Capturing epigenomes at high-resolution for insight into genome function and metabolic disease risk Fiona Allum Department of Human Genetics Faculty of Medicine McGill University, Montréal April 2019 A thesis submitted to McGill University in partial fulfillment of the requirements of the degree of Doctor of Philosophy © Fiona Allum 2019 “It does not do to dwell on dreams and forget to live” - Albus Dumbledore (J.K. Rowling) 2 Table of Contents ABSTRACT ..................................................................................................................... 6 RÉSUMÉ ......................................................................................................................... 8 LIST OF ABBREVIATIONS ......................................................................................... 10 LIST OF TABLES ......................................................................................................... 15 LIST OF FIGURES ....................................................................................................... 16 ACKNOWLEDGMENTS .............................................................................................. 18 PREFACE ...................................................................................................................... 20 Format of the Thesis ................................................................................................................. 20 Contribution of Authors ............................................................................................................ 21 Original Contribution to Knowledge ........................................................................................ 23 CHAPTER 1: INTRODUCTION .................................................................................. 26 1.1 Studying Complex Traits: Focus on Metabolic Diseases .................................................. 26 1.1.1 Pathophysiology and Socioeconomic Burden ......................................................................... 26 1.1.2 Environmental and Genetic Factors Contributing to Disease .............................................. 27 1.2 Genome-wide Association Studies ..................................................................................... 28 1.2.1 GWAS of BMI, fat distribution and lipids .............................................................................. 28 1.2.2 Limitations of GWAS ............................................................................................................... 29 1.2.3 Missing Heritability in Common Diseases ............................................................................. 30 1.2.4 Integrational Studies to Interpret Genetic Variants ............................................................. 31 1.2.5 Expression Quantitative Trait Loci Studies .......................................................................... 32 1.3 Epigenetics to Link Environment and Genetics to Disease Phenotypes ......................... 33 1.3.1 What is Epigenetics? ............................................................................................................... 33 1.3.2 Reference Epigenome Efforts .................................................................................................. 34 1.3.3 Defining Regulatory Elements ................................................................................................ 35 1.3.4 Coordinated Patterns of Epigenetic Regulation ..................................................................... 38 1.4 DNA methylation ................................................................................................................ 39 1.4.1 Roles of DNA Methylation across the Genome ...................................................................... 40 1.4.2 Methods to Study DNA Methylation ...................................................................................... 41 1.4.3 Epigenome-wide Association Studies of Common Traits ...................................................... 42 1.5 Rationale, Hypothesis and Objectives ............................................................................... 45 CHAPTER 2: CHARACTERIZATION OF METHYLOMES BY NEXT-GENERATION CAPTURE SEQUENCING .......................................................................................... 48 2.1 Bridging Statement between Chapter 1 and 2 .................................................................. 48 2.2 Title, Authors and Affiliations ........................................................................................... 50 3 2.3 Abstract ............................................................................................................................... 53 2.4 Introduction ......................................................................................................................... 54 2.5 Results ................................................................................................................................. 58 2.5.1 First-generation Capture Panel Design for MCC-Seq ........................................................... 58 2.5.2 Second-generation Panel Design for Comprehensive Profiling ............................................. 59 2.5.3 Sample-based Validation of MCC-Seq .................................................................................... 60 2.5.4 Population-based Validation of MCC-Seq .............................................................................. 62 2.5.5 Population-based Genotype Profiling by MCC-Seq ............................................................... 65 2.5.6 EWAS of TG Levels using MCC-Seq ...................................................................................... 66 2.5.7 Assessment of Loci Harbouring TG-associated CpGs ............................................................ 68 2.5.8 Follow-up of the TG-associated Loci Mapping to CD36 ......................................................... 70 2.6 Discussion ............................................................................................................................ 72 2.7 Online Methods ................................................................................................................... 76 2.7.1 First-generation Panel Design ................................................................................................ 76 2.7.2 Generation of Second-general Panel ....................................................................................... 78 2.7.3 MCC-Seq Protocol .................................................................................................................... 79 2.7.4 MCC-Seq Methylation Profiling .............................................................................................. 80 2.7.5 Illumina 450K Array Methylation Profiling .......................................................................... 81 2.7.6 Agilent SureSelect CpG Profiling and MCC-Seq Comparisons ............................................ 82 2.7.7 Trait-association Discovery Cohort ......................................................................................... 83 2.7.8 DNA Isolation ........................................................................................................................... 84 2.7.9 Identification of Hypomethylated Regions ............................................................................. 84 2.7.10 Genotyping ............................................................................................................................. 85 2.7.11 Epigenome-wide Association of TG Levels ........................................................................... 86 2.7.12 Adipocyte Nuclei Isolation ..................................................................................................... 87 2.7.13 Transposase-accessible chromatin Sequencing .................................................................... 87 2.7.14 Blood Cell Isolation ................................................................................................................ 88 2.7.15 RNA Sequencing .................................................................................................................... 89 2.8 Acknowledgements .............................................................................................................. 90 2.9 Additional Information ....................................................................................................... 92 Accession codes ................................................................................................................................. 92 Competing financial interests .......................................................................................................... 92 The Multiple Tissue Human Expression Resource Consortium .................................................... 93 2.10 Main Tables and Figures .................................................................................................. 94 2.10.1 Tables ...................................................................................................................................... 94 2.10.2 Figures .................................................................................................................................... 95 2.11 Supplementary Materials ............................................................................................... 100 2.11.1 Supplementary Tables ........................................................................................................