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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.

Orientador: Manoel Barros Bertolo / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-21T17:57:00Z (GMT). No. of bitstreams: 1 DelRio_AnaPaulaToledo_M.pdf: 1151549 bytes, checksum: 2407595c8d19b5531c6d084bba4d3238 (MD5) Previous issue date: 2012 / Resumo: Introdução: A esclerose sistêmica (ES) é uma doença autoimune caracterizada por disfunção endotelial, vasculopatia obliterativa, fibrose cutânea e visceral. Trata-se de doença poligênica complexa que se manifesta em indivíduos geneticamente predispostos com exposição a fator ambiental ou outro precipitante e seu desenvolvimento depende da interação entre processos imunológicos, vasculares e fibróticos. Estudos genéticos prévios procuram correlacionar os alelos HLA classe I e II e as manifestações clínicas da doença. A fibrose pulmonar (FP) e a hipertensão arterial pulmonar (HAP) são, atualmente, os acometimentos com maior impacto prognóstico e as principais causas de óbito nos pacientes com ES. A expressão do autoanticorpo antitopoisomerase I (anti-Scl70) é um forte preditor de FP, associada à forma difusa e a HAP está relacionada ao anticorpo anticentrômero e à forma limitada da doença. Objetivos: O objetivo deste estudo foi avaliar a participação do HLA na expressão da doença e suas manifestações clínicas de pior prognóstico (FP e HAP) em pacientes com ES em uma população miscigenada. Métodos: Foram incluídos os pacientes com ES seguidos no ambulatório de Reumatologia da Universidade Estadual de Campinas (UNICAMP) de 2008 a 2011. Os dados clínicos foram obtidos através da análise dos prontuários. A genotipagem dos alelos Classe I e II foi realizada através da técnica de amplificação pela reação em cadeia da polimerase, utilizando seqüências específicas de primers. A análise estatística incluiu o teste exato de Fisher e o teste do qui-quadrado de Pearson. Foram considerados significativos valores de p ? 0,05. A razão de prevalência foi estimada pelo método delta. Resultados Cento e quarenta e um pacientes (120 mulheres e 21 homens) foram estudados, sendo 33,3% ES difusa, 62,4% ES limitada e 4,3% ES sine scleroderma. A FP foi considerada em 61 pacientes (43,3%), os alelos HLA-A*30 e DQB1*04 foram relacionados à suscetibilidade. No entanto, os alelos HLA-DRB1*01 e DQB1*05 foram relacionados à ausência desta manifestação. A HAP foi diagnosticada em 19 pacientes (13,5%) e teve associação com HLA-B*35 e C*04, enquanto o alelo C*03 pareceu ser protetor. Conclusões: Este estudo aponta para associação de alguns alelos do HLA classe I e II às manifestações clínicas de maior morbimortalidade na ES nesta série de casos. Estes achados não foram semelhantes aos encontrados previamente em outras populações, o que evidencia múltiplos padrões genéticos na ES / Abstract: Introduction: Systemic sclerosis (SSc) is an autoimmune disease characterized by endothelial dysfunction, occlusive vasculopathy, cutaneous and visceral fibrosis. This is a complex polygenic disease that manifests in genetically predisposed individuals with environmental or other precipitating factor exposure and its development depends on the interaction between immunological, vascular and fibrotic processes. Previous genetic studies aimed to correlate class I and II HLA and the clinical manifestations of the disease. Pulmonary fibrosis (PF) and pulmonary arterial hypertension (PAH) are currently the worse prognostic features and the main causes of death in patients with SSc. The presence of anti-topoisomerase I antibody (anti-Scl70) is a strong predictor of FP, associated with diffuse SSc and PAH is related to anticentromere and the limited form of the disease. Objectives: The aim of this study was to evaluate the HLA involvement in disease expression and poor prognostic clinical features, pulmonary fibrosis (PF) and pulmonary arterial hypertension (PAH), in patients diagnosed with systemic sclerosis (SSc) in a multiethnic population. Methods: SSc patients followed 2008-2011 were included and clinical data were obtained through records review. Molecular HLA typing was performed (PCR amplification technique using sequence of specific primers). Statistical analysis included Fisher's exact test and Pearson's corrected chi-square test. P values ? 0.05 were considered significant. The prevalence ratio was estimated by delta method. Results: One hundred forty-one patients (120 women and 21 men) were studied, 33,3% dcSSc, 62,4% lcSSc and 4,3% sine scleroderma. PF was present in 61 patients (43,3%), HLA-A*30 and DQB1*04 were related to susceptibility. However, HLA-DRB1*01 and DQB1*05 alleles were protective. PAH was diagnosed in 19 (13,5%) and had association with HLA-B*35 and C*04, whereas C*03 seemed to be protective. Conclusions: Our current study documents the association of some class I and II HLA alleles with the most severe clinical manifestations in a multiethnic case series. Our findings were not absolutely similar to the previous data in other populations / Mestrado / Clinica Medica / Mestra em Clínica Médica

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2017-01-04T13:00:39Z No. of bitstreams: 2 Dissertação - Kaisson Ernane dos Santos - 2013.pdf: 2738475 bytes, checksum: 6c79ab9177dd126fa5cb677127debc2c (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2017-01-04T13:01:13Z (GMT) No. of bitstreams: 2 Dissertação - Kaisson Ernane dos Santos - 2013.pdf: 2738475 bytes, checksum: 6c79ab9177dd126fa5cb677127debc2c (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-01-04T13:01:13Z (GMT). No. of bitstreams: 2 Dissertação - Kaisson Ernane dos Santos - 2013.pdf: 2738475 bytes, checksum: 6c79ab9177dd126fa5cb677127debc2c (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2013-11-25 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The Major Histocompatibility Complex is mainly composed by genes of the adaptive immune response. In humans, part of this complex is known as the Human Leukocyte Antigens (HLA), whose genes are responsible for specific antigen presentation to effector immune cells. The classical class I HLA genes (HLA-A, -B and -C) are responsible for antigen presentation to T CD8+ cells and they constitute the most polymorphic genes in the human genome. This variability is maintained by selection mediated by microorganisms. In contrast to their classical counterparts, the non classical class I genes (HLA-G, -E and -F) present low variability and are associated with immune tolerance due to the interaction with NK and T cells inhibitor receptors. HLA-G is the most studied non classical gene, which is associated with immune response modulation, mainly during pregnancy. Considering that natural selection is acting on the HLA-G regulatory regions maintaining high heterozigosity in this region, we evaluated a nearby Alu insertion (AluyHG) correlating this Alu element with coding and 3’UTR HLA-G polymorphisms. The AluyHG insertion was particularly associated with the HLA-G haplotype known as G*01:01:01:01/UTR-1, considered a high-expressing HLA-G haplotype. The G*01:01:01:01/UTR-1/AluyHG haplotype would be the most recent HLA-G haplotypes, in spite of its high frequency in worldwide populations. / O Complexo Principal de Histocompatibilidade (MHC) é formado principalmente por genes que participam da resposta imunológica adaptativa. Entre esses genes encontramos o grupo denominado de Antígenos Leucocitários Humanos (HLA), que são responsáveis pela apresentação de antígenos específicos às células efetoras do sistema imunológico. Os genes HLA de classe I clássicos (HLA-A, -B e -C), responsáveis pela apresentação antigênica aos linfócitos T citotóxicos, são considerado como os mais polimórficos do genoma humano e de outros vertebrados. A variabilidade desses genes e elevada heterozigose é mantida por seleção mediada por microrganismos. Diferentemente dos genes clássicos, os genes HLA de classe I não clássicos (HLA-G, -E e -F) apresentam variabilidade reduzida e como função principal a tolerância imunológica, por meio de sua interação com receptores inibitórios presentes nas células NK e T. O HLA-G é o mais estudado entre esses genes e, devido sua importância como molécula imunomoduladora e sua importância em situações como gestação, e considerando evidências anteriores de seleção natural mantendo uma elevada heterozigose nas regiões regulatórias do HLA-G, avaliamos a presença de uma inserção Alu (AluyHG) próxima a este gene correlacionando os achados com a variabilidade contida nas suas regiões codificadora e 3’ não traduzida. A inserção AluyHG mostrou-se em desequilíbrio de ligação (LD) com os polimorfismos do gene HLA-G. Especificamente, o elemento inserido apresentou-se em LD com um haplótipo denominado G*01:01:01:01/UTR-1, considerado como um haplótipo de alta produção da molécula de HLA-G. Esse haplótipo aparentemente é o mais jovem entre humanos, apesar de sua elevada frequência nas populações estudadas até o momento.

Submitted by Erika Demachki (erikademachki@gmail.com) on 2014-11-11T20:37:41Z No. of bitstreams: 2 Dissertação - Leandro Prado Felício - 2013.pdf: 4839350 bytes, checksum: 07898140311917429ab55aa63ad5324e (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Erika Demachki (erikademachki@gmail.com) on 2014-11-11T20:38:05Z (GMT) No. of bitstreams: 2 Dissertação - Leandro Prado Felício - 2013.pdf: 4839350 bytes, checksum: 07898140311917429ab55aa63ad5324e (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2014-11-11T20:38:05Z (GMT). No. of bitstreams: 2 Dissertação - Leandro Prado Felício - 2013.pdf: 4839350 bytes, checksum: 07898140311917429ab55aa63ad5324e (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2013-01-31 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / The HLA-E locus is a Human Major Histocompatibility Complex (MHC) gene associated with immune-modulation and suppression of the immune response by the interaction with specific NK and T cell receptors. The HLA-E gene is considered the most conserved locus in the human HLA; however, this low variability might be a consequence of the scarce number of studies focusing this subject. In this mastering thesis we assessed the HLA-E coding and 3’ untranslated region variability in a group of individuals from Brazil and the results were evaluated together with data from the 1000Genomes Consortium. Altogether, only 28 variation sites were found in approximately 2724 bp evaluated. These variation sites were arranged into 33 haplotypes, most of them (98.2%) encoding one of the two HLA-E molecules found worldwide, i.e., the molecules associated with the allele groups E*01:01 and E*01:03. Interestingly, 85% of all haplotypes were represented by only three different sequences, each of them associated with one of the main known HLA-E coding alleles, E*01:01:01, E*01:03:01 and E*01:03:02, all of them found worldwide. This phenomenon, together with the comparisons with other primate sequences, reveals that these two main allele groups (and molecules) arose early before human speciation, and indicates that E*01:03:01 might be the oldest allele. In addition, the low nucleotide diversity found for the HLA-E coding and 3’UTR in worldwide populations suggests that the HLA-E gene is in fact a conserved gene, which might be a consequence of its key role in the modulation of the immune system. / O loco HLA-E é um gene do Complexo Principal de Histocompatibilidade Humano (MHC), cujo produto está relacionado com a modulação e supressão da resposta imunitária por meio da interação com receptores específicos das células NK e linfócitos T. O gene HLA-E é considerado o loco menos polimórfico dos genes do complexo HLA, no entanto, esta baixa variabilidade pode ser uma consequência do pequeno número de estudos realizados sobre esse tema. No presente trabalho, a variabilidade das regiões codificadoras e 3’ não traduzida do gene HLA-E foi analisada em amostras brasileiras e os resultados foram comparados com dados obtidos pelo projeto 1000Genomes. Considerando todas as populações avaliadas, apenas 28 pontos de variação foram encontrados em uma região de aproximadamente 2724-pb. Estes pontos de variação estão arranjados em 33 haplótipos diferentes, a maioria deles (98%) codificando uma das duas moléculas HLA-E frequentemente encontradas, E*01:01 e E*01:03. Ainda, 85% dos haplótipos encontrados foram representados por apenas três sequências diferentes, cada uma deles associada a um dos principais alelos da região codificadora do gene HLA-E, E*01:01:01, E*01:03:01 e E*01:03:02. Todas essas sequências foram encontradas em todas as populações avaliadas. Este fenômeno, em conjunto com as comparações envolvendo sequências de primatas, sugere que estes dois grupos de alelos principais (e moléculas) surgiram antes da especiação e dispersão humana, além de indicar que o alelo E*01:03:01 pode ser o mais antigo dentre os demais. Ainda, a baixa diversidade nucleotídica encontrada para a região codificadora e 3' NT do gene HLA-E em populações de todo o mundo sugere que este gene é, de fato, bastante conservado, provavelmente devido ao seu papel chave na modulação das respostas imunes.