published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

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 / abstract / Paediatrics and Adolescent Medicine / Master / Master of Philosophy

Wang, Xingyan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 126-131). / Abstracts in English and Chinese. / ACKNOWLEDGEMENT --- p.I / ABBREVIATIONS --- p.II / ABSTRACT --- p.V / 摘要 --- p.VIII / Chapter CHAPTER 1 --- INTRODUCTION --- p.1 / Chapter 1.1 --- CLINICAL DISEASE CAUSED BY M.TB --- p.1 / Chapter 1.1.1 --- Tuberculosis (TB) --- p.1 / Chapter 1.1.2 --- Pathogen: Mycobacteria tuberculosis (M. TB) --- p.2 / Chapter 1.2 --- HOST DEFENSE AGAINST M.TB --- p.4 / Chapter 1.2.1 --- Overview --- p.4 / Chapter 1.2.2 --- Specific pathways --- p.6 / Chapter 1.3 --- GENETIC PREDISPOSITION OF HOST TO INFECTION --- p.12 / Chapter CHAPTER 2 --- OVERVIEW AND AIM OF THIS PROJECT --- p.14 / Chapter 2.1 --- GWAS REPLICATION --- p.14 / Chapter 2.2 --- CANDIDATE GENES REVEALED IN GWAS OF OTHER GRANULOMATOUS INFLAMMATORY DISEASES (GLD) --- p.14 / Chapter 2.3 --- CHROMOSOME 17 CHEMOKINE CLUSTER REGION --- p.15 / Chapter CHAPTER 3 --- REPLICATION STUDY OF TB GWAS --- p.16 / Chapter 3.1 --- INTRODUCTION --- p.16 / Chapter 3.1.1 --- TB GWAS study --- p.16 / Chapter 3.1.2 --- Aims of this part --- p.16 / Chapter 3.2 --- MATERIAL AND METHODS --- p.17 / Chapter 3.2.1 --- Case and control samples --- p.17 / Chapter 3.2.2 --- DNA extraction --- p.18 / Chapter 3.2.3 --- Genotyping of the SNPs --- p.19 / Chapter 3.2.4 --- Statistical analysis --- p.21 / Chapter 3.3 --- RESULTS --- p.23 / Chapter 3.3.1 --- Description of studied samples --- p.23 / Chapter 3.3.2 --- Results of case-control study for replication studies of TB GWAS --- p.23 / Chapter 3.4 --- DISCUSSION --- p.28 / Chapter CHAPTER 4 --- GENETIC VARIANTS IN GRANULOMATOUS INFLAMMATORY DISEASES --- p.32 / Chapter 4.1 --- INTRODUCTION --- p.32 / Chapter 4.1.1 --- Granulomatous inflammation --- p.32 / Chapter 4.1.2 --- Diseases characterized by granulomatous inflammatory --- p.34 / Chapter 4.1.3 --- Shared immune mechanisms in GiDs --- p.38 / Chapter 4.1.4 --- Genome-wide Association Studies (GWAS) in GiD --- p.38 / Chapter 4.1.5 --- Hypothesis of this part --- p.41 / Chapter 4.2 --- MATERIAL AND METHODS --- p.43 / Chapter 4.2.1 --- Case and control samples --- p.43 / Chapter 4.2.2 --- DNA extraction --- p.44 / Chapter 4.2.3 --- Tag SNP selection --- p.44 / Chapter 4.2.4 --- Genotyping of tagging SNPs --- p.45 / Chapter 4.2.5 --- Statisitical analysis --- p.45 / Chapter 4.3 --- RESULTS --- p.55 / Chapter 4.3.1 --- Description of TB case samples --- p.55 / Chapter 4.3.2 --- Primary endpoint case-control results --- p.56 / Chapter 4.3.3 --- Secondary endpoint case-only studies results --- p.67 / Chapter 4.3.4 --- Haplotype analysis --- p.78 / Chapter 4.4 --- DISCUSSION --- p.83 / Chapter 4.4.1 --- ATG16L1 gene with TB susceptibility --- p.83 / Chapter 4.4.2 --- Associations in case-only studies (Interaction effects) --- p.83 / Chapter 4.4.2.1 --- Age and pathogenesis of TB --- p.83 / Chapter CHAPTER 5 --- STUDIES IN THE CHEMOKINE-GENE CLUSTER AND A MIRNA SNP STUDY --- p.89 / Chapter 5.1 --- INTRODUCTION --- p.89 / Chapter 5.1.1 --- Genetic susceptibility to TB in familial cases --- p.89 / Chapter 5.1.2 --- Familial studies suggested linkage at 17qll.2 --- p.89 / Chapter 5.1.3 --- Chemokines --- p.90 / Chapter 5.1.4 --- Studies of SNP rs2910164 of microRNA-146a (miRNA-146a) --- p.91 / Chapter 5.2 --- MATERIAL AND METHODS --- p.92 / Chapter 5.2.1 --- Case and control samples --- p.92 / Chapter 5.2.2 --- DNA extraction --- p.92 / Chapter 5.2.3 --- TagSNP selection --- p.92 / Chapter 5.2.4 --- Genotyping of tagging SNPs --- p.93 / Chapter 5.2.5 --- PCR-RFLP --- p.93 / Chapter 5.2.6 --- Statistical analysis --- p.94 / Chapter 5.3 --- RESULTS --- p.100 / Chapter 5.3.1 --- PCR-RFLP results of the three SNPs --- p.100 / Chapter 5.3.2 --- Description of TB case samples --- p.102 / Chapter 5.3.3 --- Primary endpoint case-control results --- p.103 / Chapter 5.3.4 --- Secondary endpoint case-only studies results of CCL genes --- p.109 / Chapter 5.4 --- DISCUSSION --- p.120 / Chapter 5.4.1 --- Genetic association of SNPs with severity of TB --- p.120 / Chapter 5.4.2 --- Smoking and immunity --- p.121 / Chapter CHAPTER 6 --- FINAL CONCLUSION AND PROSPECT FOR FUTURE WORK --- p.122 / Chapter 6.1 --- CONCLUSION --- p.122 / Chapter 6.2 --- LIMITATION OF THE STUDIES --- p.124 / Chapter 6.3 --- FUTURE WORKS AND PROSPECT --- p.125 / REFERENCES --- p.126

published_or_final_version / Paediatrics / Master / Master of Philosophy

Thesis (PhD)--University of Stellenbosch, 2002. / ENGLISH ABSTRACT: This study exploits the Mycobacterium tuberculosis H37Rv genome sequence data in the context of M. tuberculosis clinical isolates, to elucidate genetic variation, and examine the phenotypic and molecular epidemiological implications thereof. The study was initiated by investigation of the insertion sequence IS6110, the primary DNA fingerprinting probe for the molecular epidemiology of tuberculosis. The transposable element is present in variable copy number and chromosomal location in clinical isolates of M. tuberculosis strains, giving rise to extensive genetic diversity. At the inception of this study, little was known about this element in terms of the genetic identity of its surrounding regions, its chromosomal distribution, and the mechanisms contributing to genetic diversity. These shortcomings were therefore addressed by a number of approaches. Firstly, to establish their genetic identity and chromosomal distribution, IS6110 insertion sites from clinical isolates of M. tuberculosis were cloned and sequenced. This data was examined in conjunction with available genome sequence data. The results demonstrated that the majority of insertions occurred within coding regions. Furthermore, the element was shown to have a non-random chromosomal distribution, and a number of preferential integration sites were identified. Secondly, the stability of chromosomal domains flanking IS611 0 elements was investigated by utilizing the insertion site clones as hybridization probes against clinical isolates. This allowed the identification of extensive genetic variation associated with these chromosomal domains, arising from IS6110 transpositions, deletions and point mutations. These events were expressed in terms of a phylogenetic tree which demonstrated ongoing genome evolution associated with IS6110. Thirdly, to investigate the hypothesis that IS6110-mediated deletions occur via homologous recombination between adjacent elements, deletion junctions were mapped and sequenced in clinical isolates representing predecessor and descendant strains. While these results support the involvement of IS6110 as a mediator of genetic deletion, they suggest either alternative mechanisms or the existence of unidentified intermediates. The investigation of IS6110 flanking regions identified the disruption of a number of members of the PPE gene family, leading to the second main area of investigation. The PPE gene family was newly identified as a result of the M. tuberculosis genome sequencing project, and its products are speculated to be of antigenic importance. However, at the commencement of this study very little data was available regarding the biological role of PPE proteins. Therefore, to explore the phenotypic implications of PPE gene disruption, various aspects of the gene family were investigated. Firstly, phylogenetic relationships between members of the PPE family were elucidated, which suggested an evolutionary progression, and highlighted the possibility that there may be functional subdivisions within the gene family. Secondly, the extent and mechanisms of PPE gene variation were analyzed by a combination of hybridization, peR and sequence analysis. This approach revealed extensive variation associated the gene family, although different members of the family exhibit different levels of variation. Of special interest was the discovery that long tandem repeat regions (~69 bp) found within 3 members of the gene family demonstrate variation in the numbers of these tandem repeats. A third avenue of investigation focused on in vitro and in vivo PPE gene expression profiles. RT- , peR was utilized to demonstrate in vitro expression of PPE genes, while RNA:RNA in situ hybridization demonstrated the expression of PPE genes in human tissue samples. Intriguingly, in situ hybridization suggests that there is variable PPE gene expression within the human granuloma. The final approach reported here focused on the subcellular localization of one member of the PPE family, Rv1917c. A combination of cell fractionation and whole-cell antibody binding experiments suggest that the Rv 1917c protein is a cell wallassociated, surface exposed molecule. In summary, the results obtained have potential implications for the interpretation of molecular epidemiological data, support the role of IS6110 as an agent of genome evolution, and emphasize the potential for IS6110 to impact on strain phenotype. Investigation of the PPE family demonstrated that this gene family contributes to genetic variation, is expressed in vitro and in vivo and that at least one protein encoded by the gene family is cell wall associated. Together, the results obtained support the hypothesis that selected members of the PPE gene family may encode products involved in antigenic variation. / AFRIKAANSE OPSOMMING: Dié studie maak gebruik van die Mycobacterium tuberculosis H37Rv genoom volgorde data in die konteks van M. tuberculosis kliniese isolate, om genetiese variasie toe te lig en die fenotipiese en molekulêre epidemiologiese implikasies daarvan te ondersoek. Die studie het 'n aanvang geneem deur die ondersoek van die inset-volgorde /S6110, wat die primêre DNS vingerafdruk pylfragment vir die molekulêre epidemiologie van tuberkulose is. Hierdie transponerende element is in wisselende kopiegetal en chromosomale posisies teenwoordig in kliniese isolate van M. tuberculosis stamme, en gee so oorsprong aan omvangryke genetiese afwisseling. Met die aanvang van hierdie studie was min bekend omtrent hierdie element betreffende die genetiese identiteit van die areas wat die insetsels omring, die chromosomale distribusie van insetsels, asook die meganismes wat bydra tot genetiese afwisseling. Hierdie gebreke is dus deur 'n aantal benaderings aangespreek. Eerstens is IS6110 insettingsetels van kliniese M. tuberculosis isolate gekloneer en hul nukleotiedvolgorde bepaal om sodoende hul genetiese identiteit en chromosomale verspreiding vas te stel. Hierdie data is in oorleg met beskikbare genomiese volgorde data geanaliseer. Die resultate het gewys dat die meerderheid van insetsels binne koderende gebiede plaasgevind het. Verder is gewys dat hierdie element nie na willekeur deur die chromosoom versprei is nie, en 'n aantal gebiede waar insetting by voorkeur plaasvind, is geïdentifiseer. Tweedens is die stabiliteit van die chromosomale gebiede wat IS6110 elemente flankeer ondersoek deur die insettingsetel klone as pylfragmente te gebruik in hibridisasie van kliniese isolate. Dit het die identifisering van omvangryke genetiese afwisseling binne hierdie chromosomale gebiede, wat ontstaan deur IS611 0 transposisies, delesies en puntmutasies, tot gevolg gehad. Hierdie afwisselings is uitgedruk as 'n filogenetiese boom waarin die voortdurende genomiese evolusie wat geassosieer word met IS6110 gewys word. Derdens, om die teorie dat IS6110-gedrewe delesies deur middel van homoloë rekombinasie tussen naasliggende elemente plaasvind te ondersoek, is die grense van delesies gekarteer en die nukleotiedvolgorde daarvan bepaal in kliniese isolate wat voorganger- en afstammelingstamme verteenwoordig. Alhoewel die resultate die betrokkenheid van IS6110 as 'n bemiddelaar van genetiese delesie ondersteun, stel dit ook die bestaan van of alternatiewe meganismes of van onbekende intermediêre vorme voor. Ondersoek van die IS6110-flankerende gebiede het gelei tot die ontdekking van ontwrigting van 'n aantal gene wat behoort tot die PPE geenfamilie, en het so gelei tot die tweede hoof ondersoek tema. Die PPE geenfamilie is ontdek as gevolg van die M. tuberculosis genoomprojek, en dit word gespekuleer dat die produkte van hierdie gene van antigeniese belang mag wees. Daar was egter met die aanvang van hierdie studie baie min data beskikbaar omtrent die biologiese rol van die PPE proteïene. Om die fenotipiese implikasies van ontwrigting van PPE gene te ondersoek is daar dus ondersoek ingestel na verskeie aspekte van hierdie geenfamilie. Eerstens is filogenetiese verwantskappe tussen lede van die PPE familie bepaal, wat gedui het op 'n evolusionêre progressie en wat ook aangedui het dat daar moontlik funksionele onderverdelings binne hierdie geenfamilie mag bestaan. Tweedens is die omvang en meganismes van PPE geenvariasie geanaliseer deur 'n kombinasie van hibridisasie, PKR en nukleotiedvolgorde analise. Hierdie benadering het omvangryke afwisseling binne hierdie geenfamilie getoon, alhoewel verskillende lede van die familie verskillende vlakke van afwisseling demonstreer. Wat veral interessant was, was die ontdekking dat lang tandem herhalingsvolgordes (~69 bp) wat in 3 lede van hierdie geenfamilie voorkom, variasie toon in die getalle van hierdie tandem herhalingsvolgordes. 'n Derde been van ondersoek het gefokus op in vitro en in vivo PPE geen uitdrukkingsprofiele. RT-PKR is gebruik om te toon dat PPE gene in vitro uitgedruk word, terwyl RNA:RNA in situ hibridisasie getoon het dat PPE gene ook in menslike weefsel uitgedruk word. Interessant genoeg dui in situ hibridisasie daarop dat daar wisselende PPE geen uitdrukking binne die menslike granuloom voorkom. Die laaste benadering wat hier gerapporteer word fokus op die sub-sellulêre lokalisering van een lid van die PPE familie, Rv1917c. 'n Kombinasie van selfraksionering en heel-sel antiliggaam-bindingseksperimente dui daarop dat Rv1917c 'n selwand-geassosieerde molekuul is wat aan die oppervlak blootgestel word. Ter opsomming het die resultate wat bereik IS potensiële implikasies vir die interpretasie van molekulêr-epidemiologiese data, dit ondersteun die rol van IS6110 as 'n bemiddelaar van genoom evolusie en beklemtoon die potensiaal vir IS6110 om 'n invloed te hê op die fenotipe van die stam. Ondersoek van die PPE familie het getoon dat hierdie geenfamilie bydra tot genetiese afwisseling, dat dit uitgedruk word beide in vitro en in vivo en dat ten minste een lid van hierdie geenfamilie geassossieer word met die selwand. Tesame ondersteun hierdie resultate die teorie dat geselekteerde lede van die PPE geenfamilie wel produkte enkodeer wat betrokke is by antigeniese variasie.

published_or_final_version / Microbiology / Master / Master of Philosophy

M.Sc. (Biochemistry) / The vitamin D receptor (VDR) has been implicated in communicable diseases such as tuberculosis (TB), HIV infection and leprosy, as well as development or prognosis of noncommunicable diseases such as multiple sclerosis, lupus, type I diabetes mellitus, and osteoporosis. The burden of these diseases is often higher in specific population groups, for instance the higher TB burden of African as compared to White South Africans. Besides genetics, epigenetic factors play a role in differential disease susceptibility and prognosis between population groups. To uncover the cause of differential susceptibility to VDR-related diseases, it is crucial that population group-specific variations in genetic and epigenetic mechanisms that regulate VDR are identified. Increases in predictive power of in silico tools, and the recent explosion in availability of genome-wide genetic and epigenetic data made bioinformatics an attractive option to preselect regions to study in the VDR...

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.

結核病仍是全球疾病和死亡的主要原因。雖然人均新發結核病例自2003年以來一直下降，耐多藥（MDR）和廣泛耐藥（XDR）的結核病例的突然增加為全球疾病控制帶來了新的威脅。結核分枝杆菌（MTB）北京株在過去十年越来越受重視，皆因其席捲亞洲，前蘇聯，和包括美國在內的好幾個地方。北京株在動物實驗中也表現出高毒性和耐多藥的傾向。目前結核菌廣泛耐藥定義為至少對異煙肼和利福平耐藥，再加上任何氟喹諾酮類，和至少一個二線藥物。我們對這種病菌的生物知識仍然有限。在這研究，我們為來自香港和福建五株MTB北京株進行了基因組測序，其中兩株的耐藥性遠超XDR標準 - “全耐藥“（TDR）的表型。五個北京株的比較基因組學為我們提供了在北京株的毒力相關基因的啟示。一個約4 KB大小的片段被找出来了，此片段是所有已知MTB中都没有的。我們討論了此片段對MTB進化的含義。當我們研究在北京耐藥株的獨特基因變化時發現，DNA修復和香葉醇降解有關連。我們還觀察到大的缺失（D）和截斷（T）的事件，顯著高於框移位（F）的突變。此外，在TDR菌株出現的FDT事件更頻繁地涉及到最佳生長和麻風分枝杆菌的基因組中保留的基因。這方面的証據表明，MTB通過缩减進化發展極端耐藥性。適應度的顯著降低也許解釋了TDR菌株的稀缺 。 / Tuberculosis (TB) remains one of the major causes of illness and death globally. Although the number of new TB cases per capita has been falling since 2003, the emergence of multidrug resistant (MDR) and extensively drug resistant (XDR) cases of TB poses new threat to the successful worldwide control of the disease (WHO, 2008; Iseman, 2007). The Beijing lineage of Mycobacterium tuberculosis (MTB) has received much attention over the past decade due to its prevalence throughout Asia, parts of the former Soviet Union, and several other geographical locations including the United States. The strain also demonstrated hypervirulence in animal models and an increased likelihood to develop multidrug resistance. The current definition of XDR in TB is defined as resistance to at least isoniazid and rifampicin, any fluoroquinolone, and with at least one of the three second-line drugs. Here we show that our knowledge of the biology of this pathogen is still limited. We performed genome sequencing and reported the complete genomes of five Beijing isolates from Hong Kong and Fujian, of which two were shown to have drug resistance that is far beyond the current XDR standard - a "Totally Drug Resistance" (TDR) phenotype. Comparative genomics of the five Beijing isolates provided us insights into the virulence-related genes in the Beijing family. A distinct region of about 4 kb in size that are absent in all known complete genomes of MTB was also identified. The evolutionary implications of this region were discussed. When we investigated the unique genetic changes in drug resistant Beijing strains, a correlation to DNA repair and geraniol degradation was implicated. We have also observed that the big deletions (D) and truncations (T) events were significant higher when compare with frameshift (F) mutations. Moreover, the FDT events in TDR strains were more frequently found in genes that are involved in growth attenuation and retained in the genome of the Mycobacterium leprae. This evidence suggests that MTB develops its extreme drug resistance through the reductive evolution. The significant decrease in the fitness may explain the rareness of TDR strains. / Detailed summary in vernacular field only. / Leung, Ka Kit. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 93-108). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Epidemiology - a ubiquitous threat --- p.1 / Chapter 1.2 --- Surviving the Hell --- p.3 / Chapter 1.3 --- Relatives of M. tuberculosis --- p.4 / Chapter 1.4 --- The age of M. tuberculosis --- p.5 / Chapter 1.5 --- Characteristics of Beijing strains --- p.6 / Chapter 1.6 --- Drug resistance --- p.7 / Chapter 1.7 --- Genome sequencing --- p.9 / Chapter 1.7.1 --- Conventional sequencing --- p.9 / Chapter 1.7.2 --- High-throughput sequencing --- p.10 / Chapter 1.8 --- Sequence assembly --- p.11 / Chapter 1.8.1 --- De novo assembly --- p.11 / Chapter 1.8.2 --- Reference mapping --- p.12 / Chapter Chapter 2 --- Materials and Methods --- p.14 / Chapter 2.1 --- Sample preparation --- p.14 / Chapter 2.2 --- DNA extraction and genome sequencing --- p.18 / Chapter 2.3 --- Gap filling and finishing --- p.20 / Chapter 2.3.1 --- In silico gap verification --- p.20 / Chapter 2.3.2 --- Comparison among different reference mapped contigs --- p.24 / Chapter 2.3.3 --- Experimental work --- p.26 / Chapter 2.4 --- Bioinformatics analysis --- p.27 / Chapter 2.4.1 --- Genome annotation --- p.27 / Chapter 2.4.2 --- Phylogeny analysis --- p.27 / Chapter 2.4.3 --- Variation analysis --- p.28 / Chapter 2.4.4 --- In silico functionality analyses --- p.29 / Chapter Chapter 3 --- Results --- p.30 / Chapter 3.1 --- Genome features of M. tuberculosis Beijing genotype strains --- p.30 / Chapter 3.2 --- Phylogeny of M. tuberculosis Beijing genotype strains --- p.36 / Chapter 3.3 --- Evolutionary implications of a 4kb-insertion in Beijing strains --- p.40 / Chapter 3.4 --- Beijing family specific gene variations --- p.48 / Chapter 3.5 --- Drug resistance --- p.52 / Chapter Chapter 4 --- Discussions --- p.75 / Chapter 4.1 --- 4kb insertion, a potential bridge to our knowledge gap --- p.75 / Chapter 4.2 --- Beijing common and Beijing drug resistant specific variations --- p.77 / Chapter 4.3 --- Regions of deletion --- p.79 / Chapter Chapter 5 --- Conclusions --- p.82 / Chapter Chapter 6 --- Future Work --- p.84 / Chapter 6.1 --- Compensatory mutation study --- p.84 / Chapter 6.1.1 --- Database construction for drug resistance compensatory mutations --- p.85 / Chapter 6.2 --- Non-protein coding region study --- p.92