Genetic causes of mild hyperhomocysteinemia in patients with premature occlusive coronary artery diseases.

Elevated plasma homocysteine is increasingly being recognized as a risk factor for coronary artery disease (CAD). Although there is general agreement on the importance of micronutrients and genetic predisposition to elevated plasma homocysteine, the exact influence of the known prevalent mutations in genes which regulate homocysteine metabolism is not clear. We studied 376 cases of individuals with premature CAD with respect to their fasting and post-methionine load (PML) total homocysteine (tHcy) concentrations. We also determined the presence or absence of the T833C and G919A mutations of the cystathionine-beta-synthase (CBS) gene, the C677T mutation of the methylene tetrahydrofolate reductase (MTHFR) gene, and the A2756G transition of the B12 dependent methionine synthase (MS) gene. Our objectives were therefore both to confirm the relationship of plasma homocysteine with premature CAD and to examine the importance of genetic influence on both fasting and PML homocysteine. Approximately 32% of the CAD patients had fasting hyperhomocysteinemia and 16% had PML hyperhomocysteinemia. Of these, 8.5% had both forms of hyperhomocysteinemia (combined hyperhomocysteinemia). The T133C mutation in the CBS gene and the thermolabile C677T mutation in the MTHFR gene seem to play an important role in the subset of individuals with combined hyperhomocysteinemia. The A2756G transition in the MS gene is not associated with elevated plasma tHcy. Many cases (47%) of hyperhomocysteinemia are not associated with micronutrient deficiencies, impaired renal function, and/or currently known genetic mutations. Further work is needed to study whether unknown mutations, particularly those residing in the intronic sequences of the genes involved in homocysteine metabolism, other environmental factors, or interaction of gene, nutrient, and environmental factors may be the cause of currently unexplained cases of mild hyperhomocysteinemia.

Simultaneous detection and screening of T833C and G919A mutations of the cystathionine beta-synthase gene by single-strand conformational polymorphism.

OBJECTIVE: We used single-strand conformational polymorphism (SSCP) to screen for mutations at nucleotides 833 and 919 of the cystathionine beta-synthase (CBS) gene in 13 patients with homocystinuria and 11 of their relatives. METHODS: Exon 8 of genomic DNA was selectively amplified by PCR using primers derived from intronic sequences of the human CBS gene. SSCP analysis was performed on the amplified products. Genotypes identified by SSCP were confirmed by DNA sequencing and an allele-specific PCR method. RESULTS: SSCP identified 5 patterns corresponding to five genotypes. We confirmed that the different genotypes result from mutations at nucleotides 833 and 919 of the CBS gene, and that these 2 mutations account for approximately 50% of affected alleles in homocystinuria patients. CONCLUSION: Our recent elucidation of intron-exon borders and intronic sequences of the CBS gene has made possible the use of SSCP to screen for known/unknown mutations in the CBS gene. Because T833C and G919A represent the two most common mutations and both are located within exon 8 of the CBS gene, SSCP of exon 8 allows screening of the heterozygous carrier state of these mutations in a large population, to determine the importance of heterozygosity of CBS mutations as the cause of mild hyperhomocyst(e)inemia associated with premature vascular diseases.