, and therapeutic measures. Also, identifying family members that do not carry the variant is important so they can be released from the intensive surveillance. Determining which genetic variants are pathogenic and which are neutral is a major challenge in clinical genetics. The profound mechanistic knowledge on the genetics and biochemistry of MMR enables the development and use of targeted assays to evaluate the pathogenicity of variants found in suspected patients with LS. We describe different approaches for the functional analysis of MMR gene VUS and propose development of a validated diagnostic framework. Furthermore, we call attention to common misconceptions about functional assays and endorse development of an integrated approach comprising validated assays for diagnosis of VUS in patients suspected of LS.
    Human Mutation 12/2012; 33(12). DOI:10.1002/humu.22168 · 5.14 Impact Factor
  • Source
    • "Similarly, another study identified 33 of 54 MSH2 VUS that fail to restore repair in an msh2Δ strain compared to wild-type control [79]. A study of MLH1 variants in yeast demonstrated 15 out of 28 that were defective for repair activity [82]. However, in a similar assay in a different strain of yeast, the same variants displayed different repair capabilities suggesting that genetic background may affect the function of some VUS. "
    [Show abstract] [Hide abstract]
    ABSTRACT: With the discovery that the hereditary cancer susceptibility disease Lynch syndrome (LS) is caused by deleterious germline mutations in the DNA mismatch repair (MMR) genes nearly 20 years ago, genetic testing can now be used to diagnose this disorder in patients. A definitive diagnosis of LS can direct how clinicians manage the disease as well as prevent future cancers for the patient and their families. A challenge emerges, however, when a germline missense variant is identified in a MMR gene in a suspected LS patient. The significance of a single amino acid change in these large repair proteins is not immediately obvious resulting in them being designated variants of uncertain significance (VUS). One important strategy for resolving this uncertainty is to determine whether the variant results in a non-functional protein. The ability to reconstitute the MMR reaction in vitro has provided an important experimental tool for studying the functional consequences of VUS. However, beyond this repair assay, a number of other experimental methods have been developed that allow us to test the effect of a VUS on discrete biochemical steps or other aspects of MMR function. Here, we describe some of these assays along with the challenges of using such assays to determine the functional consequences of MMR VUS which, in turn, can provide valuable insight into their clinical significance. With increased gene sequencing in patients, the number of identified VUS has expanded dramatically exacerbating this problem for clinicians. However, basic science research laboratories around the world continue to expand our knowledge of the overall MMR molecular mechanism providing new opportunities to understand the functional significance, and therefore pathogenic significance, of VUS.
    Hereditary Cancer in Clinical Practice 07/2012; 10(1):9. DOI:10.1186/1897-4287-10-9 · 1.47 Impact Factor
  • Source
    • "mRNA splicing In vivo splicing assay in human cells Auclair, 2006; Sharp, 2004; Arnold, 2009 MMR activity Yeast-based chromosomeintegrated hMMR gene Vogelsang, 2009; Vogelsang, 2010 Dominant mutator effect Raevara, 2005;Takahashi, 2007; Shimodaira, 1998 Functional assay using yeast Ellison, 2001;Wanat, 2007 Utility of MLH1-deficient cells Blasi, 2006 In silico Effect of amino acid substitution on protein functions SIFT Kumar, 2009; Ng, 2003 PolyPhen Ramensky, 2002 MAPP-MMR Chao, 2008 Align GVDV Tavgtigian, 2006; Mathe, 2006 mRNA splicing NNSPLICE Sharp, 2004 "
    [Show abstract] [Hide abstract]
    Gastric Carcinoma - Molecular Aspects and Current Advances, 06/2011; , ISBN: 978-953-307-412-2
Show more