Screening for genetic variants implicated in monogenic forms of hypertension in a hypertensive urban black Free State cohort

English: Non-communicable diseases (NCDs), also known as chronic diseases of lifestyle, are the leading cause of death worldwide. Amongst the risk factors for NCDs, hypertension (blood pressure (BP) ≥140/90 mmHg) is one of the leading causes of death in South Africa. The prevalence of hypertension in an urban black population in Mangaung in the Free State is reported to be much higher than the average for South Africa. Monogenic forms of hypertension are a group of physiological disorders where the elevated BP is thought to be primarily due to a genetic component. Several genes that play a role in the sodium reabsorption pathway have been implicated in the syndromes associated with monogenic hypertension, including the chimeric CYP11B1/CYP11B2, NR3C1, HSD11B2, SCNN1B, SCNN1G, and WNK4. In a previous study involving the Mangaung population, it was found that BP correlated positively with adiposity, as well as with sodium intake. In addition, genetic analysis indicated that a genetic variant (A6986G in CYP3A5) implicated in primary hypertension, could be an independent risk factor for hypertension in 2% of the Mangaung population. It is not known, however, if genetic variants implicated in monogenic forms of hypertension could play a role in the Mangaung population. The aim of this study was to screen for genetic variants implicated in monogenic forms of hypertension in a black hypertensive cohort from Mangaung. In this study, a generic CTAB method was successfully used to extract DNA from blood spotted onto FTA® paper, which resulted in successful PCR amplification. Thereafter, a long range PCR assay was successfully optimized in order to amplify the chimeric CYP11B1/CYP11B2. Conventional PCR assays to amplify selected target regions in NR3C1 (exons 6, 7, 9, 10 and 11), HSD11B2 (exons 3, 4, and 5), SCNN1B (exon 13), SCNN1G (exon 13) and WNK4 (exons 7 and 17), were also successfully optimized. High resolution melting (HRM) analysis was optimized in an attempt to identify samples with potential sequence variants, thereby reducing the cost of sequencing. However, HRM analysis was only successful in identifying samples with sequence variants for NR3C1 exon 10, NR3C1 exon 11, and SCNN1B exon 13. As a result of the difficulty experienced identifying sequence variants using HRM analysis, it was decided to use DNA sequencing instead to screen for sequence variants in this study cohort. Long range PCR was used to screen for the presence of the chimeric CYP11B1/CYP11B2 in this hypertensive cohort. The long range PCR assay allowed the conclusive identification of the chimeric CYP11B1/CYP11B2 in at least one hypertensive individual and could potentially explain the elevated BP in this individual. However, multiple fragments were produced using the long range PCR assay. It is suspected that the high degree of similarity reported between CYP11B1 and CYP11B2 could have resulted in non-specific amplification. Using DNA sequencing, 53 sequence variants in genes implicated in monogenic forms of hypertension were identified in this hypertensive cohort. Of these, one variant (Asn767Asn) has previously been associated with glucocorticoid resistance and four variants (Arg563Gln, Thr594Met, Leu649Leu, and Ala547Ala) have previously been associated with elevated BP. Out of the 53 sequence variants identified in this study, 26 were novel. The number of sequence variants identified in genes implicated in monogenic forms of hypertension is surprisingly high. Several authors have suggested that monogenic forms of hypertension could be more common in the general population. To conclude, several sequence variants in genes implicated in monogenic forms of hypertension were identified in a hypertensive cohort from Mangaung. It was found that 84 out of 90 hypertensive individuals had one or more sequence variants in genes implicated in monogenic forms of hypertension. The data from this study suggests that monogenic forms of hypertension may play an important role in the development of hypertension in the Mangaung population.