Copy Number Variation in Han Chinese Individuals with Autism Spectrum Disorder by Matthew Joseph Gazzellone A thesis submitted in conformity with the requirements for the degree of Master of Science Department of Molecular Genetics University of Toronto © Copyright by Matthew Joseph Gazzellone 2014 Copy Number Variation in Han Chinese Individuals with Autism Spectrum Disorder Matthew Joseph Gazzellone Master of Science Department of Molecular Genetics University of Toronto 2014 Abstract Autism spectrum disorders (ASDs) are a group of neurodevelopmental conditions with a demonstrated genetic etiology. Rare copy number variations (CNVs) account for a proportion of the genetic events involved, but their contribution in non-European ASD populations has not been well studied. This thesis examines rare CNVs detected in a cohort of Han Chinese individuals with ASD. Using the Affymetrix CytoScan HD platform, we genotyped DNA from 104 ASD trios from Harbin, China. Of the probands, 8.6% had one or more de novo CNVs. Several candidate risk genes were also identified. A 24-kb duplication was found overlapping YWHAE (an ASD candidate gene). This duplication is observed at a similar frequency in cases and in population controls and is likely a benign Asian-specific copy number polymorphism. Our findings help define genomic features relevant to ASD in the Han Chinese and emphasize the importance of using ancestry-matched controls in medical genetic interpretations. ii Acknowledgments First and foremost, I would like to thank my supervisor Dr. Stephen Scherer for his mentorship over the past five years during both my undergraduate and graduate studies. It has been a privilege to come to the lab each day and work with such a brilliant scientist and genuinely good person. I greatly appreciate his confidence in me over these last five years. I would also like to thank my committee members, Dr. Freda Miller and Dr. Lucy Osborne. Their scientific advice and encouragement have been invaluable during my time as a student. I am grateful for the assistance I have received and the friendships that I have made at The Centre for Applied Genomics (TCAG). I would particularly like to thank my friend and former colleague, Dr. Anath Lionel. His advice and support have helped me become a better researcher. Many other members of the Scherer academic group and TCAG have provided assistance during the course of my time here. My profound gratitude goes to Dr. Susan Walker, Dr. Daisuke Sato, Dr. Christian Marshall, Dr. Kristiina Tammimies, Dr. Mohammed Uddin, Dr. Mehdi Zarrei, Dr. Ryan Yuen, Dr. Eric Deneault, Dr. Andy Pang, Lia D’Abate, Maggie Evans, Jenny Kaderali, Jennifer Howe, Dr. Richard Wintle, Sylvia Lamoureux, Bhooma Thiruvahindrapuram, and Sanjeev Pullenayegum. I would also like to acknowledge my friends and family who have been supportive throughout my studies. I am especially thankful for the encouragement and understanding of Nicole Lau during this degree. I would finally like to recognize my parents and sister. I thank them for instilling within me an interest in science and for their love and support during all of my educational endeavors. iii Table of Contents Acknowledgments .......................................................................................................................... iii Table of Contents ........................................................................................................................... iv List of Tables ................................................................................................................................. vi List of Figures ............................................................................................................................... vii List of Appendices ....................................................................................................................... viii Introduction ..................................................................................................................................... 1 1.1 Phenotypic Spectrum of ASD ............................................................................................. 1 1.2 Epidemiology ...................................................................................................................... 2 1.3 Genetic basis of ASD .......................................................................................................... 2 1.4 Genetic theories that may explain ASD .............................................................................. 3 1.4.1 Common Variant Model ......................................................................................... 5 1.4.2 Rare Variant of High Effect Model ........................................................................ 5 1.5 Copy Number Variation ...................................................................................................... 7 1.6 Copy Number Variation and Human Disease ..................................................................... 9 1.7 Identifying plausible candidate genes ............................................................................... 10 1.8 Neurobiology of ASD ....................................................................................................... 10 1.9 Microarray Studies ............................................................................................................ 12 1.10 ASD in the Han Chinese population ................................................................................. 13 1.11 Rationale and Objectives of the Thesis ............................................................................. 15 1.11.1 Rationale ............................................................................................................... 15 1.11.2 Hypothesis ............................................................................................................. 15 1.11.3 Objectives ............................................................................................................. 15 Disclosure ..................................................................................................................................... 16 Methods ......................................................................................................................................... 17 iv 2.1 Sample Selection ............................................................................................................... 17 2.2 Genotyping of Samples ..................................................................................................... 17 2.3 Variant Calling .................................................................................................................. 19 2.4 Population-based control datasets ..................................................................................... 19 2.5 Experimental Validation of CNV Calls ............................................................................ 23 2.6 Expression Analysis .......................................................................................................... 26 Results ........................................................................................................................................... 31 3.1 De novo Variants ............................................................................................................... 35 3.2 Rare Inherited Variants ..................................................................................................... 41 3.3 Population-Specific CNV Polymorphisms ....................................................................... 43 Discussion ..................................................................................................................................... 45 Summary and Significance ........................................................................................................... 49 Future Directions .......................................................................................................................... 53 Appendix ....................................................................................................................................... 57 References ..................................................................................................................................... 63 v List of Tables Table 1: Primers used for Expression Assays ............................................................................... 27 Table 2: Summary of de novo and rare inherited CNVs of interest in ASD probands. ................ 32 Table 3: Summary statistics of stringent CNVs larger than 20 kb ............................................... 33 vi List of Figures Figure 1: Contribution of Genetic and Non-Genetic Factors to ASD Phenotype ........................... 4 Figure 2: Ancestry Determination in ASD Cohort ....................................................................... 18 Figure 3: CNV Detection Workflow ............................................................................................. 21 Figure 4: Number of calls from each algorithm per sample ......................................................... 22 Figure 5: Agarose gel picture illustrating successful PCR amplification of FOXP2 primers ...... 24 Figure 6: qPCR assay indicating inherited loss at 10p12.33 ........................................................ 25 Figure 7: Testing of CASKIN1 and PKD1 primer sets ................................................................