Functional analysis of human MLH1 mutations in Saccharomyces cerevisiae

Department of Clinical Oncology, Institute of Development, Aging and Cancer, Tohoku University, Sendai, Japan.
Nature Genetics (Impact Factor: 29.65). 09/1998; 19(4):384-9. DOI: 10.1038/1277
Source: PubMed

ABSTRACT 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.

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Available from: Hideki Shimodaira, Jun 02, 2014
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    • "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. "
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    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 · 5.05 Impact Factor
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    • "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 "
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    Gastric Carcinoma - Molecular Aspects and Current Advances, 06/2011; , ISBN: 978-953-307-412-2
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    • "Values 44.55 are considered pathogenic. e References: 1 [Takahashi et al., 2007], 2 [Shimodaira et al., 1998], 3 [Kondo et al., 2003], 4 [Guerrette et al., 1999], 5 [Raevaara et al., 2005], 6 [Blasi et al., 2006], 7 [Avdievich et al., 2008], 8 [Nyström-Lahti et al., 2002], 9 [Wanat et al., 2007], 10 [Plotz et al., 2006], 11 [Spina et al., 2008], 12 [Trojan et al., 2002], 13 [Perera and Bapat, 2008]. For references employing more than in vitro MMR assays alone, only the result for the MMR assay has been included in this table. "
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    ABSTRACT: The hereditary colon and endometrium cancer predisposition Lynch Syndrome (also called HNPCC) is caused by a germ-line mutation in one of the DNA mismatch repair (MMR) genes. A significant fraction of the gene alterations detected in suspected Lynch Syndrome patients is comprised of amino acid substitutions. The relevance for cancer risk of these variants is difficult to assess, as currently no time- and cost-effective, validated, and widely applicable functional assays for the measurement of MMR activity are available. Here we describe a rapid, cell-free, and easily quantifiable MMR activity assay for the diagnostic assessment of variants of the MLH1 MMR protein. This assay allows the parallel generation and functional analysis of a series of variants of the MLH1 protein in vitro using readily available, or preprepared, reagents. Using this assay we have tested 26 MLH1 variants and of these, 15 had lost activity. These results are in concordance with those obtained from first-generation assays and with in silico and pathology data. After its multifocal technical and clinical validation this assay could have great impact for the diagnosis and counseling of carriers of an MLH1 variant and their relatives.
    Human Mutation 03/2010; 31(3):247-53. DOI:10.1002/humu.21180 · 5.05 Impact Factor
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