Genetic Epidemiology of Atopy and Asthma By Yize I Wan, BMedsci. (Hons) Thesis submitted to the University of Nottingham for the degree of Doctor of Philosophy May 2011 To my family, with all my love Contents ABSTRACT ..........................................................................................................................I ACKNOWLEDGEMENTS ................................................................................................ II ABBREVIATIONS ............................................................................................................III PUBLICATIONS RELEVANT TO THESIS .................................................................... IV CHAPTER 1 ........................................................................................................................ 1 GENERAL INTRODUCTION ............................................................................................ 1 1.1 ALLERGIC DISEASE ................................................................................................... 1 1.1.1 Definition ............................................................................................................. 1 1.1.2 Epidemiology ....................................................................................................... 1 1.1.3 Pathogenesis ........................................................................................................ 1 1.1.4 Allergens .............................................................................................................. 4 1.1.5 Atopy ................................................................................................................... 6 1.1.6 Clinical Manifestations of Allergic Disease........................................................... 7 1.1.7 Management ...................................................................................................... 10 1.1.8 Susceptibility to Disease ..................................................................................... 12 1.2 GENETICS OF COMPLEX DISEASE ................................................................................ 15 1.2.1 Statistical Approaches ........................................................................................ 15 1.2.2 Linkage Analysis ................................................................................................ 16 1.2.2 Association Analysis ........................................................................................... 17 1.2.3 Genome-Wide Association (GWA) Studies .......................................................... 18 1.3 GENETICS OF ALLERGIC DISEASE ................................................................................ 20 1.3.1 Genetic Studies of Allergic Disease..................................................................... 20 1.3.2 Genome–Wide Linkage (GWL) Studies ............................................................... 20 1.3.3 Meta-Analysis of GWL Studies ............................................................................ 24 1.3.4 Candidate Gene Studies ...................................................................................... 25 1.3.5 GWA Studies ...................................................................................................... 27 1.3.6 Gene-Gene Interactions ...................................................................................... 29 1.3.7 Gene-Environment Interactions .......................................................................... 29 1.4 THE „MISSING HERITABILITY‟ COMPONENT................................................................. 31 1.4.1 Deficiencies in Previous Studies ......................................................................... 31 1.4.2 Next-Generation Sequencing (NGS) .................................................................... 33 1.5 SUMMARY .................................................................................................................. 35 1.6 PROJECT AIMS ............................................................................................................ 37 CHAPTER 2 ...................................................................................................................... 38 MATERIALS AND METHODS ....................................................................................... 38 2.1 STUDY POPULATIONS AND PHENOTYPING .................................................................... 38 2.1.1 Atopy Analyses ................................................................................................... 38 2.1.2 Asthma Analyses ................................................................................................ 43 2.2 GENOTYPING AND SEQUENCING .................................................................................. 48 2.2.1 Genome-Wide SNP Genotyping .......................................................................... 48 2.2.2 Direct SNP Genotyping (Atopy Analyses)............................................................ 52 2.2.3 Targeted Re-sequencing (Asthma Analyses) ........................................................ 54 2.2.4 Dye Terminator Sequencing................................................................................ 54 2.3 BIOINFORMATICS........................................................................................................ 56 2.3.1 Online Resources ............................................................................................... 56 2.3.2 Genetics Analysis Software ................................................................................. 57 2.4 STATISTICAL ANALYSES ............................................................................................. 58 2.4.1 Principal Components Analysis (PCA) ................................................................ 58 2.4.2 Association Testing ............................................................................................ 58 2.4.3 Test Inflation ...................................................................................................... 60 2.4.4 Statistical Power ................................................................................................ 61 2.4.5 Meta-Analysis .................................................................................................... 61 2.4.6 Linkage Disequilibrium (LD) .............................................................................. 61 2.4.7 Imputation.......................................................................................................... 62 2.4.8 Significance Testing ........................................................................................... 63 2.5 CELLULAR AND MOLECULAR TECHNIQUES .................................................................. 64 2.5.1 Cell Culture ....................................................................................................... 64 2.5.2 Molecular Biology Methods ................................................................................ 66 2.6 INDIVIDUAL CONTRIBUTIONS AND COLLABORATIONS .................................................. 72 CHAPTER 3 ...................................................................................................................... 73 GENOME-WIDE ASSOCIATION STUDY TO IDENTIFY NOVEL ATOPY SUSCEPTIBILITY LOCI ................................................................................................. 73 3.1 INTRODUCTION ........................................................................................................... 73 3.2 AIM ........................................................................................................................... 74 3.3 METHODS .................................................................................................................. 75 3.3.1 Study Populations............................................................................................... 75 3.3.2 Association Testing ............................................................................................ 76 3.3.3 Statistical Significance Testing ........................................................................... 77 3.3.4 SNP Selection for Replication Studies ................................................................. 77 3.3.5 Candidate Gene Expression ................................................................................ 77 3.3.6 Evaluation of Previously Reported Atopy Genes.................................................. 78 3.4 RESULTS .................................................................................................................... 79 3.4.1 Discovery GWA Study ........................................................................................ 79 3.4.3 In silico Replication ........................................................................................... 83 3.4.4 Replication Genotyping ...................................................................................... 83 3.4.5 Total IgE Analyses ............................................................................................. 83 3.4.6 Combined Analyses ............................................................................................ 83 3.4.7 Meta-Analysis .................................................................................................... 87 3.4.8 Candidate Gene Expression ...............................................................................