Tetra primer ARMS-PCR relates folate/homocysteine pathway genes and ACE gene polymorphism with coronary artery disease
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Cardiovascular disorders and coronary artery disease (CAD) are significant contributors to morbidity and mortality in heart patients. As genes of the folate/homocysteine pathway have been linked with the vascular disease, we investigated association of these gene polymorphisms with CAD/myocardial infarction (MI) using the novel approach of tetraprimer ARMS-PCR. A total of 230 participants (129 MI cases, 101 normal subjects) were recruited. We genotyped rs1801133 and rs1801131 SNPs in 5′10′ methylenetetrahydrofolate reductase (MTHFR), rs1805087 SNP in 5′ methyltetrahydrofolate homocysteine methyltransferase (MTR), rs662 SNP in paroxanse1 (PON1), and rs5742905 polymorphism in cystathionine beta synthase (CBS). Angiotensin converting enzyme (ACE) insertion/deletion polymorphism was detected through conventional PCR. Covariates included blood pressure, fasting blood sugar, serum cholesterol, and creatinine concentrations. Our results showed allele frequencies at rs1801133, rs1801131, rs1805087 and the ACE insertion/deletion (I/D) polymorphism varied between cases and controls. Logistic regression, after adjusting for covariates, demonstrated significant associations of rs1801133 and rs1805087 with CAD in the additive, dominant, and genotype model. In contrast, ACE I/D polymorphism was significantly related with CAD where recessive model was applied. Gene–gene interaction against the disease status revealed two polymorphism groups: rs1801133, rs662, and rs1805087; and rs1801131, rs662, and ACE I/D. Only the latter interaction maintained significance after adjusted for covariates. Our study concludes that folate pathway variants exert contributory influence on susceptibility to CAD. We further suggest that tetraprimer ARMS-PCR successfully resolves the genotypes in selected samples and might prove to be a superior technique compared to the conventional approach.
KeywordsTetra primer ARMS-PCR Homocysteinemia Single nucleotide polymorphisms
The authors are grateful to Dr Haider Zaigham Baqai, Dr Asad Riaz for patient selection, Professor Dr. Wasim Ahmad, Drs Jawad Hassan, Mohammad Tariq, Salman Chishti, Mohammad Inam for their valuable comments on the manuscript, and to Dr Keyue Ding, Mayo Clinic Rochester, Minnesota USA for help with statistics. The research work was funded through a graduate scholarship grant No. 17-5-2 Ls2-50 HEC/Sch/2004/5145 awarded to the first author by Higher Education Commission (HEC), Islamabad, Pakistan.
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