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dc.contributor.advisorViljoen, C. D.
dc.contributor.authorSmith, Tanja
dc.date.accessioned2016-09-09T10:09:31Z
dc.date.available2016-09-09T10:09:31Z
dc.date.issued2016-02
dc.identifier.urihttp://hdl.handle.net/11660/4065
dc.description.abstractEnglish: 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.en_ZA
dc.description.abstractAfrikaans: Nie-oordraagbare siektes (NOS) is die hoof oorsaak van sterftes wêreldwyd. Hipertensie (bloeddruk ≥ 140/90 mmHg) is een van die risikofaktore vir die ontwikkeling van NOS en is een van die hoof oorsake van sterftes in Suid-Afrika. Volgens studies is die voorkoms van hipertensie in ‘n swart bevolking in Mangaung baie hoër as die gemiddeld vir Suid-Afrika. Monogeniese vorme van hipertensie is ‘n groep fisiologiese siektes waarin die verhoogde bloeddruk hoofsaaklik aan ‘n genetiese komponent toegeskryf word. Verskeie gene wat ‘n rol speel in die herabsorpsie van natrium is geassosieer met monogeniese vorme van hipertensie, insluitend die chimeriese CYP11B1/CYP11B2, NR3C1, HSD11B2, SCNN1B, SCNN1G en WNK4. ‘n Vorige studie in die Mangaung bevolking het ‘n positiewe korrelasie tussen bloeddruk en natrium inname gevind, sowel as indikatore van abdominale vetsug en liggaamsvet. Genetiese analise het getoon dat ‘n variant (A6986G in CYP3A5) wat voorheen met primêre hipertensie geassosieer is, moontlik ‘n onafhanklike risikofaktor vir verhoogde bloeddruk in 2% van die Mangaung populasie kan wees. Dit is onbekend of genetiese variante in gene, wat met monogeniese vorme van hipertensie geassosieer is, ‘n rol speel in die Mangaung bevolking. Die doel van hierdie studie was om genetiese variante, wat voorheen met monogeniese vorme van hipertensie geassosieer is, te ondersoek in ‘n swart bevolking van Mangaung. In die huidige studie is ‘n gemodifiseerde CTAB metode gebruik om DNA te isoleer uit bloed wat op FTA® papier versamel is en dit het tot suksesvolle PKR gelei. Verlengde PKR is geoptimiseer om die chimeriese CYP11B1/CYP11B2 teiken gebied te amplifiseer. Konvensionele PKR is suksesvol geoptimiseer om teiken gebiede in NR3C1 (eksons 6, 7, 9, 10 en 11), HSD11B2 (eksons 3, 4 en 5), SCNN1B (ekson 13), SCNN1G (ekson 13) en WNK4 (eksons 7 en 17) te amplifiseer. Hoë-resolusie-smeltkromme (HRS) analise is ook geoptimiseer om monsters waarin ‘n variant moontlik teenwoordig kan wees te identifiseer en sodoende die koste van DNS volgordebepaling te verminder. HRS kon slegs vir NR3C1 ekson 10, NR3C1 ekson 11 en SCNN1B ekson 13 suksesvol gebruik word om monsters met potensiële variante te identifiseer. Gevolglik is daar besluit om DNS volgordebepaling te gebruik om variante, wat voorheen met monogeniese vorme van hipertensie geassosieer is, in hierdie studiegroep te ondersoek. Verlengde PKR is gebruik om die teenwoordigheid van die chimeriese CYP11B1/CYP11B2, wat voorheen met ‘n monogeniese vorm van hipertensie geassosieer is, te ondersoek. Verlengde PKR het daarop gedui dat die chimeriese CYP11B1/CYP11B2 in ten minste een individu in die hipertensiewe groep teenwoordig is, wat moontlik die verhoogde bloeddruk in hierdie individu kan verduidelik. Meervoudige fragmente is egter geproduseer met verlengde PKR. Daar word vermoed dat die hoë graad van ooreenstemming tussen CYP11B1 en CYP11B2, tot niespesifieke amplifisering gelei het. Daar is 53 variante in gene, voorheen met monogeniese vorme van hipertensie geassosieer, deur middel van DNS volgordebepaling in hierdie hipertensiewe groep geïdentifiseer. Een van hierdie variante (Asn767Asn) is voorheen met glukokortikoïed weerstandigheid geassosieer, terwyl vier variante (Arg563Gln, Thr594Met, Leu649Leu en Ala547Ala) voorheen met verhoogde bloeddruk geassosieer is. Van die 53 variante wat geïdentifiseer is, is 26 nie voorheen beskryf nie. Die aantal variante wat in hierdie studie geïdentifiseer is, is verbasend hoog. Verskeie navorsers het voorgestel dat monogeniese vorme van hipertensie meer in die algemene bevolking kan voorkom. Ten slotte, verskeie variante in gene wat voorheen met monogeniese vorme van hipertensie geassosieer is, is in ‘n hipertensiewe groep van Mangaung geïdentifiseer. Daar is bevind dat een of meer variante in ‘n geen, wat voorheen met monogeniese vorme van hipertensie geassosieer is, in 84 uit 90 hipertensiewe individue teenwoordig is. Die data van hierdie studie dui daarop dat monogeniese vorme van hipertensie ‘n belangrike rol kan speel in die ontwikkeling van hipertensie in die Mangaung bevolking.af
dc.description.sponsorshipNational Research Foundation and the GMO Testing facility.en_ZA
dc.language.isoenen_ZA
dc.publisherUniversity of the Free Stateen_ZA
dc.subjectMonogenic hypertensionen_ZA
dc.subjectGenetic variantsen_ZA
dc.subjectHypertensionen_ZA
dc.subjectMangaung populationen_ZA
dc.subjectConventional polymerase chain reaction (PCR)en_ZA
dc.subjectLong range PCRen_ZA
dc.subjectDNA sequencingen_ZA
dc.subjectCommunicable diseases--South Africa--Free Stateen_ZA
dc.subjectChronic diseasesen_ZA
dc.subjectBlacks--Diseases--South Africa--Free Stateen_ZA
dc.titleScreening for genetic variants implicated in monogenic forms of hypertension in a hypertensive urban black Free State cohorten_ZA
dc.typeDissertationen_ZA
dc.rights.holderUniversity of the Free Stateen_ZA


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