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dc.contributor.advisorViljoen, C. D.
dc.contributor.authorDu Toit, Egardt
dc.date.accessioned2016-01-11T12:05:11Z
dc.date.available2016-01-11T12:05:11Z
dc.date.issued2014-08
dc.identifier.urihttp://hdl.handle.net/11660/2125
dc.description.abstractEnglish: 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.en_ZA
dc.description.abstractAfrikaans: Hipertensie is een van die grootste oorsake van sterftes en gestremdheid in die wêreld. In 95% van individue met hipertensie ontstaan die siektetoestand uit die interaksie tussen verskeie omgewingsfaktore en fisiologiese stelsels. Omgewingsfaktore wat tot „n verhoging in bloeddruk kan lei, sluit in vetsug, veroudering, hoë sout en alkohol inname, lae kalium en kalsium inname, stres, en insulien weerstandigheid. Fisiologiese stelsels wat bloeddruk reguleer sluit in die outonomiese senuweestelsel, die nierstelsel, hormonale stelsel en die kardiovaskulêre stelsel. Verskeie gene in hierdie stelsels, byvoorbeeld β1- adrenergiese reseptor (ADRB1), α-addusien (ADD1), angiotensinogeen (AGT), aldosteroon sintase (CYP11B2), CYP3A5 en G proteïen-gekoppelde kinase 4 (GRK4) is al geïmpliseer in hipertensie weens die teenwoordigheid van enkel nukleotied polimorfismes (SNPs). Die voorkoms van sulke SNPs in bloeddruk regulatoriese stelsels mag veranderde geenuitdrukking en proteïenfunksie veroorsaak. Die voorkoms van hipertensie in Suid-Afrika is ongeveer 39.9% in mans en 34.9% in vrouens. Die “Assuring Health for All in the Free State” (AHA-FS) studie het bepaal dat hipertensie in ongeveer 48.3% van die Mangaung populasie voorkom. Die AHAFS studie het ook gevind dat 37.6% en 51.2% van die individue in die studiegroep onderskeidelik oorgewig of vetsugtig was en dat hoë liggaamsmassa „n belangrike risikofaktor vir hipertensie is. Die doel van die studie was om te bepaal of gene in die simpatiese senuweestelsel, nierstelsel en hormonale stelsel „n bydrae lewer tot die hoë voorkoms van hipertensie in die Mangaung populasie. Voorheen geïdentifiseerde SNPs wat met hipertensie geassosieer is, naamlik ADRB1 (A145G en G1165C), ADD1 (G217T), AGT (G-217A, C521T en T704C), CYP11B2 (C-344T), CYP3A5 (A6986G), GRK4 (G448T, C679T en C1711T), is gegenotipeer in die AHAFS studiegroep, wat saamgestel was uit swart individue van Mangaung, Vrystaat. Ses van die 11 kandidaat SNPs was nie geassosieer met hipertensie in die swart populasie van Mangaung nie. Hierdie sluit in G1165C (ADRB1), G-217A en T704C (AGT), G448T, C679T en C1711T (GRK4). Nie een van die laasgenoemde SNPs is geassosieer met „n statistiese noemenswaardige verhoging in sistoliese of diastoliese bloeddruk nie. Die assosiasie het negatief gebly te midde van subverdeling van die studiegroep in subgroepe van ondergewig tot normale gewig en oorgewig tot vetsug. Die gebrek aan „n assosiasie tussen hierdie SNPs en hipertensiewe bloeddruk in die swart populasiegroep in Mangaung in vergelyking met ander populasiegroepe, kan toegeskryf word aan populasie verskille met betrekking tot omgewingsfaktore, etnisiteit en epistatiese patrone. Vyf SNPs is geassosieer met hipertensie in swart individue van Mangaung. Laasgenoemde behels SNPs in CYP3A5 (A6986G), ADRB1 (A145G), AGT (C521T), CYP11B2 (C- 344T) en ADD1 (G217T). Die hipertensiewe A alleel van die A6986G SNP van CYP3A5 is geassosieer met sistoliese hipertensie in homosigotiese individue in die studie. Soortgelyke bevindings van „n assosiasie tussen die A alleel en hipertensie in swart Amerikaners en Sweedse blankes bestaan. Dus lyk dit of die A alleel van die A6986G SNP kan bydra tot vatbaarheid vir hipertensie in verskillende populasies, insluitend die Mangaung populasiegroep. „n Assosiasie tussen die A145G SNP in ADRB1 is gevind tussen hipertensiewe sistoliese bloeddruk en homosigote vir die A alleel van die SNP. „n Meta-analise het bepaal dat individue wat die A alleel van die A145G SNP uitdruk, „n 24% hoër risiko het om hipertensie te ontwikkel. Die hipertensie risiko geassosieer met die A alleel van die A145G SNP was veral verhoog in oorgewig tot vetsugtige individue. Die teenwoordigheid van die hipertensiewe T alleel van die C521T SNP in AGT was geassosieer met hipertensiewe diastoliese bloeddruk in die Mangaung populasie. Soortgelyke resultate was gevind in Hutteriet, Russiese en Tartaarse populasiegroepe. Verder was die hipertensie risiko veroorsaak deur die T alleel statisties hoër in oorgewig en vetsugtige individue in die Mangaung populasie. Die resultate dui daarop dat die T alleel van C521T betrokke kan wees in veral hipertensie geassosieer met oorgewigtigheid en vetsug. Die hipertensiewe T alleel van die C-344T SNP in CYP11B2 was slegs geassosieer met hipertensiewe sistoliese en diastoliese bloeddruk in oorgewig tot vetsugtige individue van die Mangaung populasie. Die T alleel was ook geassosieer met hipertensie in „n ander studie op swart Suid-Afrikaanse individue. Die laasgenoemde studiegroep het „n oorgewig en vetsugtige liggaamsmassa gemiddeld gehad. Dit wil dus voorkom asof die T alleel van C-344T hoofsaaklik „n risikofaktor vir oorgewig en vetsug verwante hipertensie is. Interessant was die normotensiewe G alleel van ADD1 wat geïmpliseer was in vetsug verwante hipertensie, in plaas van die hipertensiewe T alleel. In teenstelling, het „n ander studie op swart Suid-Afrikaners gevind dat die T alleel geassosieer was met hipertensie. Alhoewel resultate van „n vorige studie op swart Amerikaners voorgestel het dat die T alleel beskermend teen hipertensie kan wees. Daar was ook „n voorstel dat ongeïdentifiseerde polimorfismes wat vatbaarheid vir hipertensie kan beïnvloed, in koppelingsonewewigtigheid met die G217T SNP kan wees. Indien wel, kan alleel variasie van die ander polimorfismes bydrae tot die nie-konsekwente bevindings van assosiasie studies. Verskeie individue in die populasiegroep van die AHA-FS studie kon nie gegenotipeer word vir die kandidaat SNPs nie. Pogings om konvensionele polimerase ketting reaksie (PKR) produkte te verkry van individue waar genotipering misluk het, was nie volkome suksesvol nie. In die gevalle waar PKR amplikon nie geamplifiseer kon word nie, selfs na PKR optimisering, was die gevolgtrekking dat die oorsaak nie-komplementering van veral die 3‟-kant van die priemstuk kon wees. Waar PKR amplikon suksesvol geamplifiseer was, was volgordebepaling wel moeilik as gevolg van kort fragmentgrootte. Alhoewel, gedeeltelike volgorde data het addisionele SNPs in die peilstuk bindingsareas in sommige individue aangetoon wat verantwoordelik kon wees vir die mislukte genotipering. „n Beperking van die studie was dat slegs geselekteerde SNPs, in gene geassosieer met hipertensie, gegenotipeer is in die studiegroep. Aangesien die studie nie die rol van potensiële nuwe SNPs in kandidaat gene ondersoek het nie, is dit moontlik dat addisionele ongeïdentifiseerde SNPs in die gene wel mag bydra tot hipertensie. Ten spyte van hierdie beperkings, is hierdie studie die mees omvangryke studie van sy soort op die Mangaung populasie. Toekomstige navorsing kan fokus op addisionele gene, asook die sifting vir nuwe SNPs, veral in die gene waar die ondersoekte SNPs nie met hipertensie geassosieer was in die Mangaung studie groep nie. Samevattend het die A6986G SNP in CYP3A5 geblyk om „n onafhanklike risikofaktor vir hipertensie te wees, terwyl A145G in ADRB1, C521T in AGT, C-344T in CYP11B2 en G217T in ADD1 geassosieer is met oorgewig en vetsug verwante hipertensie. Tot ons wete, is hierdie studie die mees omvattende ten opsigte van die kombinasie SNPs in gene geassosieerd met hipertensie in die swart Mangaung populasie.af
dc.language.isoenen_ZA
dc.publisherUniversity of the Free Stateen_ZA
dc.subjectHypertension -- Diagnosisen_ZA
dc.subjectBlacks -- South Africaen_ZA
dc.subjectDIssertation (M.Med.Sc. Haematology and Cell Biology--University of the Free State, 2015en_ZA
dc.titleDevelopment and application of a real-time PCR method to detect selected single nucleotide polymorphisms associated with hypertension in a black South African populationen_ZA
dc.typeDissertationen_ZA
dc.rights.holderUniversity of the Free Stateen_ZA


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