Graduate Thesis Or Dissertation
 

Phenotypic analysis of human MLH1 variants for DNA mismatch repair

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https://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/b2774087q

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  • DNA mismatch repair (MMR) is an evolutionarily conserved process used by cells to decrease mutation rates, thereby increasing genetic stability and reducing cancer risk. Germline mutations in the MMR gene MLH1 have been identified in Lynch Syndrome patients, and in many cases are suspected but not confirmed as causative for cancer predisposition. This study compared wild-type and variant hMLH1 proteins in terms of MLH1 stability and the ability to complement a MMR-deficient phenotype in cell cultures lacking endogenous MLH1. The goal was to describe the possible mechanisms of pathogenicity for individual mutations in MLH1 by identifying functional deficiencies associated with each mutation. By measuring and comparing the degradation rates of wild-type and variant hMLH1 proteins, it was determined that variants that accumulated to low steady-state levels indeed had elevated degradation rates. By using a novel pooled complementation approach, the ability of wild-type and variant hMLH1 to restore a MMR-proficient phenotype to MLH1-deficient mouse embryonic fibroblast cultures was measured. Variants with decreased MLH1 stability (eg. K616D and K618A) were incapable of inducing a MLH1-dependent cytotoxic response or decreasing spontaneous mutation frequencies to levels observed in wild-type expressing cultures. Variants with steady-state hMLH1 levels with similar to wild-type (eg. K751R and R755W) displayed variable phenotypes, depending on the individual variant’s biochemical activity. In our pooled approach, the controversial and weakly penetrant variants examined (D132H and E578G) were capable of restoring a cytotoxic response and reducing spontaneous mutation frequencies to levels comparable to wild-type transfected cultures. The study shows that the pooled approach is an effective approach for determining the phenotypic behavior of hMLH1 variants in order to identify potential mechanisms of pathogenicity.
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