Bibliography: leaves 133-152 / iv, 152 leaves, [47] leaves of plates : ill. (1 col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, 1987

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Thesis (MSc)--University of Stellenbosch, 2004. / ENGLISH ABSTRACT: The most common inherited sensory disorder that affects I in 1 000 children is severe hearing loss. In developed countries, about a third of cases have a genetic origin, 80% of which are autosomal recessive forms (DFNB). Before 1993 few genes causing hearing loss had been identified, but since then a large number of genes related to this problem have been identified. Studies indicate that the DFNBI locus, located at position 13q11-12, contributes to 20% of all childhood deafness and may have a carrier rate as high as 2.8%. There are two genes linked to DFNB 1, GJB2 and GJB6, which are the major genetic cause of non-syndromic autosomal recessive deafness. GJB2 and GJB6 encode the connexin proteins connexin 26 and 30 (Cx26 and Cx30), respectively. The specific aim of this study was to determine the role of GJB2 and GJB6 in deafness within the South African population, since there are no published results involving South African patients with non-syndromic autosomal recessive deafness. This study therefore involved the identification of mutations within the coding region of the GJB2 and GJB6 genes in the South African population and the determination of their specific allele frequencies. Another aim of this study was to analyse the effectiveness of three single-strand conformation polymorphic (SSCP) gel electrophoresis systems in the detection of GJB2 mutations, for use in a standardised diagnostic program. A total of 44 families were recruited and divided into either the familial or sporadic study group, which consisted of 16 and 28 families, respectively. Control samples were also screened from 50 Caucasians and 50 Mixed Ancestry individuals collected from the general population. To achieve the aims of this study, polymerase chain reaction (PCR) amplification followed by automated DNA sequencing of the coding regions of GJB2 and GJB6 was performed. The three SSCP systems that were tested for their effectiveness in detecting mutations within the coding region of GJB2 included mini polyacrylamide, SSCP-urea and two buffer gel electrophoresis systems. In total, six previously reported mutations (35delG, 312de1l4, W24X, M34T, V37I and W44X), a novel mutation (N62I), and four benign polymorphisms (V27I, A40A, R127H and V153I) were detected in GJB2. In the GJB6 gene only the S199T polymorphism was observed. It was determined that the most common mutations found within the Caucasian and Mixed Ancestry populations of South Africa were 35delG and 312de1l4 of GJB2. An overall detection rate of 35.227% was achieved in non-syndromic autosomal recessive deafness amongst this patient cohort. It was also observed that none of the SSCP gel electrophoresis systems were effective at detecting all of the GJB2 mutations. This could change if the systems were specifically optimised for the cornmon mutations that were identified. This study therefore, provides information that can be used in the formulation of a screenmg program for non-syndromic autosomal recessive deafness specific to the South African population. Further research should be conducted involving other genes, in addition other population groups of South Africa to provide a more comprehensive genetic diagnostic and counselling tool. / AFRIKAANSE OPSOMMING: Die mees algemene oorerflike sensoriese steuring wat 1 in 1 000 kinders affekteer is ernstige gehoorverlies. In ontwikkelde lande het omtrent een-derde van die gevalle 'n genetiese oorsprong, waarvan 80% outosomaal resessiewe vorms is (DFNB). Tot en met 1993 is min gene wat gehoorverlies veroorsaak geïdentifiseer, maar sedertdien is 'n groot aantal gene gelokaliseer en verskeie is ook al gekloneer. Studies toon dat die DFNB 1 loci, wat in posisie 13q 11-12 gevind word, 20% van doofheid in kinders veroorsaak, en dit het 'n draer frekwensie van so hoog as 2.8%. Twee gene wat koppeling met DFNBI toon, GJB2 en GJB6, is die vernaamste genetiese oorsaak van nie-sindromise autosomaal resessiewe doofheid. GJB2 en GJB6 koder vir die connexin proteïne 26 en 30 (Cx26 en Cx30), onderskeidelik. Die spesifieke doel van hierdie studie is om die rol van GJR2 en GJB6 in doofheid binne die Suid- Afrikaanse populasie te bepaal, aangesien daar tans nog geen gepubliseerde resultate omtrent Suid- Afrikaanse pasiënte met nie-sindromiese outosomaal resessiewe doofheid is nie. Hierdie studie handel dus oor die identifikasie van mutasies wat binne die koderende areas van die GJR2 en GJB6 gene voorkom in die Suid-Afrikaanse populasie, asook oor die bepaling van hulle spesifieke alleel frekwensies. Verder het hierdie studie ten doelom die effektiwiteit van drie enkel-string konformasie polimorfisme (SSCP) gel-elektroforese metodes in die opsporing van GJB2 mutasies te analiseer met die oog op toekomstige gebruik in 'n gestandardiseerde diagnostiese program. Altesaam 44 families is ingesamel en gekategoriseer in familiële of sporadiese studie-groepe met 16 en 28 families onderskeidelik. Kontrole monsters van 50 Kaukasiese en 50 Gemengde Herkoms individule uit die algemene populasie is ook getoets. Om die doeleindes van die studie te bereik is PKR amplifikasie en outomatiese DNS volgordebepaling van die koderende area van GJB2 en GJR6 gedoen. Die drie SSCP sisteme wat getoets is vir hulle effektiwiteit in die identifisering van mutasies in die koderende area van GJB2 sluit in mini poli-akrielamied, urea en twee-buffer gel elektroforese sisteme. In totaal is ses gerapporteerde mutasies (35delG, 312de114, W24X, M34T, V37I en W44X), 'n nuwe mutasie (N62I), en vier onskadelike polimorfismes (V27I, A40A, R127H en V153I) opgespoor in GJB2, maar in GJB6 is net die S199T polimorfisme waargeneem. Uit die resultate kon afgelei word dat 35deiG en 312de114 van GJB2 die mees algemene mutasies binne die Kaukasiese en Gemengde Herkoms bevolkings van Suid Afrika is. Die total ontdekking standaard van 35.227%· vir nie-sindromise autosomaal resessiewe doofheid tussen herdie patient kohort was bereik. Verder is waargeneem dat geen van die SSCP gel elektroforese metodes effektief was om al die mutasies van GJB2 op te spoor nie. Die situasie kan egter verander as die sisteme spesifiek geoptimiseer word vir die algemene mutasies wat gevind is. Hierdie studie verskaf dus inligting wat gebruik kan word in die verskaffing van 'n diagnostise program vir nie-sindromise outosomaal resessiewe doofheid wat spesifiek is vir die Suid- Afrikaanse populasie. Verdere navorsing wat ander gene en ander populasie groepe van Suid-Afrika insluit, behoort egter uitgevoer te word om uiteindelik 'n meer uitgebreide genetiese diagnostiese en raadgewing diens daar te stel.

Dissertation (PhD)--Stellenbosch University, 2007. / ENGLISH ABSTRACT: This research investigated human candidate genes for susceptibility to tuberculosis and the effect of various factors on time to sputum conversion in the admixed South African Coloured (SAC) population. Population stratification was formally tested and excluded. Population based casecontrol studies were the primary analysis method with a variety of genotyping methods. Candidate polymorphisms in RANTES, CCR5, CCR2 and SDF1, were not associated with tuberculosis susceptibility. Initially the RANTES polymorphism -403 was found to be associated with tuberculosis susceptibility but after the testing of additional samples the association was lost, illustrating the challenges with association studies. The C-type lectins DC-SIGN, encoded by the gene CD209, and L-SIGN are important pathogen-recognition receptors of the human innate immune response. Both lectins have been shown to interact with Mycobacterium tuberculosis. CD209 promoter polymorphisms, -336 and - 871, were both found to be associated with tuberculosis susceptibility. The haplotype containing CD209 -871G and -336A was strongly associated with the control group. The CD209 -336A allele has been found to be associated with increased DC-SIGN expression, which may be the underlying reason for an increased efficiency of host phagocytes. Susceptibility to tuberculosis in mice has recently been attributed to the Ipr1 gene. Eight polymorphisms in the human homologue, SP110, were investigated, including two novel polymorphisms. No significant associations were found with any of the polymorphisms investigated, including two polymorphisms that were previously found to be associated with tuberculosis susceptibility in West African populations. A cohort of 249 cases from a longitudinal study of first time pulmonary tuberculosis patients was available. The cohort was used to investigate if the vitamin D receptor gene (VDR) polymorphisms FokI, ApaI and TaqI were associated with tuberculosis susceptibility or time to sputum conversion, and to investigate other clinical and demographic factors affecting the rate of response to treatment. No association between the VDR genotype and tuberculosis was found in the case-control study. The cohort allowed for a reliable conversion time to be determined for smear (n=220) and culture (n=222). Analysis was carried out to determine which factors, including VDR FokI, ApaI, and TaqI genotypes, contribute to faster mycobacterial resolution in sputum. This was done by survival curves and Cox regression models. The results indicate that the extent of disease at diagnosis was predictive of both smear and culture conversion times in the final models. Smoking status and VDR genotype contributed independently to smear conversion time, with ApaI ‘AA’ and TaqI ‘T’ containing genotypes being predictive of a faster response to tuberculosis therapy. We can conclude that the time taken for an individual to convert to sputum negativity while on DOTS therapy, can be independently predicted by the VDR genotype. This may have implications for future immunomodulatory therapies. Identifying what contributes to susceptibility to tuberculosis will provide us with a better understanding of the human immune response to tuberculosis which may lead to the development of accurately targeted therapeutics and vaccines. / AFRIKAANSE OPSOMMING: Kandidaatgene vir die vatbaarheid vir tuberkulose en die effek van verskeie faktore op sputum oorgangstyd was in hierdie navorsingsstudie ondersoek in die Suid-Afrikaanse Kleurlingbevolking (SAC). Dié bevolking was ook getoets vir populasie-stratifikasie, waarvan daar geen bewyse gevind is nie. Populasiegebaseerde pasiënt-kontrole studies was die primêre metode van analise en verskeie genotipering metodes was gebruik. Polimorfismes in kandidaatgene soos RANTES, CCR5, CCR2 en SDF1 was nie met die vatbaarheid van tuberkulose geassosieer nie. Oorspronklik was daar ‘n assosiasie met die RANTES -403 polimorfisme, maar met die genotipering van addisionele individue het die assosiasie verdwyn. Resultate verkry vir die polimorfisme illustreer die uitdagings waaraan assosiasie studies onderworpe is. Die C-tipe lektiene DC-SIGN, wat gekodeer word deur CD209, en L-SIGN is belangrike patogeen herkenningsreseptore in die aangebore immuunreaksie. Interaksies tussen beide lektiene en Mycobacterium tuberculosis is voorheen gerapporteer. Die CD209 promoter polimorfismes, -336 en -871, was met die vatbaarheid van tuberkulose geassosieer. ‘n Haplotipe bestaande uit die CD209 -871G en -336A allele was sterk met die kontrole groep geassosieer. Die CD209 -336A alleel was geassosieer met ‘n toename in die DC-SIGN proteïen vlakke, wat moontlik ‘n onderliggende rede is vir die toename in die effektiwiteit van die gasheer se fagosiete. Vatbaarheid vir tuberkulose is onlangs toegeskryf aan die Ipr1 geen in muise. Agt polimorfismes, insluitend 2 voorheen onbekendes, was in die mens homoloog SP110 bestudeer. Geen positiewe beduidende assosiasie was met enige van die polimorfismes gevind nie ten spyte van die feit dat twee van hierdie polimorfismes voorheen met tuberkulose vatbaarheid geassosieer was in bevolkings van Wes-Afrika. ‘n Versameling van 249 TB pasiënte van ‘n longitudinale studie was beskikbaar. Dié groep was gebruik om polimorfismes FokI, ApaI and TaqI in die vitamien D reseptor geen (VDR) te bestudeer ten opsigte van vatbaarheid vir tuberkulose of sputum oorgangstyd sowel as ander kliniese en demografiese faktore wat die tempo van respons op behandeling kan affekteer. In hierdie studie was daar geen assosiasie gevind tussen die ontwikkeling van tuberkulose en die VDR genotipes nie. Die bepaling van ‘n betroubare oorgangstyd vir beide smeer en kultuur van die groep was moontlik. Analises was uitgevoer om te bepaal watter faktore bydrae tot vinniger resolusie van Mycobacteria in sputum. Resultate verkry het aangedui dat die aard van die siekte tydens diagnose voorspelbaar was van die oorgangstye van beide smeer en kultuur in die finale modelle. Die rookstatus van individue sowel as die VDR genotipes het onafhanklik bygedrae tot die oorgangstyd van die smeer, met ApaI ‘AA’ en TaqI ‘T’ bevattende genotipes wat ‘n vinniger reaksie op tuberkulose behandeling voorspel het. Ter opsomming, die tyd wat dit ‘n individu op DOTS terapie neem om na sputum negatief oor te gaan kan onafhanklik deur die VDR genotipe voorspel word. Dit kan moontlik implikasies hê vir ander immunomodulerende terapië in die toekoms. Die identifisering van faktore wat bydra tot die vatbaarheid van turberkulose sal ons in staat stel om ‘n beter begrip te hê van die immuunrespons teen tuberkulose wat moontlik kan lei tot die ontwikkeling van akkurate behandelings en inentings.

published_or_final_version / Paediatrics / Doctoral / Doctor of Philosophy

published_or_final_version / Obstetrics and Gynaecology / Master / Master of Philosophy

published_or_final_version / abstract / Clinical Oncology / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Anatomy / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Medicine / Doctoral / Doctor of Philosophy