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

[摘要] English: Hypertension is one of the leading causes of death and disability in the world. In95% of individuals with hypertension, the condition arises from the interaction ofmultiple environmental factors with physiological systems. Environmental factorsthat have been found to increase blood pressure include obesity, aging, high salt andalcohol consumption, low potassium and calcium intake, stress and insulinresistance. Physiological systems that regulate blood pressure include theautonomic nervous system, the renal system, hormonal system and thecardiovascular system. Various genes in these systems including the β1-adrenergicreceptor (ADRB1), α-adducin (ADD1), angiotensinogen (AGT), aldosterone synthase(CYP11B2), CYP3A5 and G protein-coupled receptor kinase 4 (GRK4) have beenimplicated in hypertensive blood pressure due to the presence of single nucleotidepolymorphisms (SNPs). The occurrence of such SNPs in blood pressure regulatorysystems is thought to result in altered gene expression or protein function.In South Africa, the prevalence of hypertension has been determined to beapproximately 39.9% in males and 34.9% in females. The Assuring Health for all inthe Free State (AHA-FS) study determined that the prevalence of hypertension wasapproximately 48.3% in the Mangaung population. The AHA-FS study also foundthat 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 forhypertension.The aim of this study was to determine whether genes in the sympathetic nervoussystem, renal system and hormonal systems could contribute to the high prevalenceof 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, whichcomprised black individuals from Mangaung, Free State.Six of the 11 candidate SNPs did not appear to be associated with hypertension inthe black population of Mangaung. These included G1165C (ADRB1), G-217A andT704C (AGT), G448T, C679T and C1711T (GRK4). None of the latter SNPs wereassociated with statistically significant elevations in either systolic or diastolic bloodpressure. The association remained negative even after the cohort was stratifiedinto underweight to normal weight and overweight to obese groups. The lack ofassociation between these SNPs and hypertensive blood pressure in the blackpopulation group in Mangaung compared to other population groups could beattributed to population differences in environmental factors, ethnicity, cohort sizeand/or epistasis. Five SNPs were associated with hypertension in black individualsfrom 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 withsystolic hypertension in homozygous individuals of the study. Similarly, anassociation between the A allele and hypertension in African-Americans andSwedish Caucasians has also been found. Thus, the A allele of the A6986G SNPseems to cause hypertension susceptibility in different populations, including theMangaung population group.As for the A145G SNP of ADRB1, hypertensive systolic blood pressure wasassociated with individuals that were homozygous for the hypertensive A allele of theA145G SNP. A meta-analysis determined that individuals expressing the A allele ofA145G had a 24% higher risk for developing hypertension. In the Mangaung population, hypertension risk associated with the A allele of A145G was especiallyincreased in overweight to obese individuals.The presence of the hypertensive T allele of the C521T SNP of AGT was associatedwith hypertensive diastolic blood pressure in the Mangaung population. Similarresults were found in Hutterite, Russian and Tartar population groups. Furthermore,in the Mangaung population the hypertension risk conferred by the T allele wassignificantly increased in overweight and obese individuals. This suggests that the Tallele of C521T may be involved in particularly overweight and obesity relatedhypertension.The hypertensive T allele of the C-344T SNP of CYP11B2 was only associated withhypertensive systolic and diastolic blood pressure in the overweight to obeseindividuals of the Mangaung population. In another study conducted on black SouthAfrican individuals the T allele was also associated with hypertension. The cohort ofthe 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 foroverweight and obesity related hypertension.Interestingly the normotensive G allele of ADD1 was implicated in obesity relatedhypertension, instead of the hypertensive T allele. In contrast, another study onblack South Africans found that the T allele was associated with hypertension.However, results from a previous study on African-Americans suggested that the Tallele may be protective against hypertension. It has been proposed that otherunidentified polymorphisms, which also could affect hypertension susceptibility,could be in linkage disequilibrium with the G217T SNP and that allelic variation of theother polymorphic loci could contribute to the inconsistent findings of associationstudies. Several individuals in the cohort from the AHA-FS study could not be genotyped forthe candidate SNPs. Attempts to obtain conventional PCR product for individualswhere genotyping failed did not prove entirely successful. In the cases where noPCR amplicon could be amplified even after attempts at assay optimization, it wasconcluded that primer mismatch, especially at the 3�?end of the primer may be thelikely cause. Where PCR amplicon was successfully amplified, sequencing proveddifficult due to short amplicon size. However, partial sequence data revealedadditional SNPs in some individuals in the probe binding region that would accountfor failed genotyping.A limitation of this study was that only selective SNPs in genes associated withhypertension were genotyped in the cohort. Furthermore, since the study did notinvestigate the role of potentially novel SNPs in candidate genes, it is possible thatadditional unidentified SNPs in these genes may also contribute to hypertension.Despite these limitations, this study is currently the most comprehensive of its kindon the Mangaung population. Future research could focus on additional genes aswell as screening these genes for novel SNPs, especially in the genes where theSNPs investigated were not associated with hypertension in the Mangaung cohort.In conclusion, the A6986G SNP in CYP3A5 appears to be an independent risk factorfor hypertension, whereas A145G in ADRB1, C521T in AGT, C-344T in CYP11B2and G217T in ADD1 may be associated with hypertension related to overweight andobese body weights. To our knowledge, this is the most comprehensive studyinvestigating a combination of several gene SNPs associated with hypertension in ablack Mangaung population.

[发布机构] University of the Free State