Tetra-ARMS-PCR assay development for genotyping of rs699 T/C polymorphism, its comparison with PCR-RFLP and application in a case-control association study of cardiovascular disease patients.

Genetic variations in the gene play a significant role in controlling the plasma concentration of angiotensinogen (precursor protein of bioactive octapeptide angiotensin II) and the efficacy of antihypertensive drugs. In the current study, Tetra-Amplification Refractory Mutation System-Polymerase Chain Reaction (T-ARMS-PCR) was developed for genotyping of rs699 T/C polymorphism and validated through Sanger DNA sequencing. Its efficiency was also tested using 474 human DNA samples [control, n = 181; cardiovascular disease (CVD) patients, n = 293]. Results showed that T-ARMS-PCR is superior to the commonly used PCR-Restriction Fragment Length Polymorphism (PCR-RFLP). Statistical analysis revealed that the rs699 CC genotype is more prevalent in the CVD patient group (37% vs. 28%) and rs699 C allele and CC genotype increased the risk of CVD by 1.4 and 1.9 fold, respectively. In summary, T-ARMS-PCR is the most suitable approach for quick and efficient genotyping of rs699 T/C polymorphism in a large population in resource-limited countries, Furthermore, rs699 T/C polymorphism is associated with the risk of CVD in the Punjabi Pakistani population.