Tetra-ARMS-PCR assay development for genotyping of AGT 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 AGT 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 AGT 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 AGT rs699 CC genotype is more prevalent in the CVD patient group (37% vs. 28%) and AGT 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 AGT rs699 T/C polymorphism in a large population in resource-limited countries, Furthermore, AGT rs699 T/C polymorphism is associated with the risk of CVD in the Punjabi Pakistani population.