SNP ASSOCIATIONS WITH TUBERCULOSIS SUSCEPTIBILITY IN A UGANDAN HOUSEHOLD CONTACT STUDY by ALLISON REES BAKER Submitted in partial fulfillment of the requirements For the degree of Master of Science Thesis Advisor: Dr. Catherine M. Stein Department of Epidemiology and Biostatistics CASE WESTERN RESERVE UNIVERSITY August, 2010 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of ______________________________________________________ candidate for the ________________________________degree *. 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Table of Contents Table of Contents...............................................................................................................iii List of Tables ..................................................................................................................... iv Acknowledgements............................................................................................................. v List of Commonly Used Abbreviations ............................................................................. vi Chapter 1: Literature Review.............................................................................................. 8 1.1. Genetics of Susceptibility to Tuberculosis .............................................................. 8 1.1.1. History and Epidemiology of Tuberculosis ...................................................... 8 1.1.2. Candidate Genes ............................................................................................. 10 1.1.3. Genome-wide Linkage Scans ......................................................................... 15 1.2. Methods for Fine Mapping Analysis ..................................................................... 20 1.3. Imputation.............................................................................................................. 23 Chapter 2: Specific Aims.................................................................................................. 27 2.1. Specific Aim 1 ....................................................................................................... 27 2.2. Specific Aim 2 ....................................................................................................... 27 2.3. Specific Aim 3 ....................................................................................................... 28 Chapter 3: Methods........................................................................................................... 29 3.1. Data Description .................................................................................................... 29 3.1.1. Sample............................................................................................................. 29 3.1.2. Descriptive Statistics....................................................................................... 30 3.2. Genotyping............................................................................................................. 31 3.3. Analysis Strategy ................................................................................................... 34 3.3.1. Aim 1: Candidate Gene Analysis.................................................................... 34 3.3.2. Aim 2: Fine Mapping Analysis....................................................................... 37 3.3.3. Aim 3: Imputation........................................................................................... 38 Chapter 4: Results and Discussion.................................................................................... 41 4.1. Results.................................................................................................................... 41 4.1.1. Candidate Gene Analysis................................................................................ 41 4.1.2. Fine-Mapping Analysis................................................................................... 43 4.1.3. Imputation....................................................................................................... 44 4.2. Discussion.............................................................................................................. 53 4.3. Conclusions and Future Directions........................................................................ 56 Bibliography ..................................................................................................................... 60 iii List of Tables Table 1. Descriptive Statistics........................................................................................... 34 Table 2. Candidate Gene SNPs Departing from HWE..................................................... 41 Table 3. Candidate Gene Analysis Results....................................................................... 42 Table 4. Fine Mapping SNPs Departing from HWE ........................................................ 44 Table 5. Haplotype Analysis Results for TLR2................................................................ 46 Table 6. Haplotype Analysis Results for TLR4................................................................ 46 Table 7. Haplotype Analysis Results for TLR6................................................................ 46 Table 8. Haplotype Analysis Results for TIRAP.............................................................. 46 Table 9. Results for Imputed Genotypes on Chromosome 7p.......................................... 48 Table 10. Results for Imputed Genotypes on Chromosome 20q...................................... 52 iv Acknowledgements I would like to acknowledge and thank my thesis advisor, Dr. Catherine Stein, for providing me with direction and leadership throughout my academic program. I am exceedingly grateful for the incredible mentoring, support and guidance received from Drs. Courtney Gray and Emma Larkin. Thank you also to Drs. Robert Igo and Robert Elston, and thanks to Robert Goodloe for his programming assistance and sharing in the student experience. A very special thank you is dedicated to my devoted mother and father, and to my husband, Dave, for without his endearing love and endless support, none of my success would be possible. v List of Commonly Used Abbreviations AIDS Acquired Immune Deficiency Syndrome ASW African Ancestry in Southwest USA CARD11 Caspase recruitment domain family, member 11 cM Centimorgan CTSZ Cathepsin Z GLMM Generalized Linear Mixed Model HIV Human Immunodeficiency Virus IL-1 Interleukin-1 IL-10 Interleukin-10 IL-12 Interleukin-12 IFNG1- γ Interferon Gamma HIV Human Immunodeficiency Virus HMM Hidden Markov Model HWE Hardy Weinberg Equilibrium kb Kilobasepair LD Linkage Disequilibrium LTBI Latent Mycobacterium Tuberculosis Infection LWK Luhya in Webuye, Kenya MAF Minor Allele Frequency Mb Megabasepair MC3R Melanocortin 3 Receptor MKK Maasai in Kinyawa, Kenya Mtb Mycobacterium tuberculosis NOS2A Nitric Oxide Synthase 2A NRAMP1 Natural-Resistance-Associated Macrophage Protein 1 PPD Purified Protein Derivative QC Quality Control QTL Quantitative Trait Locus SLC11A1 Solute Carrier Family 11, Member 13 SNP Single Nucleotide Polymorphism TB Tuberculosis TBSCPB Tuberculosis Susceptibility Variable TDT Transmission Disequilibrium Test TLR-2 Toll-Like Receptor-2 TLR-4 Toll-Like Receptor-4 TNF Tumor Necrosis Factor TNF-α Tumor Necrosis Factor-α TST Tuberculin Skin Test UG Uganda YRI Yoruba in Ibadan, Nigeria vi SNP Associations with Tuberculosis Susceptibility in a Ugandan Household Contact Study Abstract by ALLISON REES BAKER The World Health Organization reports that over 9 million new cases of tuberculosis (TB) are diagnosed each year, killing between 1.6 and 2 million individuals worldwide. TB is an infectious disease caused by the bacterium Mycobacterium tuberculosis (Mtb), and reports indicate that only 10% of individuals infected with Mtb actually advance to disease. Genetic linkage and association analyses have established several chromosome regions involved in TB susceptibility. This study examines the association of TB susceptibility with a selection of biologically relevant markers, a chromosome 7 region identified through a previous genome scan, and association with imputed genotypes. Across chromosomes 7 and 20, 564 Ugandan individuals were genotyped at 1,417 SNPs. None of the candidate genes or fine mapping SNPs were found significantly associated with TB susceptibility (P > 0.10). Five imputed SNPs were significant at the P = 0.01 level. Suggested future work includes GWAS and resequencing analyses. vii Chapter 1: Literature Review 1.1. Genetics of Susceptibility to Tuberculosis 1.1.1. History and Epidemiology of Tuberculosis The World Health Organization (WHO) reports that over 9 million new cases of tuberculosis (TB) are diagnosed each year, killing between 1.6 and 2 million individuals worldwide (World Health Organization 2009). TB is an infectious disease caused by the bacterium Mycobacterium tuberculosis (Mtb), but reports indicate that only 10% of individuals infected with Mtb actually advance to disease (Murray et al. 1990). The pathogenesis of