FUNCTIONAL GENOMIC ANALYSIS OF NOVEL MICRODELETIONS AND MICRODUPLICATIONS ASSOCIATED WITH INTELLECTUAL DISABILITY by Chansonette Badduke M.Sc., Andrews University, 2008 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Pathology and Laboratory Medicine) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) December 2015 © Chansonette Badduke, 2015 Abstract Intellectual disability (ID) is a diagnosis given to persons who have life-long cognitive and adaptive impairments that begin early in life. ID affects about 1-3% of the population. Extremely small chromosome losses and gains, called microdeletions and microduplications respectively (or collectively Copy Number Variants, CNVs), are the cause of ID in ~15% of cases and their identification has helped to pinpoint genomic regions that contain ID-genes. The objective of my PhD research was to search for ID candidate genes in subjects with ID, focusing on the functional genomic analysis of genes from CNVs and in the rest of the genome. I studied individuals with unique de novo pathogenic CNVs at chromosomal position 2p15-16.1 or with familial CNVs at chromosomal position 1q21.1. I used a multi-faceted approach that included the study of candidate genes’ 1) expression, 2) sequence variants, 3) knock down consequence in C. elegans and 4) imprinting potential. My results showed that the best candidate genes from the 2p15-16.1 CNV are XPO1, USP34 and REL because their expression is reduced in individuals with deletions. In case of the 1q21.1 CNV, I identified two candidate genes (CHD1L and PRKAB2) from the CNV that had altered expression and cellular function. I also identified a pathogenic sequence change in ATF6 in individuals with a familial 1q21.1 duplication. ATF6 is located outside the 1q21.1 CNV and is part of the Endoplasmic Reticulum (ER) stress response pathway which may contribute to the phenotypic variability in this family. Finally, I identified 3 CNVs in children with ID that overlap putative imprinted regions. The results of my study therefore led to the identification of genes which could contribute to ID as their function is altered in patients with the CNV or their characteristics suggest that they can be sensitive to copy number changes. This work contributes to an improved ii understanding of how CNVs and additional genetic changes in the rest of the genome can lead to ID. iii Preface All studies involving human subjects were approved by the University of British Columbia Clinical Research Ethics Board (C01-0509). Chapter 2 contains the results of multiple studies done by me and in collaboration with others. Extraction of clinical information from publications and charts for individuals with 2p15- 16.1 deletions was initially done by Dr. Ying Qiao (research associate) and was checked by me, Dr. Evica Rajcan-Separovic, and Dr. Suzanne Lewis. Chromosomal Microarrays (CMAs) for our 2p15-16.1 deletion cases were run in house by Sally Martell (technician), Ying Qiao or by me, or were provided by clinical cytogenetics service including Royal Columbian Hospital’s Cytogenetics Laboratory. Breakpoint comparison, genomic overlap, generation of gene frequencies and addition of haploinsufficiency scores was done by me. Exome sequencing for one subject was done by Otogenetics. I performed the initial analysis of variants in the 2p15-16.1 region based on Otogenetics data and subsequently supervised data analysis using Golden Helix software (performed by my co-op student Flamingo Tang). The Illumina Expression BeadChip array (HumanRef-8 v3.0) was run by the CFRI core facility. I generated the background- corrected intensity values for each probe using GenomeStudio software (Illumina). Subsequent normalization and expression/copy number correlation analysis was done in collaboration with Dr. Paul Pavlidis (UBC). After normalization, I generated the expression ratios and calculated the fold changes for all genes. Lymphoblasts were transformed on a service basis, and the resulting lymphoblastoid cell lines (LBCs) were grown and maintained by me. RNA and protein extractions for downstream analysis were done by me. QMPSF confirmation of small CNVs in Case No. 2 was done by Sally Martell with my advice on where to position primers. Microsatellites were designed and parent of origin experiments were run by me. iv Immunohistochemistry using anti-XPO1 and anti-USP34 was done by the histochemistry lab (Department of Pathology and Laboratory Medicine, UBC). Image analysis was done with the help of neuropathologist, Dr. Chris Dunham. Real time qPCR for XPO1 and USP34 was done by me as were the Western blots for XPO1. Western blots for c-REL and USP34 were performed by Dr. Jiadi Wen (research associate) and in Dr. Marc O’Driscoll’s lab respectively. The animal model experiments in C. elegans were done in Dr. Harald Hutter’s lab. The RNAi experiments and design of the constructs for the transgenic strains were performed by me. Jessie Jie Pan engineered the working constructs and did the microinjections, Dr. Harald Hutter did the image capture and supervised my image analysis. Lastly, all bioinformatics analyses in this chapter, including the use of WebGestalt for functional enrichment analysis, were done by me. The manuscript deriving from this work and subsequent extension of it using additional patients and a zebrafish knock-out model is in preparation for publication and I share the first co-authorship with Hani Bagheri. The majority of work from Chapter 3 was published in my first-author publication (Harvard et al., 2011). Published results included genomic and clinical assessment of 3 families with 1q21.1 CNVs and whole genome expression experiments for 2 families. The Illumina Expression BeadChip array (HumanRef-8 v3.0) was run by the CFRI core facility. I performed data analysis from the gene expression data in collaboration with Dr. Paul Pavlidis (UBC) who ran the gene expression copy number correlation analysis and Mr. Eloi Mercer who ran the statistical tests for the over-representation analysis. Functional analysis for two candidate genes from the 1q21.1 CNV with highest expression/copy number correlation in patient cells (CHD1L and PRKAB2) was performed in the laboratory of our collaborator Dr. Mark Driscoll (University of Sussex, UK). Subsequent to the paper, whole exome data for members of 2 families carrying a v 1q21.1CNV was generated at BGI and I played a major role in data processing and analysis that resulted in the identification of variants in two genes (ATF6 and DARS1) that warranted follow- up including confirmation by Sanger sequencing and real time qPCR evaluation of gene expression. I supervised the Sanger confirmation of variants (done by my co-op student, Flamingo Tang) and did the qPCR experiments to confirm gene expression. Western blotting for ATF6 was conducted in Dr. Alan Volchuk’s lab at the University of Toronto. Finally, I performed an extensive literature analysis in order to identify genes involved in ER stress and used these lists to do a global analysis of genes expression for genes from the ER stress response pathway using my whole genome expression data in members of 2 families (A & C). I also assessed the response of patient lymphoblast cells to ER stress induced by two chemicals (tunicamycin and thapsigargin). Chapter 4 contains the results of a FISH-RT study that was designed and performed by me. I extracted known and predicted imprinted genes from multiple online databases and compared their positions with CNVs from individuals in our ID cohort in order to identify candidate imprinted regions for FISH-RT studies. Dr. Evica Rajcan-Separovic and I both counted replication timing patterns for each FISH-RT experiment. Putative imprinted differentially methylated regions (DMRs) were provided by Dr. Courtney Hanna based on her work in Dr. Wendy Robinson’s Lab and I performed CNV/DMR overlap analysis to identify CNVs that contain putative imprinted DMRs. Finally, a comparison analysis of the DMR fraction in putatively pathogenic CNVs from individuals with ID (de novo and familial) and in randomly generated genomic regions was run in collaboration with Mr. Eloi Mercer. vi Table of Contents Abstract .......................................................................................................................................... ii Preface ........................................................................................................................................... iv Table of Contents ........................................................................................................................ vii List of Tables .............................................................................................................................. xiv List of Figures ...............................................................................................................................xv List of Abbreviations ................................................................................................................ xvii Acknowledgements .................................................................................................................. xviii Dedication ................................................................................................................................... xix Chapter 1: Introduction ................................................................................................................1