A common amino acid polymorphism of methylenetetrahy-drofolate reductase is associated with hyperhomocysteinemia and predisposition to cardiovascular disease. It is hypothesized that mutations of other enzymes of homocysteine metabolism may have similar impact. The focus of this study will be on two enzyme systems of homocysteine remethylation. One is methionine synthase (MS) which requires two gene products: the MS apoenzyme (deficient in cblG disorder) and a reducing system associated with MS function (cbIE disorder). The second is betaine-homocysteine methyltransferase (BHMT). The investigators have cloned cDNAs for human MS and BHMT and identified mutations and a polymorphism of the MS apoenzyme.
Their specific aims are: 1) Identify amino acid polymorphisms in the MS (cblG, cbIE) and BHMT genes (cbIE to be cloned). This will be performed by SSCP analysis for the detection of mutations from RT-PCR or genomic DNA PCR products. 2) Clone the cbIE cDNA by functional complementation or homology-based RT-PCR (used successfully for the MS cbIG cDNA cloning). The complementation method will involve transformation of an E. coli methionine auxotroph, already expressing human MS, with a human cDNA library. Successful complementation will require the cblE gene product to permit growth in methionine-free medium. The homology method makes use of conserved sequences identified between flavodoxin/flavodoxin reductase of prokaryotes and eukaryotic flavoproteins to specify degenerate oligonucleotides for RT-PCR based cloning. 3) Express polymorphisms in human cell or E. coli expression systems to assess impact of mutations. Mutant fibroblasts will be the recipient for cDNA-mediated complementation assays. Expression of cDNAs in E. coli will permit biochemical analysis of purified protein. These studies will include introducing mutations into E. coli MS for analysis in conjunction with the flavodoxin system. 4) Examine impact of polymorphisms on homocysteine and nutrient levels. Identified polymorphisms will be screened in human subjects to determine if mutations are associated with abnormal homocysteine or nutrient (folate, B12) levels. Evaluate impact of polymorphisms on cardiovascular disease. Coronary artery disease patients will be evaluated for genotype status for polymorphisms shown to be associated with altered metabolities to determine if genotype frequencies vary from control groups. These studies, will determine the importance of genotype in the metabolic causes of hyperhomocysteinemia and risk of cardiovascular disease.
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