Development and application of a real-time PCR method to detect selected single nucleotide polymorphisms associated with hypertension in a black South African population

English: Hypertension is one of the leading causes of death and disability in the world. In 95% of individuals with hypertension, the condition arises from the interaction of multiple environmental factors with physiological systems. Environmental factors that have been found to increase blood pressure include obesity, aging, high salt and alcohol consumption, low potassium and calcium intake, stress and insulin resistance. Physiological systems that regulate blood pressure include the autonomic nervous system, the renal system, hormonal system and the cardiovascular system. Various genes in these systems including the β1-adrenergic receptor (ADRB1), α-adducin (ADD1), angiotensinogen (AGT), aldosterone synthase (CYP11B2), CYP3A5 and G protein-coupled receptor kinase 4 (GRK4) have been implicated in hypertensive blood pressure due to the presence of single nucleotide polymorphisms (SNPs). The occurrence of such SNPs in blood pressure regulatory systems is thought to result in altered gene expression or protein function. In South Africa, the prevalence of hypertension has been determined to be approximately 39.9% in males and 34.9% in females. The Assuring Health for all in the Free State (AHA-FS) study determined that the prevalence of hypertension was approximately 48.3% in the Mangaung population. The AHA-FS study also found that 37.6% and 51.2% of individuals in the study cohort were overweight or obese, respectively, and that high body mass could be an important risk factor for hypertension. The aim of this study was to determine whether genes in the sympathetic nervous system, renal system and hormonal systems could contribute to the high prevalence of hypertension in the Mangaung population. Previously identified SNPs associated with hypertension in ADRB1 (A145G and G1165C), ADD1 (G217T), AGT (G-217A, C521T and T704C), CYP11B2 (C-344T), CYP3A5 (A6986G) and GRK4 (G448T, C679T and C1711T) were genotyped in a cohort of the AHA-FS study, which comprised black individuals from Mangaung, Free State. Six of the 11 candidate SNPs did not appear to be associated with hypertension in the black population of Mangaung. These included G1165C (ADRB1), G-217A and T704C (AGT), G448T, C679T and C1711T (GRK4). None of the latter SNPs were associated with statistically significant elevations in either systolic or diastolic blood pressure. The association remained negative even after the cohort was stratified into underweight to normal weight and overweight to obese groups. The lack of association between these SNPs and hypertensive blood pressure in the black population group in Mangaung compared to other population groups could be attributed to population differences in environmental factors, ethnicity, cohort size and/or epistasis. Five SNPs were associated with hypertension in black individuals from Mangaung. The latter included SNPs in CYP3A5 (A6986G), ADRB1 (A145G), AGT (C521T), CYP11B2 (C-344T) and ADD1 (G217T). The hypertensive A allele of the A6986G SNP of CYP3A5 has been associated with systolic hypertension in homozygous individuals of the study. Similarly, an association between the A allele and hypertension in African-Americans and Swedish Caucasians has also been found. Thus, the A allele of the A6986G SNP seems to cause hypertension susceptibility in different populations, including the Mangaung population group. As for the A145G SNP of ADRB1, hypertensive systolic blood pressure was associated with individuals that were homozygous for the hypertensive A allele of the A145G SNP. A meta-analysis determined that individuals expressing the A allele of A145G had a 24% higher risk for developing hypertension. In the Mangaung population, hypertension risk associated with the A allele of A145G was especially increased in overweight to obese individuals. The presence of the hypertensive T allele of the C521T SNP of AGT was associated with hypertensive diastolic blood pressure in the Mangaung population. Similar results were found in Hutterite, Russian and Tartar population groups. Furthermore, in the Mangaung population the hypertension risk conferred by the T allele was significantly increased in overweight and obese individuals. This suggests that the T allele of C521T may be involved in particularly overweight and obesity related hypertension. The hypertensive T allele of the C-344T SNP of CYP11B2 was only associated with hypertensive systolic and diastolic blood pressure in the overweight to obese individuals of the Mangaung population. In another study conducted on black South African individuals the T allele was also associated with hypertension. The cohort of the latter study furthermore had an overweight to obese body mass index average. It therefore appears that the T allele of C-344T could primarily be a risk factor for overweight and obesity related hypertension. Interestingly the normotensive G allele of ADD1 was implicated in obesity related hypertension, instead of the hypertensive T allele. In contrast, another study on black South Africans found that the T allele was associated with hypertension. However, results from a previous study on African-Americans suggested that the T allele may be protective against hypertension. It has been proposed that other unidentified polymorphisms, which also could affect hypertension susceptibility, could be in linkage disequilibrium with the G217T SNP and that allelic variation of the other polymorphic loci could contribute to the inconsistent findings of association studies. Several individuals in the cohort from the AHA-FS study could not be genotyped for the candidate SNPs. Attempts to obtain conventional PCR product for individuals where genotyping failed did not prove entirely successful. In the cases where no PCR amplicon could be amplified even after attempts at assay optimization, it was concluded that primer mismatch, especially at the 3‟ end of the primer may be the likely cause. Where PCR amplicon was successfully amplified, sequencing proved difficult due to short amplicon size. However, partial sequence data revealed additional SNPs in some individuals in the probe binding region that would account for failed genotyping. A limitation of this study was that only selective SNPs in genes associated with hypertension were genotyped in the cohort. Furthermore, since the study did not investigate the role of potentially novel SNPs in candidate genes, it is possible that additional unidentified SNPs in these genes may also contribute to hypertension. Despite these limitations, this study is currently the most comprehensive of its kind on the Mangaung population. Future research could focus on additional genes as well as screening these genes for novel SNPs, especially in the genes where the SNPs investigated were not associated with hypertension in the Mangaung cohort. In conclusion, the A6986G SNP in CYP3A5 appears to be an independent risk factor for hypertension, whereas A145G in ADRB1, C521T in AGT, C-344T in CYP11B2 and G217T in ADD1 may be associated with hypertension related to overweight and obese body weights. To our knowledge, this is the most comprehensive study investigating a combination of several gene SNPs associated with hypertension in a black Mangaung population.