Thesis (MScMedSc (Biomedical Sciences. Molecular Biology and Human Genetics))--Stellenbosch University, 2008. / A South African family of Mixed Ancestry presented with a rapidly progressive dementia and a movement disorder which affected a number of individuals across three generations. The initial symptoms included personality changes and tremors that escalated to severe dementia and eventually a completely bedridden state. It was determined that the mean age at onset was in the third decade of life and affected individuals died within 10-15 years after the onset of symptoms. The aim of the present study was to elucidate the genetic cause of the disorder in this family and to further investigate the patho-biology of the disease. Mutations that could possibly cause the observed phenotype in this family were screened for. These included loci implicated in Huntington’s disease, Parkinson’s disease, Dentatorubral-Pallidoluysian Atrophy, Spinocerebellar ataxias (types 1, 2, 3, 6, and 7), Huntington’s disease-like 2 (HDL2) and several mitochondrial disorders. Single-strand Conformation Polymorphism (SSCP) analysis and direct sequencing were used to detect possible mutations while genotyping on an ABI genetic analyser was used to detect disorders caused by repeat expansions. Haplogroup and Short Tandem Repeats (STRs) analyses of the Y-chromosome and mitochondrial DNA of one affected family member was used to determine the family’s genetic ancestry. Reverse transcriptase polymerase chain reaction (RT- PCR) and complementary DNA (cDNA) analyses of the Junctophlin-3 (JPH3) gene was performed to provide information on the expression profile of this gene. After the exclusion of several genetic loci it was shown that this family had HDL2. This is a rare disease caused by a CAG/CTG repeat expansion in an alternatively spliced version of the JPH3 gene. HDL2 occurs almost exclusively in individuals of Black African ancestry. The genetic ancestry data suggested that the family member was most likely of South African Mixed Ancestry making this the first reported family of South African Mixed Ancestry with HDL2. A pilot study investigated the repeat distribution amongst three South African sub-populations in order to determine whether there was a bias in the repeat distribution that possibly predisposes Black Africans to develop the disease. The results showed a statistically significant difference (P= 0.0014) in the distribution of the repeats between the Black African and Caucasian cohorts. However, no conclusions could be drawn as to whether Black Africans harboured larger repeats that predisposes them to developing HDL2. The expanded repeat is located in an alternatively spliced version of the JPH3 mRNA. Interestingly, this repeat is not present in the mouse homologue of the gene although the rest of the genomic sequence is highly conserved across the human, mouse and chimpanzee genomes. Using foetal brain cDNA and PCR primers designed to be specific for different JPH3 isoforms, independent confirmation of the presence of two JPH3 mRNA transcripts (the full length and a shorter alternatively spliced version) was provided. In the absence of brain tissue from an HDL2-affected individual, it was investigated whether both JPH3 mRNA transcripts could be detected in lymphocytes. Using RNA isolated from the transformed lymphocytes of two HDL2-affected family members, real-time PCR was attempted. These experiments produced inconclusive results and required further optimisation. Further RT-PCR experiments for JHP3 expression in different tissues (brain and other) obtained from HDL2-affected individuals would be of interest. The present study identified the first Mixed Ancestry family with HDL2. This family will now be able to request genetic counselling and pre-symptomatic testing for all at-risk family members. Aspects of this study provided independent confirmation of characteristics of the mutated gene. More research on HDL2 will be crucial in understanding the pathogenesis of this disease.

Thesis (PhD)--Stellenbosch University, 2004. / Bibliography / ENGLISH ABSTRACT: Heart failure due to cardiomyopathy or cardiac conduction disease is a major cause of mortality and morbidity in both developed and developing countries. Although defined as separate clinical entities, inherited forms of cardiomyopathies and cardiac conduction disorders have been identified that present with overlapping clinical features and/or have common molecular aetiologies. The objective of the present study was to identify the molecular cause of progressive familial heart block type II (PFHBII), an inherited cardiac conduction disorder that segregates in a South African Caucasian Afrikaner family (Brink and Torrington, 1977). The availability of family data tracing the segregation of PFHBII meant that linkage analysis could be employed to identify the chromosomal location of the disease-causative gene. Human Genome Project (HGP) databases have provided additional resources to facilitate the identification of positional candidate genes. Clinical examinations were performed on individuals of the PFHBII-affected family, and, where available, clinical records of subjects examined in a previous study by Brink and Torrington (1977) were re-assessed. Retrospective data suggested redefining the classification of PFHBII. Subsequently, linkage analysis was used to test described dilated cardiomyopathy (DCM), hypertrophic cardiomyopathy (HCM) and cardiac conduction-causative loci on chromosomes 1, 2, 3, 6, 7, 9, 11, 14, 15 and 19 for their involvement in the development of PFHBII. Once a locus was mapped, bioinformatics tools were applied to identify and prioritise positional candidate genes for mutation screening. The retrospective and prospective clinical study redefined PFHBII as a cardiac conduction and DCM-associated disorder and simultaneously allowed more family members to be traced. Fortuitously, candidate loci linkage analysis mapped the PFHBII locus to chromosome 1q32, to a region that overlapped a previously described DCM-associated disorder (CMD1D), by the generation of a maximum pairwise lod score of 3.13 at D1S3753 (theta [θ]=0.0) and a maximum multipoint lod score of 3.7 between D1S3753 and D1S414. However, genetic fine mapping and haplotype analysis placed the PFHBII-causative locus distal to the CMD1D locus, within a 3.9 centimorgan (cM) interval on chromosome 1q32.2-q32.3, telomeric of D1S70 and centromeric of D1S505. Bioinformatics analyses prioritised seven candidate genes for mutation analysis, namely, a gene encoding a potassium channel (KCNH1), an extracellular matrix protein (LAMB3), a protein phosphatase (PPP2R5A), an adapter protein that interacts with a cytoskeletal protein (T3JAM), a putative acyltransferase (KIAA0205) and two genes encoding proteins possibly involved in energy homeostasis (RAMP and VWS59). The PFHBII-causative mutation was not identified, although single sequence variations were identified in four of the seven candidate genes that were screened. Although the molecular aetiology was not established, the present study defined the underlying involvement of DCM in the pathogenesis of PFHBII. The new clinical classification of PFHBII has been published (Fernandez et al., 2004) and should lead to tracing more affected individuals in South Africa or elsewhere. The identification of a novel disease-causative locus may point toward the future identification of a new DCM-associated aetiology, which, in turn, might provide insights towards understanding the associated molecular pathophysiologies of heart failure. / AFRIKAANSE OPSOMMING: Hartversaking as gevolg van kardiomiopatie of kardiale geleidingsiekte is ‘n hoof-oorsaak van mortaliteit and morbiditeit in beide ontwikkelde en ontwikkelende lande. Alhoewel gedefinieer as verskillende kliniese entiteite is oorerflike vorms van kardiomiopatie en kardiale geleidingsstoornisse geïdentifiseer met oorvleuelende kliniese eienskappe en/of molukulêre oorsake. Die doelwit van hierdie studie was om die molukulêre oorsaak van progressiewe familiële hartblok tipe II (PFHBII), ‘n oorerflike kardiale geleidingsstoornis, wat in ‘n Suid-Afrikaanse Kaukasiër familie segregeer (Brink en Torrington, 1977), te identifiseer. Die beskikbaarheid van familie data, beteken dat koppelingsanalise gebruik kan word om die chromosomale posisie van die siekte-veroorsakende geen te identifiseer. Menslike Genoom Projek (MGP) databanke het addisionele hulpbronne beskikbaar gestel om die identifikasie van posisionele kandidaat gene te vergemaklik. Kliniese ondersoeke is uitgevoer op PFHBII-geaffekteerde familielede, en waar beskikbaar is kliniese rekords van persone, wat in ‘n vorige studie deur Brink en Torrington (1977) geassesseer was, herontleed. Retrospektiewe data-analise het die kliniese herdefinisie van PFHBII voorgestel. Daarna is koppelingsanalise gebruik om dilateerde kardiomiopatie (DKM), hipertrofiese kardiomiopatie (HKM) en kardiale geleidingssiekte-veroorsakende loki op chromosoom 1, 2, 3, 6, 7, 9, 11, 14, 15 en 19 te ondersoek vir hul moontlike bydrae tot die ontwikkeling van PFHBII. Toe die lokus gekarteer was, is bioinformatiese ondersoeke gebruik om posisionele kandidaat gene te identifiseer en prioritiseer vir mutasie analise. Die retrospektiewe en prospektiewe kliniese ondersoek het PFHBII herdefinieer as ‘n geleidingsstoornis en DKM-verbonde siekte, en terselfde tyd het dit gelei tot die opsporing van nog familielede. Toevallig het kandidaat loki-analise die PFHBII lokus op chromosoom 1q32 gekarteer, na ‘n gebied wat met ‘n voorheen-beskyfde DKM-verbonde stoornis (CMD1D) oorvleuel, met die opwekking van ‘n makisimum paargewyse lod-getal van 3.13 by D1S3753 (theta [θ] = 0.0) en ‘n maksimum multipunt lod-getal van 3.7 tussen D1S3753 en D1S414. Genetiese fynkartering en haplotipe-analise het die PFHBII-veroorsakende lokus afwaards van die CMD1D lokus geplaas, in ‘n 3.9 centimorgan (cM) gebied op chromosoom 1q32.2-q32.3, telomeries van D1S70 en sentromeries van D1S505. Bioinformatiese analise het daarnatoe gelei dat sewe kandidaat gene vir mutasie analise geprioritiseerd is, naamlik, gene wat onderskeidelik ‘n kalium kanaal (KCNH1), ‘n ekstrasellulêre matriksproteïen (LAMB3), ‘n proteïen fosfatase (PPP2R5A), ‘n aansluiter proteïen wat met ‘n sitoskilet proteïen bind (T3JAM), ‘n asieltansferase (KIAA0205) en twee gene moontlik betrokke in energie homeostase (RAMP en VWS59) enkodeer. Die PFHBII-veroorsakende geen is nie geïdentifiseer nie, alhoewel enkele volgorde-wisselings geïdentifiseer is in vier van die sewe geanaliseerde kandidaat gene. Alhowel die molekulêre oorsaak van die siekte nie vasgestel is nie, het die huidige studie die onderliggende betrokkenheid van DKM in die pathogenese van PFHBII gedefinieer. Die nuwe kliniese klassifikasie van PFHBII is gepubiliseer (Fernandez et al., 2004) en sal lei tot die identifisering van nog geaffekteerde persone in Suid Afrika of in ander lande. Die identifikasie van ‘n nuwe siekte-verbonde lokus mag lei tot die toekomstige identifikasie van ‘n nuwe DKM-verbonde genetiese oorsaak wat, opsig self, dalk insig kan gee in die molekulêre patofisiologie van hartversaking.

Thesis (PhD)--University of Stellenbosch, 2011. / ENGLISH ABSTRACT: The tuberculosis (TB) incidence has increased in Southern Africa and the situation is worsened by the emergence of drug-resistant Mycobacterium tuberculosis strains. Molecular biological techniques have been used to understand the disease dynamics of TB. In a series of studies we describe the use of these techniques to understand the disease dynamics of TB in Southern Africa. Using spoligotyping and IS6110-restriction fragment length polymorphism (RFLP) to characterize M. tuberculosis strains from TB patients in Zimbabwe, we identified a genotype causing a disproportionate number of TB cases. The genotype belonged to the Latin American Mediterranean (LAM) lineage and we named it the Southern Africa1 (SAF1) family and later renamed it SAF1/RDRio, also reflecting its predominance in South America. To establish if this family of strains was predominant elsewhere in Southern Africa, genotypes were compared to those from Western Cape, South Africa and Zambia. The SAF1/RDRio strains were highly prevalent in Zambia but were only a minor fraction of the strains in South Africa. The geographical distribution of SAF1/RDRio strains was determined in Gweru, Zimbabwe, and was found to be spread in high incidence areas. From these two studies it was hypothesized that certain host and bacterial factors were associated with disease due to SAF1/RDRio. Subsequently potential risk factors and clinical outcomes of disease due to SAF1/RDRio strains were explored. An association was found with smoking and cavitary pulmonary disease suggesting that SAF1/RDRio caused a more severe and highly transmissible formof TB Using IS6110-RFLP, principal genetic grouping, spoligotyping, IS6110 insertion-site mapping and variable-number tandem repeats (VNTR) typing, low IS6110 copy clade (LCC) identified in Zimbabwe were characterized and compared to the strains from Cape Town, South Africa and other regions. The LCC strains from Cape Town, South Africa, were found to have close evolutionary relationship with strains from Zimbabwe and other regions and were widely distributed suggesting they play an important role in the global TB epidemic. Observations from these studies and those from other studies led to the hypothesis that specific genotypes of M. tuberculosis predominate in regions of Southern Africa. To gain an insight on the population structure of M. tuberculosis strains in Southern Africa, spoligotyping and/or IS6110-RFLP data from eight countries were compared. This is the first study to describe the M. tuberculosis population structure in Southern Africa. Distinct genotypes were associated with specific geographic regions. These findings have important implications for TB diagnostics, anti-TB drug and vaccine development. The population structure of multidrug-resistant (MDR), pre-extensively drug-resistant (pre-XDR) and extensively drug-resistant (XDR) M. tuberculosis isolates from provinces in South Africa was also determined. This is again the first study to describe the population structure of drug-resistant M. tuberculosis in South Africa. The results also showed geographic localization of genotypes and an association with resistance class. However, decreasing strain diversity was observed as the isolates evolved from MDR-TB to XDR-TB suggesting selection for the specific genotypes. These findings highlight the importance of identifying genetic markers in drug-resistant strains, to enhance early detection of those at risk of developing XDR-TB. / AFIKAANSE OPSOMMING: Die voorkoms van tuberkulose (TB) in Suider Afrika word vererger deur stamme van Mycobacterium tuberculosis wat weerstandig is teen die beskikbare anti-tuberkulose middels. Molekulêre tegnieke word gebruik om in hierdie reeks studies die dinamika van TB in Suider Afrika te ondersoek Deur spoligotipering en IS6110 restriksie fragment lengte polimorfisme (RFLP) tegnieke te gebruik om M. tuberculosis stamme van pasiente in Zimbabwe te beskryf, het ons ‘n genotipe gevind wat ‘n buitengewone aantal TB gevalle veroorsaak het. Hierdie genotipe is deel van die internasionaal beskryfde Latyns Amerikaase en Meditereense (LAM) stam familie. Ons het dit die Suider Afrikaanse Familie1 (SAF1) genoem, maar later hernoem na SAF1/RDRio, omdat dieselfde genotipe in ook volop is in Suid Amerika. Om vas te stel of hierdie familie ook oorheesend is in die res van Suider Afrika, is dit vergelyk met beskikbare databasisse van die Wes-Kaap, Suid-Afrika en Zambië. Alhoewel SAF1/RDRio in die Wes-Kaap gevind is, dra dit slegs tot ‘n mindere mate by tot die plaaslike TB epidemie. Aan die anderkant kom SAF1/RDRio baie algemeen in Zambië voor. ‘n Verdere studie wys ook dat die SAF1/RDRio familie eweredig en wyd verspreid voorkom in hoë insidensie gebiede in Gweru, Zimbabwe. Vanuit die bevindings van hierdie 2 studies, kan ons aflei dat sekere gasheer- en bakteriële eienskappe geassosieer is met SAF1/RDRio-TB-infeksie. Hierna is potensiële risiko faktore en kliniese uitkomste van siekte as gevolg van infeksie met SAF1/RDRio ondersoek. ‘n Assosiasie met rook en kaviterende pulmonale infeksie is gevind,wat daarop dui dat SAF1/RDRio erger vorm van TB veroorsaak en hoogs oordraagbaar is. Deur gebruik te maak van IS6110- (RFLP), hoof groep groepering, spoligotipering, IS6110 invoegings kaartering en veranderlike getal tandem herhaling (VNTR) tipering kon lae IS6110 invoeginsgetal (LCC) stamme van Kaapstad, Zimbabwe en ander gebiede vergelyk word. Al die LCC stamme in die studie is evolusionêr naby verwant aan mekaar en is wyd verspreid, wat dui op hulle belangrike rol in die wêreldwye TB epidemie. Waarnemings in hierdie asook ander studies het tot die hipotese gely dat spesifieke genotipes van M. tuberculosis dominant is in verskillende gebiede van Suider Afrika. Om meer insig tot die populasie samestelling van M. tuberculosis stamme in Suider Afrika in te win is spoligotipes en RFLP-data van 8 lande vergelyk. Hierdie is die eerste studie om die populasie samestelling van M. tuberculosis in Suider Afrika te beskryf en is belangrike fir toekomstige ontwikkeling van nuwe TB diagnose tegnieke, anti-TB middels en TB entstowwe. Die populasie samestelling van multiweerstandige (MDR), pre-ekstreme weerstandige (pre-XDR) en ekstreme weerstandige (XDR) M. tuberculosis van verskillende provinsies in Suid-Afrika is ook bepaal. Hierdie studie is ook die eerste wat die populasie samestelling van weerstandige M. tuberculosis in Suid-Afrika beskryf. Die resultate wys geografiese lokalisering van genotipes en ‘n assosiasie met weerstandigheidsklas. ‘n Afname in stam diversiteit soos die isolate van MDR-TB tot XDR-TB ontwikkel, dui op seleksie van spesifieke genotipes. Hierdie bevinding lê die klem op die belangrikheid van die identifisering van genetiese merkers in weerstandige stamme om die risiko vir die ontwikkeling van XDR-TB te verminder deur vroë deteksie.

Thesis (PhD )--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: Diagnostic delay is regarded as a major contributor to the continuous rise in tuberculosis (TB) cases and the emergence and transmission of multidrug-resistant tuberculosis (MDR-TB) and extensively drug resistant tuberculosis (XDR-TB). It is therefore essential that more rapid diagnostic methods are developed. Molecular-based assays have the potential for the rapid species-specific diagnosis of TB and associated drug-resistances directly from clinical specimens. We investigated whether high resolution melting analysis (HRM) could enable the rapid diagnosis of TB and associated drug resistance, since the HRM apparatus and reagents are relatively inexpensive and the methodology can easily be implemented in high incidence, low income regions. Application of this methodology allowed for the rapid identification of mycobacterial lymphadenitis from fine-needle aspiration biopsy (FNAB) samples in 2 studies. This was done by targeting the region of deletion 9 (RD9), present in M. tuberculosis and M. canettii, but absent from all other members of the complex. However, the sensitivity of the method was low (51.9% and 46.3%, respectively) when compared to the reference standard (positive cytology and/or positive culture). Despite this limitation our method was able to provide a rapid diagnosis in more than half of the infected patients with a relatively high specificity (94.0% and 83.3%, respectively). We therefore proposed a diagnostic algorithm allowing the early treatment of patients with both HRM and cytology results indicative of mycobacterial disease. We developed the Fluorometric Assay for Susceptibility Testing of Rifampicin (FAST-Rif) which allowed the rapid diagnosis of MDR-TB by detecting rifampicin (RIF) resistance mutations in the rpoB gene with a sensitivity and specificity of 98% and 100%, respectively. The FAST-Rif method was easily adapted to detect ethambutol (EMB) resistance due to mutations in the embB gene with a sensitivity and specificity of 94.4% and 98.4% respectively, as compared to DNA sequencing. The FAST-EMB method was a significant improvement over the inaccurate culture based method. We identified a strong association between EMB resistance (and pyrazinamide resistance) and MDR-TB and subsequently advised modifications to the current (2008) South African National TB Control Programme draft policy guidelines. Due to the potential for amplicon release, we adapted the FAST-Rif and FAST-EMB methods to a closed-tube one-step method using the detection of inhA promoter mutations conferring isoniazid (INH) resistance as a model. The method (FASTest-inhA) was able to identify inhA promoter mutations with a sensitivity and specificity of 100% and 83.3%. These mutations are of particular interest as they confer low level INH resistance and cross-resistance to ethionamide (Eto). Since inhA promoter mutations are strongly associated with XDR-TB in the Western and Eastern Cape Provinces of South Africa, data generated by the recently implemented GenoType® MDRTBPlus assay may allow individualised treatment regimens to be designed for a patient depending on their INH mutation profile. Our proposed treatment algorithm may be particularly useful in XDR-TB cases, for which only few active drugs remain available. Since current diagnostic methods all carry advantages and disadvantages, a combination of phenotypic and genotypic-based methodologies may be the best scenario while awaiting superior methods. / AFRIKAANSE OPSOMMING: Die onvermoë om tuberkulose (TB), multi-weerstandige tuberkulose (MDR-TB) en uiters weerstandige tuberkulose (XDR-TB) vinnig te diagnoseer, is ‘n belangrike oorsaak vir die volgehoue toename en verspreiding daarvan. Dit is noodsaaklik dat diagnostiese toetse wat vinniger resultate oplewer, ontwikkel word. Molukulêre toetsing het die potensiaal om vinnig spesie-spesifieke diagnoses van TB en die weerstandigheid teen TB-medikasie te lewer. Hierdie studie wil vasstel of hoë-resolusie smeltingsanalise (HRS) ‘n vinnige diagnose van TB en die weerstandigheid teen TB-medikasie kan oplewer aangesien die relatiewe lae koste van reagense en apparaat, asook die minimale infrastruktuur en vaardighede wat vir dié toets benodig word, dit uiters geskik maak vir pasiënte in gebiede met ‘n hoë TB-insidensie en lae inkomste. Die toepassing van die HRS-metode op fynnaald-aspiraatbiopsies in twee afsonderlike studies, het gelei tot die vinnige identifisering van mikrobakteriële-limfadenitis. Dit is bemiddel deur die gebied van delesie 9 (RD9) teenwoordig in Mycobacterium tuberculosis en M. canettii, maar afwesig in al die ander lede van die kompleks, te teiken. Die sensitiwiteit van die metode was (51.9% en 46.3%, vir die twee studies onderskeidelik) in vergelyking met die verwysingstandaard (positiewe sitologie en/of positiewe kultuur). Ten spyte van dié beperking was ‘n vinnige diagnose in meer as die helfte van geïnfekteerde pasiënte met ‘n redelike hoë spesifisiteit (94.0% en 83.3%, onderskeidelik) moontlik. ‘n Diagnostiese algoritme wat gebaseer is op die resultate van die HRS en sitologie-toetse, is voorgestel om pasiënte vroeër te behandel. ‘n Fluorometriese toets (FAST-Rif) is ontwikkel vir die vinnige diagnose van MDR-TB deur mutasies in die rpoB-geen op te spoor met ‘n hoë sensitiwiteit en spesifisiteit (98% en 100%, onderskeidelik). Hierdie mutasies is verantwoordelik vir weerstandigheid teen die antibiotikum rifampicin (FAST-Rif) en word beskou as ‘n vinnige diagnose vir MDR-TB. Die FAST-Rif metode kon maklik aangepas word om mutasies in die embB-gene, verantwoordelik vir weerstandigheid teen die antibiotikum ethambutol (EMB), op te spoor. Die FAST-EMB-metode het ‘n sensitiwiteit en spesifisiteit van 94.4% en 98.4% onderskeidelik getoon in vergelyking met DNS volgordebepaling. Die FAST-EMB-metode was ‘n betekenisvolle verbetering op die onakkurate kultuurgebaseerde metodes. ‘n Sterk korrelasie tussen EMB-weerstandigheid (en weerstandigheid teen pyrazinamide) en MDR-TB is geïdentifiseer. Vervolgens is veranderinge aan die Suid-Afrikaanse Nasionale TB-beheerprogram se Konsepbeleidsgids (2008) voorgestel. Om die potensiële vrylating van amplikone te verhoed, is die FAST-Rif en FAST-EMB aangepas tot ‘n enkelstap geslote buissisteem deur gebruik te maak van die opsporing van inhA promotormutasies wat weerstandigheid teen isoniazid (INH) veroorsaak. Die metode het ‘n sensitiwiteit en spesifisiteit van 100% en 83.3% onderskeidelik, getoon. Hierdie mutasies veroorsaak laevlak weerstandigheid teen INH, maar ook kruisweerstandigheid teen ethionamide (Eto). Aangesien daar ‘n sterk verbintenis tussen inhA-promotormutasies en XDR-TB in die Oos en Wes-Kaapprovinsies van Suid-Afrika is, kan data van die GenoType® MDRTBPlus-toets moontlik gebruik word om ‘n meer geïndividualiseerde behandeling te ontwerp afhangende van die pasiënt se INH-mutasieprofiel. Ons behandelingsalgoritme is veral geskik vir XDR-TB pasiënte vir wie daar weinig aktiewe antibiotika beskikbaar is. Huidige diagnostiese metodes het almal voor- en nadele, dus bied ‘n kombinasie van fenotipiese en genotipiese metodes moontlik die beste oplossing totdat beter metodes ontwikkel word.