GENOME-WIDE ASSOCIATION STUDIES OF CHILDHOOD BONE HEALTH by Kelly Amanda Johnson BS, University of Maryland, College Park, 2014 Submitted to the Graduate Faculty of the Department of Human Genetics in the Graduate School of Public Health in partial fulfillment of the requirements for the degree of Master of Science and Master of Public Health University of Pittsburgh 2016 UNIVERSITY OF PITTSBURGH GRADUATE SCHOOL OF PUBLIC HEALTH This thesis was presented by Kelly Amanda Johnson It was defended on April 5, 2016 and approved by Committee Chair: John R. Shaffer, PhD Assistant Professor, Department of Human Genetics Graduate School of Public Health, University of Pittsburgh Committee Member: Andrea L. Durst, MS, DrPH, LCGC Assistant Professor and Assistant Director of Genetic Counseling Program Department of Human Genetics Graduate School of Public Health, University of Pittsburgh Committee Member: Allison L. Kuipers, PhD Visiting Assistant Professor, Department of Epidemiology Graduate School of Public Health, University of Pittsburgh ii Copyright © by Kelly Johnson 2016 iii John R. Shaffer, PhD GENOME- WIDE ASSOCIATION STUDIES OF CHILDHOOD BONE HEALTH Kelly Johnson, MS/MPH University of Pittsburgh, 2016 ABSTRACT Osteoporosis is a major public health concern characterized by low bone mineral density (BMD) and deterioration of bone tissue, causing increased bone fragility and risk of fracture. Though current research has focused primarily on bone health in the elderly, early bone health, including peak BMD attainment, is a strong predictor of bone health later in life. Twin and family studies have demonstrated a strong genetic component in peak BMD, though the specific genes influencing variation in bone development are largely unknown. Moreover, the question of whether the genes influencing bone health during childhood are the same as those influencing bone health later in life is currently unknown. Therefore, to identify variants and genes implicated in childhood bone health, we performed separate genome-wide association studies (GWAS) for ten bone health phenotypes (bone mineral content [BMC] and BMD of the hip, spine, and head, BMC of the whole body, and four measures of hip geometry) in 296 Caucasian children aged 5 years (mean = 5.3) who were enrolled in the Iowa Bone Development Study. Linear regression while adjusting for sex, height, and weight was used to test 548,051 genetic polymorphisms and 7.4 million imputed variants for evidence of association. Genomic regions showing statistical association were scrutinized for relevant gene functions related to bone biology. Five genome- wide significant (P≤5x10-8) and 30 suggestive (P<10-6) loci were identified in total. Implicated genes may represent significant roles in the converging pathways that regulate BMD, embryonic bone development, and bone remodeling. Furthermore, understanding the genetic determinants of iv bone health during childhood may have implications across the lifespan. Though osteoporosis is usually viewed as an age-related disorder, risk of osteoporosis is impacted much earlier in life, including phases of bone mineral acquisition during youth. Therefore, the public health significance of this study is that identifying the genetic factors contributing to early skeletal health may ultimately lead to screening programs, which identify children with a genetic predisposition to bone disease. This allows for targeted interventions to optimize bone health in adolescence, promote management of bone health across the lifespan, and lower risk for osteoporosis later in life. v TABLE OF CONTENTS PREFACE ................................................................................................................................. XIII 1.0 INTRODUCTION ........................................................................................................ 1 1.1 BACKGROUND .................................................................................................. 2 1.1.1 Bone Function and Composition .................................................................... 2 1.1.2 Bone Cells ......................................................................................................... 3 1.1.3 Bone Mineral Density and Content ................................................................ 5 1.1.3.1 Bone Densitometry in Children and Adolescents............................... 6 1.1.4 Peak Bone Mass ............................................................................................... 6 1.1.5 Genetic Regulators of Bone Biology ............................................................... 7 1.1.5.1 Candidate Genes Implicated in Previous Literature ......................... 7 1.1.5.2 Previous Published GWAS of Bone Health ........................................ 9 1.1.5.3 Polymorphisms Known to Influence BMD ....................................... 10 1.1.5.4 Genetic Syndromes Associated with Low BMD ............................... 11 1.1.5.5 Agonistic Pleiotropy ............................................................................ 12 1.1.6 Environmental Factors and Regulators of Bone Health ............................ 13 1.1.6.1 Nutrients .............................................................................................. 13 1.1.6.2 Physical Activity .................................................................................. 15 1.1.6.3 Hormones ............................................................................................. 16 1.1.7 Osteoporosis ................................................................................................... 18 1.1.7.1 Osteoporosis and Public Health Burden ........................................... 18 1.1.7.2 Risk Factors for Osteoporosis ............................................................ 20 vi 1.1.7.3 Current Interventions for Osteoporosis ............................................ 21 1.2 PUBLIC HEALTH SIGNIFICANCE OF GWAS OF CHILDHOOD BONE PHENOTYPES ................................................................................................................... 23 1.3 RESEARCH QUESTIONS AND SPECIFIC AIMS ...................................... 24 1.3.1 Research Questions........................................................................................ 24 1.3.2 Specific Aims .................................................................................................. 25 2.0 METHODS ................................................................................................................. 26 2.1 STUDY POPULATION .................................................................................... 26 2.2 BONE PHENOTYPES ...................................................................................... 27 2.3 COVARIATES ................................................................................................... 28 2.4 GENOTYPING AND IMPUTATION ............................................................. 28 2.5 PRINCIPAL COMPONENTS OF ANCESTRY ............................................ 29 2.6 STATISTICAL ANALYSIS AND RESULTS ANNOTATION .................... 30 3.0 RESULTS ................................................................................................................... 33 3.1 BONE MINERAL DENSITY GWAS .............................................................. 35 3.1.1 Hip Bone Mineral Density GWAS ............................................................... 35 3.1.2 Spine Bone Mineral Density GWAS ............................................................ 40 3.1.3 Head Bone Mineral Density GWAS ............................................................. 41 3.2 BONE MINERAL CONTENT GWAS ............................................................ 42 3.2.1 Hip Bone Mineral Content GWAS .............................................................. 42 3.2.2 Spine Bone Mineral Content GWAS ........................................................... 44 3.2.3 Head Bone Mineral Content GWAS ............................................................ 45 3.2.4 Whole Body (Excluding Head) Bone Mineral Content GWAS ................. 47 vii 3.3 BONE GEOMETRY GWAS ............................................................................ 49 3.3.1 Femoral Neck Cross-Sectional Area GWAS ............................................... 50 3.3.2 Femoral Neck Section Modulus GWAS ...................................................... 52 3.3.3 Femoral Neck Width GWAS ........................................................................ 58 4.0 DISCUSSION ............................................................................................................. 60 4.1 LIMITATIONS .................................................................................................. 67 4.2 FUTURE WORK ............................................................................................... 69 4.2.1 A Priori Research .......................................................................................... 69 4.2.2 Genetic Risk Score ......................................................................................... 70 4.2.3 Longitudinal Data .......................................................................................... 71 4.3 CONCLUSIONS ................................................................................................ 71 5.0 PUBLIC HEALTH APPLICATION ....................................................................... 73 5.1 PREVIOUS EXAMPLES OF PUBLIC HEALTH GENETIC SCREENING PROGRAMS