Abstract for presentation at 11th International Congress of Human Genetics

Functional characterization of hMLH1 missense mutations using yeast and in vitro assays

  • Masanobu Takahashi, Department of Clinical Oncology, IDAC, Tohoku University, Japan
  • Hideki Shimodaira, Department of Clinical Oncology, IDAC, Tohoku University, Japan
  • Chikashi Ishioka, Department of Clinical Oncology, IDAC, Tohoku University, Japan

    • Purpose: Hereditary non-polyposis colorectal cancer (HNPCC) is one of the most common familial cancer syndrome caused by germline mutations in mismatch repair (MMR) genes, such as hMLH1, hMSH2, hMSH6 and hPMS2. Since germline missense hMLH1 mutations are frequently detected, functional analysis has been needed to predict whether these mutations are pathogenic. In the present study, we examined the larger number of hMLH1 mutations than our previous study and other studies, in the yeast and the in vitro MMR assay, to predict the pathogenecity of these missense mutations and to elucidate the genotype-phenotype relationships.
    Methods: We chose 99 missense, one 3-bp deletion and one nonsense hMLH1 mutants found in individuals with HNPCC or sporadic colorectal cancers that were documented mainly in the InSight or the Swiss-Prot database. These mutants were examined in the two types of functional assays. First, by the yeast assays using three different reporters, each mutant phenotype was determined to retain the dominant mutator effect of the expressed human MLH1 to interfere the yeast MMR system as reported previously. Second, by in-vitro MMR assay, the ability for the hMLH1 mutant expressed in the MMR-deficient HCT116 cells to restore MMR activity of the cell extracts was calculated.
    Results: Among a majority of mutants, similar phenotypes were observed between the yeast and the in vitro assay. The MMR activity of the wild-type MLH1 was 79.7+-7.8 % (average +- SD) whereas the MLH1-null was 0%. Each hMLH1 mutant had a different ability to restore in vitro MMR activity. Mutants found in families fulfilling Amsterdam criteria tended to show loss of function phenotype compared with those found in families not fulfilling Amsterdam criteria.
    Discussion: We examined 101 hMLH1 mutations using two functional assays and characterized their pathogenicity. We propose that this functional evaluation can provide more insights into the genotype-phenotype relationships.

    Conference Organiser - ICMS Pty Ltd