STUDY OF GENOMIC COPY NUMBER VARIATION IN EQUINE HEALTH AND DISEASE A Dissertation by SHARMILA GHOSH Submitted to the Office of Graduate and Professional Studies of Texas A&M University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Chair of Committee, Terje Raudsepp Committee Members, Ernest Gus Cothran Penny K. Riggs Bhanu P. Chowdhary James Cai Head of Department, Evelyn Castiglioni August 2014 Major Subject: Biomedical Sciences Copyright 2014 Sharmila Ghosh ABSTRACT This is a study of copy number variations (CNVs) in the horse genome to gain knowledge about the role of CNVs in equine biology, and their contribution to complex diseases and disorders. We constructed a 400K whole-genome tiling array and applied it for the discovery of CNVs in 38 normal horses of 16 diverse breeds, and the Przewalski horse. Altogether, 258 CNV regions (CNVRs) were identified across all autosomes, chrX, and chrUn. The CNVRs comprised 1.3% of the horse genome with chr12 being most enriched. American Miniature Horses had the highest and American Quarter Horses the lowest number of CNVs in relation to Thoroughbred references. The Przewalski horse was similar to native ponies and draft breeds. About 20% of CNVRs were intergenic, while 80% involved 750 annotated genes with molecular functions predominantly in sensory perception, immunity, and reproduction. The findings were integrated with previous CNV studies in the horse to generate a composite genome-wide dataset of 1476 CNVRs. Of these, 301 CNVRs were shared between studies, while 1174 were novel and require further validation. Integrated data revealed that only 41 out of over 400 breeds of the domestic horse have been analyzed for CNVs, whereas this study added 11 new breeds. The composite CNV dataset served as a foundation for the discovery of variants contributing to Recurrent Airway Obstruction (RAO) and XY disorders of sexual development (DSDs), such as cryptorchidism and XY sex reversal. In 16 RAO affected ii horses 363 CNVRs were identified, of which 31 were novel and not found in healthy horses. A deletion in SPI2 and SERPINA1 was studied in detail because the genes are involved in respiratory diseases in human. In horses with XY DSDs, over 50 novel CNVRs were identified including deletions of functional interest in the pseudoautosomal region and the ATRX gene. A potentially causative homozygous deletion in chr29 disrupting AKR1C genes with functions in sex hormone metabolism was shared between a cryptorchid and two sex reversal horses. The findings effectively improved the knowledge about CNVs in horses, in health and disease, and generated resources for future studies. iii ACKNOWLEDGEMENTS I would like to thank my committee chair, Dr. Raudsepp, and my committee members, Dr. Cothran, Dr. Cai, Dr. Chowdhary, and Dr. Riggs, for their guidance and support throughout the course of this research. I want to thank my friends, Anindita Das, Nandina Paria, Anuradha Ghosh, Anrini Majumder who always helped me to go through all kind of situations. I also like to thank my colleagues, Pranab Jyoti Das, Felipe Avila, Fahad Alshanbari, Daisy Johnson, Priyanka Kachroo, Jana Caldwell, Joana Rocha, Alex Trott, Jan Janecka, and Samantha Steelman and the department faculty and staff for making my time at Texas A&M University a great experience. I also like to extent my gratitude to Ryan Doan, for his guidance on a very important part of my research. A special thanks to Dr. Jane Welsh, who helped a lot in difficult situations. Finally, thanks to my parents and brother for their encouragement, patience, and love. iv NOMENCLATURE CNV Copy Number Variants CNVR Copy Number Variable Region aCGH array Comparative Genomic Hybridization qPCR quantitative PCR FISH Fluorescent in situ Hybridization RAO Recurrent Airway Obstruction DSD Disorder of Sexual Development CO Cryptorchids v TABLE OF CONTENTS Page ABSTRACT .......................................................................................................................ii ACKNOWLEDGEMENTS .............................................................................................. iv NOMENCLATURE ........................................................................................................... v TABLE OF CONTENTS .................................................................................................. vi LIST OF FIGURES ........................................................................................................ viii LIST OF TABLES ............................................................................................................. x CHAPTER I INTRODUCTION AND LITERATURE REVIEW ................................... 1 The horse: domestication, breeds, importance ............................................................... 1 The horse genome .......................................................................................................... 2 Horse genome mapping .................................................................................................. 3 The genome sequence of the domestic horse ................................................................. 6 The development and application of advanced genome analysis tools ........................ 10 Genomics of equine diseases and traits ........................................................................ 16 Genomic Copy Number Variation (CNV) ................................................................... 20 Present status of CNV research in the horse ................................................................ 46 Prospective equine complex traits for CNV research .................................................. 49 Goals of this study ........................................................................................................ 70 CHAPTER II GENOMIC COPY NUMBER VARIATION IN EQUINE POPULATIONS ............................................................................................................... 72 Introduction .................................................................................................................. 72 Material and methods ................................................................................................... 75 Results ....................................................................................................................... 89 Discussion .................................................................................................................. 108 CHAPTER III ANALYSIS OF GENOMIC COPY NUMBER VARIATION IN EQUINE RECURRENT AIRWAY OBSTRUCTION .................................................. 121 Introduction ................................................................................................................ 121 Material and methods ................................................................................................. 124 Results ..................................................................................................................... 133 vi Page Discussion .................................................................................................................. 148 CHAPTER IV IDENTIFICATION OF CNVS IN EQUINE DISORDERS OF SEXUAL DEVELOPMENT (DSDS) – CRYPTORCHIDISM AND XY SRY- POSITIVE SEX REVERSAL ........................................................................................ 153 Introduction ................................................................................................................ 153 Material and methods ................................................................................................. 156 Results ..................................................................................................................... 164 Discussion .................................................................................................................. 181 CHAPTER V CONCLUSIONS AND FUTURE WORK ............................................. 187 REFERENCES ............................................................................................................... 190 APPENDIX .................................................................................................................... 284 vii LIST OF FIGURES Page Figure 1. Different mechanisms of CNV formation. ....................................................... 23 Figure 2: The molecular and genetic events in mammalian sex determination. .............. 58 Figure 3: Synthesis of dihydrotestosterone via the classic and alternative pathways. ..... 60 Figure 4: Genome-wide distribution of CNVs in self-to-self hybridization. ................... 90 Figure 5: Male-to-female aCGH for the X chromosome. ................................................ 91 Figure 6: Comparative DLRSD values. ........................................................................... 91 Figure 7: A CNVR map of the horse genome. ................................................................. 93 Figure 8: Confirmation of homozygous deletion CNVs by qualitative PCR. .................. 98 Figure 9: Chromosome-wise distribution of CNVRs in the horse genome. .................... 99 Figure10: GO classifications of copy number variable genes in horses. ....................... 100 Figure 11: A summary diagram for the CNVs identified in the horse genome. ............ 102 Figure 12: Validation of CNVRs by qPCR. ................................................................... 106 Figure 13: Validation of a copy number gain