The Genetic and Epigenetic Determinants of Osteoporosis John Allan Morris Department of Human Genetics Faculty of Medicine McGill University, Montréal November 2018 A thesis submitted to McGill University in partial fulfillment of the requirements of the degree of Doctor of Philosophy © John Allan Morris 2018 1 Table of Contents Abstract/Résumé ........................................................................................................................... 8 Abstract ...................................................................................................................................... 8 Résumé ....................................................................................................................................... 9 List of Abbreviations .................................................................................................................. 11 List of Tables ............................................................................................................................... 17 List of Figures .............................................................................................................................. 18 Acknowledgments ....................................................................................................................... 20 Preface .......................................................................................................................................... 25 Contribution of Authors ......................................................................................................... 25 Original Contribution to Knowledge .................................................................................... 29 Chapter 1: Introduction ............................................................................................................. 31 1.1 Osteoporosis and bone mineral density .......................................................................... 31 1.2 Studying bone mineral density instead of bone fracture ............................................... 31 1.3 Different measurement sites of bone mineral density ................................................... 32 1.4 Genetic determinants of bone mineral density ............................................................... 32 1.5 Fine-mapping of genetic loci for causal variation .......................................................... 34 1.6 Integrative analyses with functional genomics data ...................................................... 35 1.7 Epigenetic determinants of bone mineral density .......................................................... 36 1.8 Rationale, objectives and hypothesis ............................................................................... 37 Chapter 2 ..................................................................................................................................... 39 Preface: Bridge Between Chapter 1 and Chapter 2 ............................................................ 39 2.1 Abstract .............................................................................................................................. 43 2 2.2 Introduction ....................................................................................................................... 44 2.3 Results ................................................................................................................................ 46 2.3.1 Genome-wide association study of eBMD................................................................ 46 2.3.2 Effects on fracture ...................................................................................................... 49 2.3.3. Shared genetic factors............................................................................................... 50 2.3.4 Gene Prioritization..................................................................................................... 50 2.3.5 GPC6 Findings ........................................................................................................... 53 2.4 Discussion........................................................................................................................... 55 2.5 Online Methods ................................................................................................................. 59 2.5.1 Measurement of eBMD, fracture and weight in UK Biobank ............................... 59 2.5.2 Preparation, quality control and genetic analysis in UK Biobank samples ......... 60 2.5.3 Estimation of genome-wide significance threshold ................................................. 62 2.5.4 Approximate conditional association analysis......................................................... 63 2.5.5 Estimation of variance explained by significant variants and SNP heritability .. 63 2.5.6 Linkage disequilibrium score regression ................................................................. 64 2.5.7 Gene prioritization and pathway analysis ............................................................... 65 2.5.8 Prioritising candidate genes and possible causal variants at each eBMD locus .. 66 2.5.9 Genetically modified animals used for functional validation ................................ 68 2.5.10 OBCD methods......................................................................................................... 68 2.5.11 Gene expression in primary human and mouse osteoblasts ................................ 70 2.5.12 Gene expression in murine osteocytes .................................................................... 71 2.5.13 Gene expression in mouse osteoclasts .................................................................... 72 2.5.14 URLs.......................................................................................................................... 72 3 2.6 Acknowledgements ........................................................................................................... 73 2.8 Tables and Figures ............................................................................................................ 74 2.8.1 Tables .......................................................................................................................... 74 2.8.2 Figures ......................................................................................................................... 76 2.9 Supplementary Tables and Figures ................................................................................. 82 Chapter 3 ..................................................................................................................................... 83 Preface: Bridge Between Chapter 2 and Chapter 3 ............................................................ 83 3.1 Abstract .............................................................................................................................. 88 3.2 Introduction ....................................................................................................................... 89 3.3 Results ................................................................................................................................ 92 3.3.1 GWAS for eBMD and Fracture................................................................................ 92 3.3.2 Sex Heterogeneity....................................................................................................... 93 3.3.3 Coding Variants ......................................................................................................... 94 3.3.4 Fine-Mapping Associated Loci ................................................................................. 95 3.3.5 Comparing Fine-Mapped SNPs for Biological Activity ......................................... 96 3.3.6 Mapping Fine-Mapped SNPs to Target Genes & Enrichment for Positive Control Genes .................................................................................................................................... 96 3.3.7 Mapping Fine-Mapped SNPs to Osteocyte-Signature Genes ................................ 98 3.3.8 A Large-Scale High Throughput Murine Knockout Screening Program ............ 99 3.3.9 In-Depth Characterization of DAAM2 .................................................................. 100 3.3.10 Additional Novel Candidate Bone Genes ............................................................. 102 3.4 Discussion......................................................................................................................... 106 3.5 Online Methods ............................................................................................................... 110 4 3.5.1 Curating osteoporosis associated outcomes in the UK Biobank study ............... 110 3.5.2 Ancestry assignment ................................................................................................ 112 3.5.3 Identification of unrelated samples for LD reference estimation and X chromosome analyses........................................................................................................ 113 3.5.4 Genome-wide association analysis .......................................................................... 113 3.5.5 Fracture replication meta-analysis ......................................................................... 115 3.5.6 Approximate conditional association analysis....................................................... 115 3.5.7 Estimation of variance explained by significant variants