THE ASSOCIATION OF TOLL-LIKE RECEPTOR POLYMORPHISMS WITH HUMAN IMMUNODEFICIENCY VIRUS INFECTION IN NORTH AMERICANS by BARNE WILLIE Submitted in partial fulfillment of the requirement for the degree of Master of Science Dissertation Advisor: Dr. Peter A Zimmerman (PhD) Biology Case Western Reserve University May 2014 Case Western Reserve University School of Graduate Studies We hereby approve the thesis/dissertation of BARNE WILLIE ………………………………………………………………………………………………………………… candidate for the Master in Science degree*. (signed) Roy Ritzmann (PhD) …………………………………………………………………………………………………………………… (Chair of the committee) Peter A Zimmerman (PhD) …………………………………………………………………………………………………………………… Daniel Tisch (PhD) …………………………………………………………………………………………………………………… Christopher Cullis (PhD) …………………………………………………………………………………………………………………… (date) 21st March 2014 ………………………………………………… * We also certify that the written approval has been obtained for any proprietary material contained therein. II Dedication I would take this precious time that I have to dedicate this work to my Bikpela Papa - Naiyo Niniko and Bikpela Ankol - Phillip Felix Buri. I know you twos will be proud to see me through this time of my life journey. But now, I could only wish you twos were here to witness this. Miss you twos so much, and what more can I say...? I shall see you twos one day. III Table of Content List of Tables ............................................................................................................... VII List of Figures ............................................................................................................. VIII Acknowledgement ....................................................................................................... IX List of Abbreviations ..................................................................................................... X Thesis Abstract ............................................................................................................ XII 1.0 Introduction ............................................................................................................ 1 1.1. General Immunology ........................................................................................... 1 1.3. Innate Immunity .................................................................................................. 3 1.4. Toll-Like Receptors .............................................................................................. 6 1.5. TLR1-9 properties ................................................................................................ 8 1.5.1. TLR1 (cluster of differentiation 281-CD281) ................................................ 8 1.5.2. TLR2 (cluster of differentiation 282-CD282) ................................................ 9 1.5.3. TLR3 (cluster of differentiation 283-CD283) ................................................ 9 1.5.4. TLR4 (cluster of differentiation 284-CD284) .............................................. 10 1.5.5. TLR5 (cluster of differentiation 285-CD285) .............................................. 10 1.5.6. TLR6 (cluster of differentiation 286-CD286) .............................................. 11 1.5.7. TLR7 (cluster of differentiation 287-CD287) .............................................. 11 1.5.8. TLR8 (cluster of differentiation 288-CD288) .............................................. 12 1.5.9. TLR9 (cluster of differentiation 289-CD289) .............................................. 12 1.5.10. TLR10 (cluster of differentiation 290-CD290) .......................................... 13 1.6. Human TLR Groupings ....................................................................................... 13 IV 1.7. Chemistry of ligand binding .............................................................................. 14 1.8. PAMPs Recognition by TLRs .............................................................................. 15 1.9. TLR signaling pathways ..................................................................................... 17 1.10. SNPs, gene expression and functionality ........................................................ 19 1.11. SNP analysis-Genome Wide Association Studies ............................................ 21 1.12. TLR genes SNPs and disease association ......................................................... 22 1.13. Thesis Objectives ............................................................................................. 27 2.0 Methods ................................................................................................................ 28 2.1. Selection of TLR gene SNPs ............................................................................... 28 2.2. Sample preparation for genotyping .................................................................. 29 2.3. Genotyping assay .............................................................................................. 30 2.3.1. Golden Gate Vera Code Bead Express Platform ......................................... 30 2.3.2. Establishing the Bead Express Genotyping Assay ...................................... 31 2.4. Sample ............................................................................................................... 32 2.5. Genotype data processing ................................................................................ 33 2.6. Statistical association analysis .......................................................................... 35 3.0 Result .................................................................................................................... 37 3.1. Batch Effect (Plate - Plate) ................................................................................ 37 3.2. Hardy Weinberg Equilibrium Test (HWE) .......................................................... 37 3.3. Allele frequencies .............................................................................................. 37 3.4. Statistical analysis - Logistic regression ............................................................. 39 3.4.1. Unadjusted ................................................................................................. 39 3.4.2. Adjusted: Stratified by race with HIV status as covariant .......................... 40 V 3.4.3. Haplotype analysis ...................................................................................... 41 3.5. Linkage Disequilibrium analysis ........................................................................ 42 4.0 Discussion .............................................................................................................. 45 4.1. Logistic regression, Linkage Disequilibrium and Haplotype analysis ................ 47 4.2. Biological importance of statistical significant SNPs in this study .................... 51 4.2.1. TLR1 rs5743618 .......................................................................................... 51 4.2.2. TLR1 rs5743551 .......................................................................................... 52 4.2.3. TLR4 rs10759932 ........................................................................................ 53 4.2.4. TLR6 rs5743810 .......................................................................................... 53 4.2.5. TLR6 rs1039559 .......................................................................................... 54 4.2.6 Biological summary ...................................................................................... 54 5.0 Conclusion ............................................................................................................. 56 Reference .................................................................................................................... 58 VI List of Tables Table 1: Differences between Innate and Adaptive Immunity ...................................... 2 Table 2: Innate immunity germ line encoded receptors-the pattern recognition receptors, PAMPs and function. .................................................................................... 5 Table 3: TLR and associated PAMPs ............................................................................. 15 Table 4: TLRs and associated adaptor recruitment ..................................................... 18 Table 5: Summary of studies that have been done and what was found with infection/disease.......................................................................................................... 24 Table 6: Individual demography in the study .............................................................. 33 Table 7: Fishers Exact t-test plate to plate variation results ....................................... 37 Table 8: Observed allele frequency in HIV status and within each population........... 38 Table 9: Logistic regression statistical analysis results showing only significant alleles at p < 0.05 .................................................................................................................... 39 Table 10: Additive and Dominant genetic model test statistical analysis results showing significant alleles at p < 0.05 in AfA population ............................................ 40 Table 11: Additive and Dominant genetic model test statistical analysis results showing significant alleles at p < 0.05 in CA population ............................................. 41 Table 12: Haplotype analysis results showing only significant alleles at p < 0.05 in CA population in CA group