Background: Coronary Artery Disease (CAD) represents the leading cause of mortality and morbidity worldwide despite declines in the prevalence of environmental risk factors. This trend has drawn attention to the risk conferred by genetic variation. Twin and linkage studies demonstrate a profound hereditary risk for CAD, especially in young individuals. Rare genetic variants conferring high risk for extreme disease phenotypes can provide invaluable insight into novel mechanisms underlying CAD development. Methods: Whole exome sequencing was performed to characterize rare protein-altering variants in 52 early-onset CAD (EOCAD) patients encompassing the DECODE study. The enrichment of Mendelian dyslipidemias in EOCAD was assessed through interrogation of pathogenic mutations among known lipid genes. The identification of novel genetic CAD associations was conducted through case-only and case-control approaches across all protein-coding genes using rare variant burden and variance component tests. Lastly, beta coefficients for significant risk genes from the European population in the Early-onset Myocardial Infarction (EOMI) cohort (N=552) were used to construct calibrated, single-sample rare variant gene scores (RVGS) in DECODE Europeans (N=39) and a local European CAD-free cohort (N=77). Results: A 20-fold enrichment of Familial hypercholesterolemia mutation carriers was detected in EOCAD cases compared to CAD-free controls (P=0.005). Association analysis using EOMI Europeans revealed exome-wide and nominal significance for two known CAD/MI genes: CELSR2 (P=1.1x10-17) and APOA5 (P=0.001). DECODE association revealed exome-wide and nominal significance for genes involved in endothelial integrity and immune cell activity. RVGS based upon beta coefficients of significant CAD/MI risk genes were significantly increased in DECODE (z-score=1.84; p=0.03) and insignificantly decreased among CAD-free individuals (z-score=-1.61; p=0.053). Conclusion: Rare variants play a pivotal role in the development early CAD through Mendelian and polygenic mechanisms. Construction of RVGS that are calibrated against population and technical biases can facilitate discovery of single-sample and cohort-based associations beyond what is detectable using standard methods. / Thesis / Master of Science (MSc)

A hepatite auto-imune (HAI) é uma doença inflamatória crônica do fígado, de etiologia desconhecida, que acomete preferencialmente mulheres, com destruição progressiva do parênquima hepático e que, sem tratamento imunossupressor, evolui freqüentemente para cirrose. É uma doença rara na infância, com menos de 10% dos pacientes com doença hepática crônica, porém de alta mortalidade. Caracteriza-se pela presença de hipergamaglobulinemia, auto-anticorpos não órgãos-específicos e infiltrado inflamatório portal linfoplasmocitário. Cerca da metade dos pacientes atendidos no Instituto da Criança, apresenta também níveis elevados de IgE, sem causas aparentes como parasitoses ou atopia. A suscetibilidade genética à doença está principalmente associada a genes que codificam as moléculas de histocompatibilidade (HLA). A presença do HLA-DR é importante, mas não suficiente para o desenvolvimento dessa doença rara, fazendo inclusive supor um forte componente externo/ambiental no desencadeamento da doença. Genes recém descritos, localizados na região de classe III do Complexo Principal de Histocompatibilidade (CPH) e ligados ao controle da resposta imune, em especial, alguns próximos à junção com a região de classe I, têm sido investigados como \"loci\" secundários para o desenvolvimento de doenças auto-imunes. A forte associação da HAI com genes na região do MHC, bastante comuns na população, aliada a incidência muito baixa da doença, leva à questão da presença de genes adicionais de suscetibilidade, que, junto com o HLA-DR, seriam responsáveis pela suscetibilidade genética observada. Dessa forma, o HLADRB1* 13, além de um fator por si, também pode ser um marcador da região cromossômica, ou seja, de um haplótipo específico e que, portanto, carrega mais de um gene de suscetibilidade. Estudamos polimorfismos, tipo SNP, de xx genes próximos ao HLA-DRB1, como TNFA, LTA, NFKBIL1 e BAT1, buscando haplótipos de susceptibilidade à doença, em pacientes HAI-1 (n=105) e controles sadios (n=227). O haplótipo ancestral 8.1 que inclui HLA-DRB1*03 e o alelo raro na posição -308 do gene TNFA estava aumentado (p=0.0005). Já o alelo HLA-DRB1*13, presente na maioria dos pacientes, não mostrou haplótipo específico associado. Também avaliamos genes de citocinas envolvidas na produção de IgE, elevado em parte dos pacientes HAI-1. Na comparação com controles, a freqüência dos SNPs IL- 4+33 e IL13+110, localizados em genes vizinhos, apresentou aumento estatisticamente significante nos pacientes, sugerindo haver um grupo gênico adicional no cromossomo 5q31 envolvido na susceptibilidade à HAI / Autoimmune hepatitis (AIH) is an inflammatory chronic liver disease of unknown etiology found predominantly in females, leading when untreated, to cirrhosis. It is a rare disease in the childhood, corresponding to about 10% of patients with chronic hepatitis, but exhibits high mortality. It is characterized by hipergammaglobulinemia, organ nonspecific circulating autoantibodies, and an inflammatory liver-infiltrating lymphocytes and plasma cells. Almost half of patients investigated at Instituto da Criança had increased plasma IgE levels, without any apparent cause such as parasite infestation or atopy. Genetic predisposition to AIH has been mainly linked to genes coding for HLA class II molecules. HLA-DR is important but not sufficient to explain this rare disease, suggesting there is an external/environmental component triggering the disease. Recently, several genes in the class III region of MHC, linked to immune responses, especially near the junction with the MHC class I region have been investigated as secondary loci for autoimmune disease susceptibility. The strong association of AIH with genes in the MHC region, common in the population, but a disease with the very low incidence, suggests additional genes linked with HLA-DR could add to the disease susceptibility. So, HLA-DR*13 besides being a factor by itself, could also be a chromosomal region marker, taking part of a specific haplotype carrying more than one susceptibility gene. We studied single nucleotide polymorphisms (SNP) in genes near HLA-DRB1, like TNFA, LTA, NFKBIL1 and BAT1 searching for disease susceptibility haplotypes, in HAI-1 patients (n=105) compared to healthy controls (n=227). The ancestral haplotype 8.1, which includes HLA-DRB1*03 and the rare TNFA allele at position -308 was increased (p=0.0005). However, HLA-DRB1*13, though present in the majority of the patients, did not show any specific haplotype associated to it. xxii We also analyzed cytokine genes involved in IgE production, which is increased in a part of the AIH type 1 patients. In comparison with controls, the frequency of the SNPs IL-4+33 and IL13+110 was significantly increased, suggesting the existence of an additional gene cluster in chromosome 5q31 involved in the susceptibility to AIH

Yung Chung Ming Edmund. / Thesis submitted in: September 2005. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 44-54). / Abstracts in English and Chinese. / Title Page / Contents / Abstract / 摘要 / List of Figures and tables / Abbreviations / Text / References / Chapter Chapter 1: --- General Introduction / Chapter 1.1 --- An overview to the study of CTLA-4 gene in childhood Graves' disease (GD) / Chapter 1.1.1 --- "Graves' disease 226}0ؤ features, incidence, aetiology and pathogenesis page" / Chapter 1.1.1.1 --- GD features 226}0ؤ from clinical to laboratory --- p.1 / Chapter 1.1.1.2 --- GD incidence - from adult to children --- p.2 / Chapter 1.1.1.3 --- GD aetiology - from environment to genes --- p.3 / Chapter 1.1.1.4 --- GD pathogenesis - from auto-antigen to autoantibody --- p.4 / Chapter 1.1.2 --- CTLA-4 gene study in Graves' disease --- p.5 / Chapter 1.1.3 --- Conclusion --- p.6 / Chapter 1.2 --- "Objectives, hypothesis and planning of the study" --- p.7 / Chapter 1.2.1 --- Objectives --- p.7 / Chapter 1.2.2 --- Hypothesis --- p.7 / Chapter 1.2.3 --- Planning --- p.7 / Chapter Chapter 2: --- Literature Review --- p.8 / Chapter 2.1 --- The CD28 / CTLA-4: B7 co-stimulatory pathway and Graves' disease --- p.8 / Chapter 2.1.1 --- Overview of co-stimulation and T cell activation --- p.8 / Chapter 2.1.2 --- Overview of the CD28 gene --- p.9 / Chapter 2.1.3 --- Overview of the CTLA-4 gene --- p.10 / Chapter 2.1.4 --- Co-stimulation and Graves' disease --- p.13 / Chapter 2.2 --- The study of CTLA-4 gene polymorphism in Graves' disease --- p.14 / Chapter Chapter 3: --- Methodology --- p.16 / Chapter 3.1 --- Recruitment of subjects --- p.16 / Chapter 3.1.1 --- Recruitment of cases --- p.16 / Chapter 3.1.2 --- Recruitment of controls --- p.16 / Chapter 3.1.3 --- Ethical approval --- p.17 / Chapter 3.2 --- Peripheral blood collection and genomic DNA preparation --- p.17 / Chapter 3.2.1 --- Peripheral blood collection --- p.17 / Chapter 3.2.2 --- White blood cell harvesting --- p.17 / Chapter 3.2.3 --- White blood cell digestion --- p.17 / Chapter 3.2.4 --- DNA extraction --- p.17 / Chapter 3.3 --- Polymerase Chain Reaction (PCR) amplification of CTLA-4 gene exon one --- p.18 / Chapter 3 .4 --- PCR-Restriction Fragment Length Polymorphism (PCR-RFLP) analysis of CTLA-4 gene codon 17 A/G dimorphism --- p.19 / Chapter 3.5 --- PCR-Single Strand Conformational Polymorphism (PCR-SSCP) analysis of of CTLA-4 gene codon 17 A/G dimorphism --- p.21 / Chapter 3.5.1 --- Preparation of SSCP gel and buffer --- p.21 / Chapter 3.5.2 --- ´5ةend labelling of forward PCR primer --- p.21 / Chapter 3.5.3 --- Preparation of PCR fragment for SSCP analysis --- p.21 / Chapter 3.5.4 --- SSCP analysis --- p.22 / Chapter 3.5.5 --- Autoradiography --- p.22 / Chapter 3.6 --- Sequence confirmation of the SSCP fragment by PCR cycle sequencing --- p.22 / Chapter 3.6.1 --- Preparation of sequencing template from SSCP fragment --- p.22 / Chapter 3.6.2 --- PCR cycle sequencing --- p.23 / Chapter 3.6.3 --- Preparation of cycle sequencing products for electrophoresis --- p.23 / Chapter 3.6.4 --- Sequencing by capillary electrophoresis (CE) --- p.24 / Chapter 3.7 --- Statistical analysis --- p.24 / Chapter Chapter 4: --- Results and Data Analysis --- p.26 / Chapter 4.1 --- Results --- p.26 / Chapter 4.1.1 --- Demographic data of case and control subjects --- p.26 / Chapter 4.1.2 --- PCR amplification of CTLA-4 gene exon one --- p.26 / Chapter 4.1.3 --- PCR-RFLP analysis of CTLA-4 gene codon 17 A/G dimorphism locus --- p.26 / Chapter 4.1.4 --- PCR-SSCP analysis of CTLA-4 gene codon 17 A/G dimorphism locus --- p.29 / Chapter 4.1.5 --- PCR cycle sequencing of the SSCP fragments --- p.31 / Chapter 4.2 --- Data analysis --- p.32 / Chapter 4.2.1 --- Overview of data --- p.32 / Chapter 4.2.2 --- CTLA-4 exon one polymorphism analysed with respect to sex --- p.32 / Chapter 4.2.3 --- CTLA-4 exon one polymorphism in patients with Graves' disease and controls --- p.34 / Chapter Chapter 5: --- Discussion --- p.36 / Chapter Chapter 6: --- Summary and Conclusions --- p.42

A hepatite auto-imune (HAI) é uma doença inflamatória crônica do fígado, de etiologia desconhecida, que acomete preferencialmente mulheres, com destruição progressiva do parênquima hepático e que, sem tratamento imunossupressor, evolui freqüentemente para cirrose. É uma doença rara na infância, com menos de 10% dos pacientes com doença hepática crônica, porém de alta mortalidade. Caracteriza-se pela presença de hipergamaglobulinemia, auto-anticorpos não órgãos-específicos e infiltrado inflamatório portal linfoplasmocitário. Cerca da metade dos pacientes atendidos no Instituto da Criança, apresenta também níveis elevados de IgE, sem causas aparentes como parasitoses ou atopia. A suscetibilidade genética à doença está principalmente associada a genes que codificam as moléculas de histocompatibilidade (HLA). A presença do HLA-DR é importante, mas não suficiente para o desenvolvimento dessa doença rara, fazendo inclusive supor um forte componente externo/ambiental no desencadeamento da doença. Genes recém descritos, localizados na região de classe III do Complexo Principal de Histocompatibilidade (CPH) e ligados ao controle da resposta imune, em especial, alguns próximos à junção com a região de classe I, têm sido investigados como \"loci\" secundários para o desenvolvimento de doenças auto-imunes. A forte associação da HAI com genes na região do MHC, bastante comuns na população, aliada a incidência muito baixa da doença, leva à questão da presença de genes adicionais de suscetibilidade, que, junto com o HLA-DR, seriam responsáveis pela suscetibilidade genética observada. Dessa forma, o HLADRB1* 13, além de um fator por si, também pode ser um marcador da região cromossômica, ou seja, de um haplótipo específico e que, portanto, carrega mais de um gene de suscetibilidade. Estudamos polimorfismos, tipo SNP, de xx genes próximos ao HLA-DRB1, como TNFA, LTA, NFKBIL1 e BAT1, buscando haplótipos de susceptibilidade à doença, em pacientes HAI-1 (n=105) e controles sadios (n=227). O haplótipo ancestral 8.1 que inclui HLA-DRB1*03 e o alelo raro na posição -308 do gene TNFA estava aumentado (p=0.0005). Já o alelo HLA-DRB1*13, presente na maioria dos pacientes, não mostrou haplótipo específico associado. Também avaliamos genes de citocinas envolvidas na produção de IgE, elevado em parte dos pacientes HAI-1. Na comparação com controles, a freqüência dos SNPs IL- 4+33 e IL13+110, localizados em genes vizinhos, apresentou aumento estatisticamente significante nos pacientes, sugerindo haver um grupo gênico adicional no cromossomo 5q31 envolvido na susceptibilidade à HAI / Autoimmune hepatitis (AIH) is an inflammatory chronic liver disease of unknown etiology found predominantly in females, leading when untreated, to cirrhosis. It is a rare disease in the childhood, corresponding to about 10% of patients with chronic hepatitis, but exhibits high mortality. It is characterized by hipergammaglobulinemia, organ nonspecific circulating autoantibodies, and an inflammatory liver-infiltrating lymphocytes and plasma cells. Almost half of patients investigated at Instituto da Criança had increased plasma IgE levels, without any apparent cause such as parasite infestation or atopy. Genetic predisposition to AIH has been mainly linked to genes coding for HLA class II molecules. HLA-DR is important but not sufficient to explain this rare disease, suggesting there is an external/environmental component triggering the disease. Recently, several genes in the class III region of MHC, linked to immune responses, especially near the junction with the MHC class I region have been investigated as secondary loci for autoimmune disease susceptibility. The strong association of AIH with genes in the MHC region, common in the population, but a disease with the very low incidence, suggests additional genes linked with HLA-DR could add to the disease susceptibility. So, HLA-DR*13 besides being a factor by itself, could also be a chromosomal region marker, taking part of a specific haplotype carrying more than one susceptibility gene. We studied single nucleotide polymorphisms (SNP) in genes near HLA-DRB1, like TNFA, LTA, NFKBIL1 and BAT1 searching for disease susceptibility haplotypes, in HAI-1 patients (n=105) compared to healthy controls (n=227). The ancestral haplotype 8.1, which includes HLA-DRB1*03 and the rare TNFA allele at position -308 was increased (p=0.0005). However, HLA-DRB1*13, though present in the majority of the patients, did not show any specific haplotype associated to it. xxii We also analyzed cytokine genes involved in IgE production, which is increased in a part of the AIH type 1 patients. In comparison with controls, the frequency of the SNPs IL-4+33 and IL13+110 was significantly increased, suggesting the existence of an additional gene cluster in chromosome 5q31 involved in the susceptibility to AIH