Genetic Factors Underlying Disease and Performance Traits in Standardbreds A DISSERTATION SUBMITTED TO THE FACULTY OF THE UNIVERSITY OF MINNESOTA BY Annette Marie McCoy IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY Molly E. McCue, Adviser and Troy N. Trumble, Co-Adviser May 2014 © 2014 Annette Marie McCoy Acknowledgements Thank you to everyone who has supported me throughout this process. This is not a journey that I could have taken alone – nor would I have wanted to. My life is richer for having known and worked with you all. My advisors and committee members: Thank you all for your unflagging support and encouragement throughout my time here. I have learned more from you than I could have imagined. In particular: Dr. Molly McCue: Thank you for giving me the freedom to grow and the support to be successful. I am unquestionably a better scientist, writer, and teacher because of your guidance, but knowing that you care about me as a person has been your greatest gift. Dr. Troy Trumble: You help me to remember that I can be both a surgeon and a scientist. Thank you so much for supporting me in my goal to do just that. Dr. James Mickelson: A willing ear and a critical eye – thank you for sharing both with me without reservation whenever I needed them. Dr. Cathy Carlson: Thank you for making this all possible – without your belief in me as a T32 candidate, it would have been much harder to even get in the door. Dr. Denis Clohisy: Thank you for pinch hitting in the ninth inning. I so appreciate your generosity in being a part of all this despite your already packed schedule. My lab mates and fellow graduate students: Thank you for letting me bounce ideas off of you, commiserate with you when things got frustrating, celebrate successes with you, and learn from you every day. i Our fabulous technicians: Thank you for showing me the ropes and being patient when I asked the same questions over and over. A special thank you to Shea Anderson as well as our undergraduates/lab assistants Leah Freilich and Nicole Tate for providing technical support for the projects reported in this thesis. To my family: You may not have entirely understood why I wanted to get a PhD when I was “already a doctor,” but you never stopped believing in me or encouraging me. Thank you for a lifetime of support and love. In particular, thank you to my wonderful husband Jeffrey Fox – you have been my rock through the highs and lows of the past few years, and without your commitment to me and my goals, I never would have made it this far. Thank you for telling me that I could do it, even when I wasn’t sure I could. Finally, to my son Luke, who arrived halfway through this adventure: you are the light of my life, and your smile reminds me that no matter what happens at work, my most important job is always waiting for me at home. ii Dedication Willard George Sampson (November 1927 – December 2013) and Elizabeth Ann Kimmel Sampson Dearest grandparents, you helped to teach me how to read a book, sew a seam, solve algebraic equations. More than that, you engendered a love of learning new things and a belief that I could accomplish any goal that I set for myself. For as long as I can remember, you have been interested in what I was doing and how it fit into the world, asking insightful questions and listening with respect to my responses. I could never ask for better role models. So, even though Grandpa is not here to read it, I dedicate this culmination of “what I’ve been doing” to you both with love. iii Abstract Annette M. McCoy 342 words Many diseases and performance characteristics of the horse are considered to be “complex” traits because they are influenced by both genetic and environmental factors. Furthermore, many are polygenic in nature, reflecting the combined effects of multiple genes. Traditional methodological approaches, such as family linkage analysis and candidate gene sequencing are not ideal for identifying the multiple interacting alleles underlying complex/polygenic traits. An alternative investigational approach is needed that can account for environmental risk factors, issues related to population structure in large study cohorts, and epistatic interactions. In the work presented here, whole-genome approaches, including genome-wide association (GWA) analysis, whole-genome sequencing (WGS), and high-throughput genotyping, were used to investigate the genetic factors underlying three complex traits in Standardbred horses, a breed primarily used for harness racing. These were 1) osteochondrosis (OC; a disease of young horses in which the cartilage at the end of long bones does not form normally); 2) pacing (an alternative pattern of locomotion); and 3) performance (using speed as the phenotype). GWA analysis identified chromosomal regions of association for all three traits of interest, although the significance of the findings for speed was marginal, reflecting the challenge of appropriately phenotyping a complex trait such as performance. WGS performed in eighteen horses identified thousands of variants within chromosomal regions of association identified for OC and pacing, of which a small fraction were predicted to have functional effect. These variants were prioritized and a subset was selected for high-throughput genotyping in the study cohorts (180 horse phenotyped for OC, 500 phenotyped for gait). A few of the markers selected for OC were moderately associated with disease status, while the majority of the markers selected for gait were highly associated with this trait. A crucial next step for interpreting these data will be trying to understand the potential interactions between markers, using a combination of pathway analysis and random forest analysis. Knowledge of gene variants that affect complex traits in the horse – and how they interact with each other – may help reduce the incidence of disease and assist selection for desirable characteristics. iv Table of Contents List of Tables viii List of Figures xi Chapter 1 Introduction and Literature Review 1 History and Use of the Standardbred Horse 2 Factors Affecting Performance in the Standardbred Horse 4 The Importance of Osteochondrosis in the Standardbred Horse 6 Challenges to Mapping Complex Traits in the Horse 8 Hypotheses and Specific Aims 14 Part I: Genetic Risk Factors Underlying Osteochondrosis in the Standardbred Chapter 2 Articular Osteochondrosis: A Comparison of Naturally-Occurring Human 20 and Animal Disease Summary 22 Introduction 24 Disease Terminology 25 Clinical Aspects of Osteochondrosis in Humans and Animals 26 Prevalence of Osteochondrosis 28 Proposed Pathogenesis and Risk Factors 29 Evidence for Disturbances of Endochondral Ossification Leading to 35 Osteochondrosis Shared Aspects of Human and Animal Osteochondrosis 38 Conclusion 40 Chapter 3 Short- and Long-Term Racing Performance of Standardbred Pacers 50 and Trotters After Early Surgical Intervention for Tarsal Osteochondrosis Summary 52 Introduction 54 Materials and Methods 55 Results 59 Discussion 64 v Supplemental Methods 79 Supplemental Results 86 Chapter 4 A Genome-Wide Association Study of Tarsal Osteochondrosis in North 111 American Standardbreds Summary 113 Introduction 115 Materials and Methods 120 Results 124 Discussion 126 Chapter 5 Validation of Imputation Between Equine Genotyping Arrays 148 Background 150 Methods 150 Results/Conclusions 150 Supplemental Material 152 Chapter 6 Investigation of Putative Risk Alleles for Osteochondrosis in the Horse 168 Summary 170 Introduction 171 Materials and Methods 173 Results 180 Discussion 182 Part II: Genetic Determinants of Gait and Performance in the Standardbred Chapter 7 Investigation of Putative Modifying Variants Underlying Pacing in the 203 Standardbred Summary 205 Introduction 207 Materials and Methods 209 Results 216 Discussion 221 vi Chapter 8 Investigation of Genetic Determinants of Performance in the Standardbred 244 Horse Summary 246 Introduction 248 Materials and Methods 251 Results 254 Discussion 258 Chapter 9 Conclusions and Future Directions 271 Conclusions 272 Future Directions 278 References 285 Appendix 1: Sequencing Histone Deacetylase 4 (HDAC4) 315 Appendix 2: Approval for inclusion of previously published material 325 vii List of Tables Chapter 1 Table 1: Prevalence of OC by breed and predilection site 18 Chapter 2 Table 1: Disease names for OC at different anatomical locations 42 Chapter 3 Table 1: Summary of 2-year-old performance for all horses 72 Table 2: Summary of 2-year-old performance for pacers and trotters 73 Table 3: Summary of 2-year-old performance for OC-affected horses 74 Table 4: Multiple regression results for number of starts at 2 years 75 for OC-affected horses Supplemental Table 1: Summary of 2- through 5-year old performance 76 Supplemental Methods Table 1: Outcome and predictor variables included in regression models 85 Supplemental Results Table 1: Multiple regression, 2-year-old, all horses 87 Table 2: Yearling sales price ANOVA, all horses 89 Table 3: Comparison of 2-year-old performance between study and 89 random cohorts Table 4: OC status ANOVA, all horses 90 Table 5: Multiple regression, 2-year-old, pacers 90 Table 6: Yearling sales price ANOVA, pacers 92 Table 7: Multiple regression, 2-year-old, trotters 92 Table 8: Yearling sales price ANOVA, trotters 94 Table 9: Multiple regression, OC lesion location and distribution 94 Table 10: DIRT ANOVA, OC-affected horses 95 Table 11: MM ANOVA, OC-affected horses 95 Table 12: LTR ANOVA, OC-affected horses 95 Table 13: Bilateral