ABSTRACT In southern Africa, oculocutaneous albinism (OCA) is the most common autosomal recessive disease amongst black Africans, occurring with a prevalence of approximately 1 in 3 900 individuals. OCA occurs in southern African Caucasoids with a frequency that reflects the European origins of this population, approximately 1 in 20-30 000. OCA type 1 is caused by mutations in the tyrosinase gene on chromosome 11q. Tyrosinase mutations occur in the Caucasoid population but are extremely rare in black Africans. OCA type 2 is caused by mutations in the P gene on chromosome 15q. P gene mutations occur in both the black and Caucasoid populations. A sub-type of OCA2 seen in black individuals, brown OCA (BOCA), is also caused by mutations at the P gene locus. A mutation screen was undertaken to identify disease-causing mutations in a group of OCA subjects from Sub-Saharan Africa. A common P gene mutation had been identified in the black population, a 2.7 kb intragenic deletion, accounting for 78% of P gene mutations in this group. No common tyrosinase mutations have been identified to date, in any population. A cohort of OCA subjects from South Africa, Lesotho, Zambia and the Central African Republic (CAR) were available for study in our laboratory. All subjects were screened for the 2.7 kb deletion mutation. Subjects homozygous for this mutation were excluded from further study. Subjects where one or two mutations remained to be identified were included in a mutation screen (63 blacks and 9 Caucasoids). Depending on the clinical categorisation of the type of albinism, subjects were screened for P gene mutations only (black OCA2) or were screened for P gene mutations and tyrosinase mutations (BOCA, unclassified black OCA and unclassified Caucasoid OCA). All 72 subjects were screened for P gene mutations and ten putative pathogenic mutations were identified. In the group of black OCA2 patients, four mutations which are likely to be pathogenic were found: A334V, 614delA, 683insT and 727insG. Mutations were identified in four individuals with an unusual hypopigmentation phenotype: E678K was found in the homozygous state in an individual from the CAR. A second individual was found to be a compound heterozygote for the I370T and the L688F mutations. A third individual was found to be heterozygous for the I370T mutation. Three P gene mutations were found in the Caucasoid sample: IVS 14-2 (a→g), V350M and P743L. No further mutations were identified in the BOCA sample. The P gene screen comprised 72 subjects, but 40 were heterozygous for the 2.7 kb deletion, therefore (144 minus 40 alleles) 104 alleles remained to be identified. Identification of 12/104 alleles means that a further 11.5% of the unknown P gene mutations are now accounted for. Thirty three of the 72 subjects were included in a further mutation screen – at the tyrosinase locus. Four mutations were identified, all in the Caucasoid group. Compound heterozygosity was shown in two individuals, one carrying the mutations, E294K and A490D and the other, P431T and T373K. Following mutation analysis of the P gene, it was apparent that a proportion of mutations did not lie in the coding region of the gene and it was proposed that some of the remaning unidentified mutations might be found in the 5’control or promoter region of the gene. At that time, sequence data for the region upstream of the P gene was not known, and so an attempt was made to clone the 5’region of the P gene. Two approaches were adopted – a bacterial artificial chromosome (BAC) known to contain this region was subcloned; and secondly, an inverse PCR experiment was undertaken. Neither experiment was successful in generating P gene promoter sequence. Variation at the P locus was investigated in a second context. This region of chromosome 15q was implicated as a host susceptibility locus for the infectious disease, tuberculosis (TB). A case-control study was undertaken to compare the frequencies of five intragenic, polymorphic markers in the P gene: the 2.7 kb deletion, the R305W polymorphism and the microsatellite markers, D15S1533, D15S1536 and D15S1537, between a group of black South African TB patients from Gauteng and healthy community controls and between a group of mixed-ancestry (Coloured) TB patients and healthy controls from the Western Cape region. Presence or absence of the 2.7 kb deletion mutation does not appear to influence susceptibility to TB in either the black or Coloured population samples studied here. The W allele of the R305W polymorphism is significantly (p<0.05) more common in the black patient group than in the black control group, suggesting it may be in linkage disequilibrium with a disease susceptibility allele. Microsatellite marker analysis showed that, in the black population, allele 18 at the D15S1533 locus is significantly (p<0.05) associated with susceptibility to TB. In the Cape Coloured population, alleles 20 and 27 at the D15S1533 locus, allele 12 at the D15S1536 locus and allele 16 at the D15S1537 locus are over represented in the patient group suggesting they may be markers for increased susceptibility to TB. Further, in the Coloured population alleles 12, 13 and 15 at the D15S1537 locus showed significant (p<0.05) association with normal controls and may be in linkage disequilibrium with protective or resistance alleles. The results of this study support the proposal of a TB susceptibility locus on chromosome 15q. OCA-causing mutations have been identified, but many remain elusive. Further characterisation of this region will give us a better understanding of the biological consequences of variation both within and around the P locus.

Thesis (Masters Diploma (Medical Technology))-- Cape Peninsula University of Technology, 1992 / Studies that document the higher incidence of tuberculosis as well as the variable efficacy of the BCG vaccine in Black, compared to White, populations have alluded to resistance or susceptibility to tuberculosis being genetically controlled.The HIA system has been associated with many diseases involving an immune aetiology. It has been shown that T cell receptor genes have limited restriction fragment length polymorphisrns, serving to create a variation in the repertoire of expressed T cell receptor genes. These repertoire differences may play a fundamental role in disease susceptibility.A study was therefore undertaken to establish whether linkage exists between the HIA system or the T cell receptor genes and a putative susceptibility gene for tuberculosis.Polymorphisrns of these genetic markers were examined in three Cape Coloured multiplex families, affected individuals having culture-positive pulmonary tuberculosis.HLA haplotypes were derived from serological typing of peripheral leucocytes from each individual. B-Iymphoblastoid cell lines were established from each family member. DNA was then extracted and digested with a variety of restriction endonucleases. After gel electrophoresis and Southern blotting, the DNA fragments were probed with a panel of T cell receptor cDNA probes, revealing the allelic polymorphisms.Linkage analysis was done using the Liped computer programme and Lad scores were determined for each marker locus using various genetic models. Haplotypes were also established for the T cell receptor genes and used in the linkage analysis.Although most of the Lad scores fell within the indeterminate range, a cumulative Lod score of 1.79 was obtained from the allele generated by the EcoRV/a2 enzyme/probe combination under a recessive model with 50% penetrance. This represents odds of about 52: 1 in favour of linkage between the T cell receptor a gene and a putative susceptibility gene to tuberculosis.

Thesis (MScMedSc (Biomedical Sciences))--University of Stellenbosch, 2010. / Thesis presented in partial fulfilment of the requirements for the degree Master of Medical Science (Human Genetics) at the University of Stellenbosch. / Bibliography / ENGLISH ABSTRACT: The infectious disease tuberculosis (TB) remains the leading cause of death worldwide by a single infectious agent, despite significant advances in biomedical sciences. The idea that host genetics plays a role in the development of disease was proposed by Haldane in 1949. The observation that only 10% of immunocompetent individuals develop disease while others are able to successfully contain it, further suggests that host genetics plays an important role. TB is thus a complex disease, with the causative bacterium, Mycobacterium tuberculosis, host genetic factors and environment all contributing to the development of disease. To date several genes have been implicated in TB susceptibility, albeit with small effect. Genome-wide association studies (GWAS) offer the means to identify novel susceptibility variants and pathways through their ability to interrogate polymorphisms throughout the genome without being limited by our understanding of the immune processes involved in TB infection and disease progression. TB and sarcoidosis are both granulomatous diseases, and we therefore hypothesized that the genes and their associated variants identified in recent GWAS conducted in West Africa for TB, and Germany for sarcoidosis, could alter susceptibility to TB in the South African Coloured (SAC) population. In the sarcoidosis GWAS, ANXA11 was shown to alter susceptibility to sarcoidosis; whereas in the TB GWAS, CADM1 was found to alter susceptibility to TB. This study tested the association with TB of 16 polymorphisms in 5 potential TB host susceptibility genes in the SAC population. A well designed case-control study was employed, using the TaqMan® genotyping system to type the various polymorphisms. Any polymorphism that was found to be significantly associated with susceptibility to TB was then subjected to further analysis to determine the functional effect of the polymorphism. Promoter methylation patterns were also investigated in ANXA11 as another mechanism to elucidate its role in TB susceptibility. A 3’ UTR ANXA11 polymorphism was found to be strongly associated with susceptibility to TB, including 3 haplotypes. The gene expression analysis identified differential transcriptional levels between individual with the different genotypes, with individuals homozygous for the A-allele exhibiting a 1.2-fold increase in gene expression relative to those homozygous for the G-allele. Methylation analysis however found no differences between cases and controls. In addition, 16 novel polymorphisms were also identified, 15 of which occurred in the 3’UTR of ANXA11. The mechanism of action of ANXA11 in TB susceptibility is hypothesised to be in the area of endocytosis, autophagy or apoptosis. A weak association was noted with one of the 5’ UTR polymorphisms of CADM3, which did not hold up to further analysis in the GWAS study, and no functional work was therefore done. This work facilitates our understanding of the role of host genetics in susceptibility to TB and adds to the growing amount of information available. Proper understanding of the role that host genetics plays in TB susceptibility could result in better treatment regimens and prediction of individuals who are at a greater risk of developing TB, a disease that still kills millions of individuals annually. / AFRIKAANSE OPSOMMING: Tuberkulose is verantwoordelik vir meer sterftes as enige ander aansteeklike siekte, ten spyte van die voortuitgang wat die Biomediese Wetenskappe tans beleef. In 1949 het Haldane voorgestel dat die genetiese samestelling van die gasheer ‘n rol speel in vatbaarheid vir aansteeklike siektes. Vir tuberkulose word hierdie aanname gesteun deur die feit dat slegs 10% van individue wat geïnfekteer word aktiewe simptome ontwikkel, terwyl 90% die siekte suksesvol sal afweer. Tuberkulose is dus ‘n komplekse siekte wat veroorsaak word deur Mycobacterium tuberculosis, maar wat beïnvloed word deur genetiese sowel as omgewingsfaktore. Verskeie gene is al geïdentifiseer wat ‘n rol speel in vatbaarheid vir tuberkulose, tog is hul invloed betreklik klein. Genoom-wye assosiasiestudies (GWAS) bied unieke geleenthede vir die identifisering van nuwe polimorfismes wat genetiese vatbaarheid kan beïnvloed. Hierdie tegniek kan die hele genoom fynkam, sonder dat enige vooropgestelde idees oor die immuunrespons teen tuberkulose ‘n invloed sal hê. Tuberkulose en sarkoïdose is albei siektes wat die vorming van granulomas veroorsaak. Verskeie gene met hul geassosieerde variante is geïdentifiseer in ‘n onlangse GWAS, wat gefokus het op populasies in Wes-Afrika en Duitsland. Ons hipotese was dat die polimorfismes wat in hierdie studie geïdentifiseer is, ‘n invloed kan hê op genetiese vatbaarheid vir TB in die Suid-Afrikaanse Kleurlingbevolking (SAK). Die sarkoïdose GWAS het bevind dat ANXA11 vatbaarheid vir die siekte beïnvloed, terwyl CADM1 in die tuberkulose GWAS geïdentifiseer is. Die studie het die assosiasie tussen 16 variante en tuberkulose vatbaarheid ondersoek in die SAK populasie. Die variante strek oor 5 potensiële tuberkulose vatbaarheidsgene. Goedbeplande pasiënt-kontrole assosiasiestudies is gedoen en die polimorfismes is gegenotipeer deur gebruik te maak van die TaqMan® genotiperingsisteem. Enige polimorfisme wat beduidend met tuberkulose geassosieer was, is verder geanaliseer om die moontlike funksionele invloed daarvan te bepaal. Promotormetileringspatrone van ANXA11 is ook geanaliseer, om ‘n addisionele meganisme in tuberkulose vatbaarheidheid te ondersoek. Na genotipering van die polimorfismes is ‘n 3’ UTR ANXA11 variant geïdentifiseer wat beduidend met tuberkulose vatbaarheid geassosieer was. Drie haplotipes is ook geïdentifiseer. Geenuitdrukkingsanalise het aangedui dat verskille in transkripsie vlakke voorkom in individue met verskillende genotipes. Individue wat homosigoties was vir die A-alleel het ‘n verhoging van 1.2 in geenuitdrukking gehad, relatief tot individue wat homosigoties was vir die G-alleel. Metileringsanalise het egter geen verskil aangedui tussen pasiënte en kontroles nie. Addisioneel, is 16 nuwe variante ontdek, waarvan 15 in die 3’UTR van ANXA11 geleë was. Die meganisme waarmee ANAX11 genetiese vatbaarheid vir tuberkulose beïnvloed, blyk in die area van endositose, apoptose of outofagie, te wees. ‘n Swak assosiasie is gevind vir ‘n 5’ UTR variant van CADM3 en is nie verder opgevolg in die GWAS nie. Gevolglik is geen funksionele studies op hierdie polimorfisme gedoen nie. Hierdie studie dra by tot ons kennis oor die rol wat die genetiese samestelling van die gasheer speel in vatbaarheid vir tuberkulose. Indien die rol van mensgenetika in tuberkulose vatbaarheid korrek verstaan word, kan behandeling van die siekte verbeter word en kan individue wat ‘n hoër risiko loop om tuberkulose te ontwikkel geïdentifiseer word.

Thesis (PhD)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Tuberculosis (TB) disease results in approximately 2 million deaths annually and is the leading cause of death due to a single infectious agent. Previous studies have indicated that host genetics play an important role in the development of TB. This together with pathogen and environmental factors intensifies the complexity of this disease. The Major Histocompatibility Complex (MHC) and Leukocyte Receptor Complex (LRC) comprise several genes which are known to be important modulators of the host immune response. The human leukocyte antigen (HLA) class-I genes of the MHC are involved in the presentation of pathogenic antigens on the surfaces of infected cells, while the killer cell immunoglobulin-like receptors (KIRs) of the LRC are involved in the recognition of self and non-self cells. Natural Killer (NK) cells through their KIRs are thus able to kill non-self cells through recognition of the class-I molecules expressed. Additionally, HLAs and KIRs are extremely polymorphic and differ markedly across populations of different ethnicities. Here we studied these genes and their polymorphisms in the South African Coloured (SAC) population to determine their involvement in susceptibility to TB, susceptibility to disease caused by specific Mycobacterium tuberculosis subtypes, and understanding their ancestral contribution to the SAC with regards to the development of TB. We showed that the KIR3DS1 gene and KIR genotypes with five or more activating KIRs, and the presence of 3DS1, protected against the development of active TB in the SAC population. Several HLA class-I alleles were identified as susceptibility factors for TB disease. With regards to genes of the MHC and LRC, several loci were found to alter susceptibility to TB in the SAC population, including MDC1, BTNL2, HLA-DOA, HLA-DOB, C6orf10, TAP2, LILRA5, NCR1, NLRP7 and the intergenic regions between HLA-C/WASF5P and LAIR1/TTYH1. We showed that the Beijing strain occurred more frequently in individuals with multiple disease episodes, with the HLA-B27 allele lowering the odds of having an additional episode. Associations were identified for specific HLA types and disease caused by the Beijing, Latin America-Mediterranean (LAM), Low-Copy Clade (LCC), and Quebec strains. HLA types were associated with disease caused by strains from the Euro-American or East Asian lineages, and the frequencies of these alleles in their sympatric human populations identified potential co-evolutionary events between host and pathogen. Finally, we showed that the SAC population is the most diverse SA population with regards to HLA alleles and KIR genotypes, as would be expected given the admixture of the SAC. Based on the HLA allele class-I profiles across SA populations, we noted that the Ag85BESAT- 6, Ag85B-TB10.4 and Mtb72f vaccines currently undergoing clinical trials would have low efficacy across most SA populations. We showed that the MHC and LRC regions in SAC healthy controls are predominantly of European ancestry, and that SAC TB cases are more closely related to Khoisan and black SA population groups. Our work highlights the importance of investigating both host and pathogen genetics when studying TB disease development and that understanding the genetic ancestral contributions to the SAC population can contribute to the identification of true and novel TB causing variants. / AFRIKAANSE OPSOMMING: Tuberkulose (TB) is jaarliks verantwoordelik vir ongeveer 2 miljoen sterftes en is die hoofoorsaak van dood as gevolg van „n aansteeklike siekte. Vorige navorsingstudies het aangedui dat die genetiese samestelling van die gasheer „n beduidende rol speel in die ontwikkeling van TB. Die kompleksiteit van hierdie siekte word vererger deur die betrokkenheid van die gasheer genoom sowel as bakteriële en omgewings faktore. Die Major Histocompatibility Complex (MHC) en Leukocyte Receptor Complex (LRC) bestaan uit verskeie gene wat die gasheer immuunrespons verstel. Die human leukocyte antigen (HLA) klas I gene van die MHC is betrokke by die aanbieding van patogeniese antigene op die oppervlak van geïnfekteerde selle, terwyl die killer cell immunoglobulin-like receptors (KIRs), geleë in die LRC, betrokke is by die herkenning van eie en vreemde selle. NK selle, deur middel van hul KIRs, kan dus vreemde selle uitwis aangesien hulle die uitgedrukte klas I molekules kan herken. Beide HLA en KIRs is hoogs polimorfies en verskil beduidend tussen etniese groepe. In hierdie studie is die bogenoemde gene en hul polimorfismes in die Suid Afrikaanse Kleurling bevolking (SAC) ondersoek om vas te stel tot watter mate dit genetiese vatbaarheid vir TB, asook vatbaarheid vir TB wat deur spesifieke Mycobacterium tuberculosis subtipes veroorsaak word, beïnvloed. Daar is ook gepoog om te verstaan hoe die voorouerlike bydrae van hierdie gene die SAC met betrekking tot TB vatbaarheid affekteer. Die resultate van die studie het aangedui dat die KIR3DS1 geen en KIR genotipes met vyf of meer aktiewe KIRs en die teenwoordigheid van 3DS1, die SAC bevolking beskerm teen die ontwikkeling van aktiewe TB. Verskeie HLA klas I allele is geïdentifiseer as vatbaarheidsfaktore vir TB. Talle lokusse van die MHC en LRC gene is ook as vatbaarheidsfaktore vir TB in die SAC bevolking geïdentifiseer, insluitende MDC1, BTNL2, HLA-DOA, HLA-DOB, C6orf10, TAP2, LILRA5, NCR1, NLRP7 en die intergeniese areas tussen HLA-C/WASF5P en LAIR1/TTYH1. Die studie het aangedui dat die Beijing stam meer voorkom in individue wat verskeie kere TB gehad het en dat die HLA-B27 alleel die kanse om „n verdere episode te hê, verlaag het. Assosiasies is geïdentifiseer tussen spesifieke HLA tipes en siekte veroorsaak deur die Beijing, LAM, LCC, en Quebec TB stamme. HLA tipes was geassosieer met siekte veroorsaak deur TB stamme van Euro-Amerikaanse en Oos-Asiëse afkoms. Die frekwensies van hierdie allele, in hul ooreenstemmende mensbevolkings, dui op „n potensïele koevolusionêre gebeurtenis tussen die gasheer en patogeen. Die studie het ook vasgestel dat die SAC populasie die mees diverse SA bevolking is met betrekking tot die HLA allele en KIR genotipes, soos verwag sou word gegewe die gemengde genetiese herkoms van die SAC. Gebaseer op die HLA allele klas I profiel van verskillende SA bevolkings merk ons op dat die Ag85B-ESAT-6, Ag85B-TB10.4 en Mtb72f vaksiene, wat huidiglik kliniese toetsing ondergaan, nie so effektief in die meeste SA bevolkings sal wees nie. Die studie het ook bewys dat die MHC en LRC streke in gesonde SAC kontroles, grootliks afkomstig was van „n Europese nalatenskap en dat die SAC TB gevalle meer verwant is aan die Khoisan en swart SA bevolkings. Hierdie studie beklemtoon die noodsaaklikheid om beide gasheer en patogeen genetika te bestudeer wanneer die ontwikkeling van TB ondersoek word en dat die verstaan van die genetiese voorouerlike bydrae van die SAC bevolking kan bydra tot die identifisering van ware en nuwe TB-veroorsakende variante.

In der vorliegenden Arbeit wurden neue Untersuchungsmöglichkeiten mit dem BacT/Alert 3D Mykobakterien-Testsystem erprobt. Erstens wurden Untersuchungen durchgeführt, um die Testkonzentrationen für Protionamid (PTH) und Linezolid (LIZ) für die standardmäßige Empfindlichkeitstestung von M. tuberculosis (Mtb) mit dem BacT/Alert 3D-System festzulegen. Dazu wurden die MHK-Werte für 32 Mtb-Stämme bestimmt: Referenzstamm Mtb H37Rv, sensible Patientenstämme, Patientenstämme mit verschiedenen Resistenzen (u. a. PTH-Resistenz) sowie eigens für die Arbeit isolierte LIZ-resistente Mutanten. Die PTH-MHK betrug für 20 von 21 sensiblen Mtb-Stämmen einschließlich des Referenzstammes Mtb H37Rv 0,125 - 1 mg/l (0,25 mg/l bei 11 von 21 Stämmen). Lediglich ein Stamm mit Resistenz gegenüber Isoniazid, Ethambutol und Streptomycin fiel mit einer etwas erhöhten PTH-MHK von 2 mg/l auf. Sechs PTH-resistente Stämme (z. T. mit anderen Resistenzen gegenüber Erstrang-Antituberkulotika) zeigten PTH-MHK von 4 - 16 mg/l. Die Gruppen der PTH-sensiblen und resistenten Stämme zeigten ein bimodales Verteilungsmuster, das mit einem Schwellenwert von 2 mg PTH/l gut zu differenzieren ist. Für die standardmäßige Durchführung der Empfindlichkeitstestung gegenüber PTH mit dem BacT/Alert 3D-System empfehlen wir deshalb eine PTH-Testkonzentration von 2 mg/l. Die LIZ-MHK betrug für 20 sensible Mtb-Stämme (inklusive Referenzstamm Mtb H37Rv) und sieben Stämme mit verschiedenen Resistenzen gegenüber Erstrang-Antituberkulotika 0,25 - 2 mg/l (0,5 mg/l bei 17 von 27 Stämmen). Für die vier isolierten LIZ-resistenten Mutanten betrug die LIZ-MHK 8 - 16 mg/l. Es zeigt sich auch bei der Verteilung der LIZ-MHK ein bimodales Verteilungsmuster; die Gruppen der sensiblen und resistenten Stämme sind gut zu differenzieren. Wir empfehlen für die standardmäßige Durchführung der Empfindlichkeitstestung gegenüber LIZ mit dem BacT/Alert 3D-System eine LIZ-Testkonzentration von 4 mg/l. Die festgestellten MHK-Werte von PTH und LIZ und die vorgeschlagenen Testkonzentrationen entsprechen Ergebnissen aus der Literatur, die mit ähnlichen Methoden erhoben wurden. Zweitens wurden mit dem BacT/Alert 3D-System Untersuchungen zur Kombinationstestung von Antituberkulotika bei Mtb und Stämmen des MAC-Komplexes durchgeführt, bisher liegen keine Publikationen für Untersuchungen von Wirkstoff-Kombinationen bei Mykobakterien mit diesem System vor. Es wurde geprüft, ob die MHK eines Antituberkulotikums durch die Zugabe einer subinhibitorischen Menge eines anderen Antituberkulotikums verändert wird. Bei Mtb wurden dazu folgende Kombinationen geprüft: Rifampicin (RMP) + LIZ, Moxifloxacin + LIZ, Isoniazid + PTH, RMP + PTH, PTH + LIZ. In keinem Fall konnten signifikante Effekte beobachtet werden. Ein tendenziell synergistischer Effekt der PTH-RMP-Kombination beim Stamm Mtb H37Rv (Reduktion der RMP-MHK um eine Stufe) wurde durch die Analyse der Wachstumskinetik des Stammes unterstützt. Bei zufällig ausgewählten Stämmen des MAC-Komplexes wurde die Kombination Ciprofloxacin (CIP) + Ethambutol (EMB) geprüft. Es zeigte sich bei sieben von zehn Stämmen eine Reduzierung der CIP-MHK um mindestens drei Stufen bei Zugabe einer subinhibitorischen Konzentration von EMB. Dieser synergistische Effekt wurde bereits in den 1990er Jahren mit einer ähnlichen Methode festgestellt, allerdings ohne die Stämme des MAC-Komplexes zu differenzieren (Arbeitsgruppe von S. Hoffner). Interessanterweise handelte es sich bei den von uns untersuchten Stämmen, bei denen dieser synergistische Effekt nachgewiesen wurde, um M. avium-Stämme. Diese Problematik sollte weiter verfolgt werden, da sich daraus Konsequenzen für die Therapieempfehlung ergeben könnten.