Identification of causal rare variants in an extended pedigree with Obsessive-Compulsive Disorder By Rageen Rajendram A thesis submitted in conformity with the requirements for the degree of Master of Science In the Graduate department of the Institute of Medical Science University of Toronto © Copyright by Rageen Rajendram (2014) Identification of causal rare variants in an extended pedigree with Obsessive-compulsive disorder Master of Science, 2014 Institute of Medical Science, University of Toronto Abstract Obsessive-Compulsive Disorder (OCD) is a common, heritable and etiologically heterogeneous neuropsychiatric disorder. Despite twin and family studies supporting genetic determinants in OCD, the discovery of causal variants remains elusive. To identify causal variants, an extended pedigree consisting of multiple affected individuals with early- onset OCD was studied. Genome-wide linkage analysis was performed using both parametric and non-parametric approaches. Whole exome sequencing was conducted on two of the most distantly related affected individuals who shared a haplotype. Variants were filtered based on their presence in both individuals, rare frequency (MAF≤0.05), and location within linkage regions. Genes previously implicated in OCD were also screened. Several putatively damaging low/rare frequency variants were identified in genes involved in immune function (TRAFD1) and glutamate pathways (DLG1, CPQ, SLC1A1). Our findings identified variants within genes/pathways known to be associated with OCD and demonstrated the ability of our methodology to identify rare variants in common complex diseases. ii Acknowledgements This thesis is dedicated to the families and children who live with Obsessive-Compulsive Disorder every day. Thank you for donating your time and energy so we can better understand your condition. First and foremost, I would like to thank Dr. Paul Arnold for taking me on as a graduate student and for providing me with this amazing opportunity to learn and grow. I am also indebted to Christie Burton, S-M Shaheen, Julie Coste, Laura Park, Vanessa Sinopoli, Bingbin Li, and Lauren Erdman from the Arnold Lab for their support over the past two years. Next, I would like to acknowledge my family for their tremendous support and for teaching me to follow my heart without ever giving up. Thank you to my mother and father for giving me much needed guidance and my brother Praveen for giving me a path to follow. I will always be grateful for all you have done and continue to do for me. To my closest friend, thank you for cheering me on and making these two years some of the best of my life. I am glad to have made a discovery that will last a lifetime. I am thankful for the support of Christian Marshall, Susan Walker, Matt, Anath Lionel, and Stephen Scherer from the Scherer Lab. I am also grateful for the support of Darci Butcher, Dasha Grafodatskya, Yi-an Chen, Sanaa Choufani and Rosanna Weksberg from the Weksberg Lab. Thank you to the IMS Students’ Association, and the staff at IMS for all their support and guidance. A special thanks to 216 who have been with me since day one. Finally, thank you to my committee members Dr. Andrew Paterson and Dr. John Vincent for your guidance through all aspects of this project. iii Contributions Dr. Paul Arnold was involved initial study design, and funding acquisition. Dr. Gregory Hanna assessed the family’s phenotype and acquired DNA for genetic studies at the University of Michigan. S-M Shaheen and Julie Coste assisted with sample acquisition, and DNA extraction. I prepared all samples for genotyping and whole exome sequencing at The Center for Applied Genomics (TCAG) at SickKids. I also developed most of the analysis pipeline, analyzed all data and interpreted results. Dr. Andrew Paterson and Nicole Roslin assisted with genome-wide linkage analysis and Christian Marshall, Lynette Lau, and Susan Walker assisted with whole exome sequencing analysis. I conducted primer design, and PCR for sequencing validation of WES variants. Christie Burton helped with thesis editing, defense preparation, and mentorship. The McLaughlin Centre at the University of Toronto funded this study. I was supported by an Institute of Medical Science Entrance Scholarship, CIHR Masters award and a SickKids Research Training Competition Award. iv Table of Contents ABSTRACT………………………………………………………………………...………………..……………………..ii ACKNOWLEDGMENTS………………………………………………………………….………….…………..……iii CONTRIBUTIONS……………………………………………………………………………….……...…..….………iv TABLE OF CONTENTS………………………………………………………..……………...…..…………………...v LIST OF ABBREVIATIONS……………………………………………………….…..…………………………..viii LIST OF FIGURES………………………..…………………………………………………………..…………………ix LIST OF TABLES……………………………………………………………………………………...………….………x 1 Chapter 1: OCD, where we are today ....................................................................................................... 1 1.1 Obsessive-Compulsive Disorder, a common complex disease .............................................. 1 1.1.1 Prevalence and Symptomology ........................................................................................... 1 1.1.2 Assessment ................................................................................................................................. 2 1.1.3 Comorbidities............................................................................................................................. 3 1.1.4 Distinguishing between early-onset and late-onset OCD ......................................... 3 1.2 Genetics of OCD ....................................................................................................................................... 4 1.2.1 Heritability .................................................................................................................................. 4 1.2.2 Linkage studies in OCD ........................................................................................................... 7 1.3 Candidate Genes, GWAS, and the Glutamate Hypothesis ..................................................... 14 1.3.1 SLC1A1 (EAAC1/EAAT3) ................................................................................................... 14 1.3.2 Genome-wide association studies ................................................................................... 16 1.3.3 Copy-number variations in OCD ...................................................................................... 20 1.3.4 Environmental factors ......................................................................................................... 21 v 1.4 Genetic Heterogeneity in OCD: a case for rare variants ........................................................ 22 1.5 Next Generation Sequencing ........................................................................................................... 26 1.5.1 Enrichment and Sequencing of DNA .............................................................................. 28 1.5.2 SNP Calling Pipeline ............................................................................................................. 29 1.5.3 Annotation................................................................................................................................ 29 1.6 Approaches for Identifying Rare variants in complex diseases ........................................ 30 1.7 Hypothesis .............................................................................................................................................. 33 2 Methodology................................................................................................................................................... 35 2.1 Subject Ascertainment and Diagnosis ......................................................................................... 35 2.2 Genotyping and Linkage Analysis ................................................................................................. 41 2.2.1 SNP Quality Control .............................................................................................................. 43 2.2.2 Linkage Analysis .................................................................................................................... 43 2.3 CNV Analysis .......................................................................................................................................... 45 2.4 Whole Exome Sequencing ................................................................................................................ 45 2.4.1 Variant filtration .................................................................................................................... 45 2.5 Validation by Sanger Sequencing .................................................................................................. 48 2.6 Preliminary Screening of SLC1A1 variant (rs34342853) in an additional cohort of OCD cases and controls ..................................................................................................................... 48 3 Results .............................................................................................................................................................. 50 3.1 Linkage Analysis................................................................................................................................... 50 3.1.1 Parametric Linkage Analysis ............................................................................................. 50 3.1.2 Non-parametric