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.

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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.
    Full-text · Article · Dec 2012 · Human Mutation
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    • "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. "
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    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.
    Full-text · Article · Jul 2012 · Hereditary Cancer in Clinical Practice
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    • "This system is unique in that a single copy of the MMR gene was introduced per cell which was placed under the control of the yeast promoter, providing uniformity in the genetic analysis. A simple yeast assay was developed to detect and evaluate pathogenic mutations in MLH1 by suppression of the dominant mutator effect of human MLH1 expressed in S. cerevisiae[81]. Heterozygous loss of function missense mutations in human MLH1 were also identified by the assay. In a subsequent study by the same group, 101 MLH1 variants were examined for the dominant mutator effect in vivo by three independent yeast-based assays and in vitro by a plasmid-based MMR assay using extracts. "
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    ABSTRACT: Model systems have been extremely useful for studying various theories of aging. Studies of yeast have been particularly helpful to explore the molecular mechanisms and pathways that affect aging at the cellular level in the simple eukaryote. Although genetic analysis has been useful to interrogate the aging process, there has been both interest and debate over how functionally conserved the mechanisms of aging are between yeast and higher eukaryotes, especially mammalian cells. One area of interest has been the importance of genomic stability for age-related processes, and the potential conservation of proteins and pathways between yeast and human. Translational genetics have been employed to examine the functional roles of mammalian proteins using yeast as a pliable model system. In the current review recent advancements made in this area are discussed, highlighting work which shows that the cellular functions of human proteins in DNA repair and maintenance of genomic stability can be elucidated by genetic rescue experiments performed in yeast.
    Full-text · Article · Feb 2012 · DNA repair
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