2002 Fiscal Year Final Research Report Summary
Functional analysis of DNA mismatch protein hMLH1 using germline mutants
| Project/Area Number |
13680756
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Molecular biology
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| Research Institution | Tohoku University |
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Principal Investigator |
FUKUSHIGE Shinichi Tohofcu Univ., Sch. Mat, Dept.Mol. Path., Lecturer, 大学院・医学系研究科, 講師 (90192723)
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| Project Period (FY) |
2001 – 2002
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| Keywords | DNA mismatch repair / HNPCC / hMLH1 / hPMS2 / missense mutation / two-hybrid assay |
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| Research Abstract |
Hereditary nonpolyposis cotorectal cancer (HNPCC) is the most common form of familial cotorectal cancer and is associated with germline mutations in one of the DNA mismatch repair genes. Among these genes, the hMLH1 gene is the most frequently damaged, and high frequency of the missense mutation (about onethird of all the mutations) is a particularly characteristic feature. Therefore, it is important to clarify whether or not these missense mutations are in fact pathogenic. In this study, we have developed an assay to distinguish between pathogenic mutations and polymorphisms inb hMLH1 variants. We first surveyed the database (http://www.nfdht.nl) presented by the International Collaborative Group on Hereditary Nonpolyposis Colorectal Cancer (ICG-HNPCC) and chose 23 missense variants of the hMLH1 protein. We used the yeast two-hybrid assay with hPMS2 or hEX01 as bait proteins to measure the function of the protein and found that 18 (78%) of 23 variants showed significant decreased β-gal activities in comparison with the wild type hMLH1. Two of the remaining five variants were thought to be rare polymorphisms. Hence the yeast two-hybrid assay is an extremely simple and useful tool for evaluating the biological significance of the variant hMLH1 proteins. We further analyzed 14 truncating mutations (11 frameshtft and 3 nonsense mutations) and one in-frame 3-bp deletion; these mutants were chosen because these still remain some or most of the Cterminai portion for hPMS2-interacting domain. AH of these 15 mutants also showed extremely low levels of β-gal activities. Our present results suggest that these dysfunctions are due to either lack in the interacting domain with associating proteins at the C-terminus or con formationai changes caused mainly by the N-terminal abnormalities. In conclusion, the yeast two-hybrid assay is a simple and reliable system for accurate diagnosis of the hMLH1 alterations.
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Research Products
(4 results)
