THE PATHOGENICITY OF COPY NUMBER VARIANTS IN CHILDREN WITH INTELLECTUAL DISABILITY by Farah R Zahir B.Sc., Genetics, Biochemistry, Microbiology, Bangalore University, 2003 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Medical Genetics) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) August 2011 © Farah R Zahir 2011 Abstract Intellectual disability affects 1-3% of individuals globally, and, for half the cases, the cause is unknown. Recent studies using whole genome microarray genomic hybridization have shown that submicroscopic genomic imbalance causes intellectual disability in at least 10% of idiopathic cases with normal conventional cytogenetic analysis. I established genotype- phenotype correlations for de novo copy number variants detected by previous whole genome array genome hybridization studies performed by our group in children with intellectual disability. These genotype-phenotype correlations show that genomic imbalance of genes belonging to the epigenetic regulatory category, among others, are causative of intellectual disability. I hypothesized that dosage changes in the broad functional category of genes encoding epigenetic regulatory proteins are more likely to be pathogenic for intellectual disability than dosage changes in other kinds of genes. Epigenetic regulatory proteins include those with DNA methylation, histone modification or chromatin remodeling activity. I have selected all known genes encoding epigenetic regulatory proteins and defined probes to interrogate these candidate genes for copy number alteration as part of a custom targeted microarray design that selectively investigates all candidate genes associated with intellectual disability. We have conducted comparative genome hybridization on 177 patients with idiopathic intellectual disability using this array and on both normal parents of each affected child. We identified and independently validated 16 cases with de novo CNVs involving the epigenetic regulatory candidates. 7 of the 16 CNVs involve the same exon of the JARID2 gene, while the other 9 CNVs affect different genes. I discuss genotype-phenotype correlations for these cases and show that epigenetic perturbation by way of disruption of genes that encode epigenetic regulators is an important cause for intellectual disability. ii Preface This dissertation is comprised of both published and unpublished material as follows; Chapter 2, 3 and 4 are verbatim reproductions of published manuscripts. The papers detail genotype-phenotype correlations for four patients identified to have previously unknown genetic defects by studies undertaken at the Medical Genetics Research Unit in collaboration with the British Columbia Genome Sciences Center and headed by Dr. Jan M. Friedman. This study and the subsequent genotype-phenotype correlations studies arising from this work that are detailed below were approved by the University of British Columbia Clinical Research Ethics Board (certificate number C04-0537). For chapter 2, ‗Novel deletions of 14q11.2 associated with intellectual disability and similar minor anomalies in three children‘ (Journal of Medical Genetics. 2007 Sep;44(9):556-61), chapter 3, ‗A patient with vertebral, cognitive and behavioural abnormalities and a de novo deletion of NRXN1α’ (Journal of Medical Genetics. 2008 Apr;45(4):239-43) and chapter 4, ‘A novel de novo 1.1 Mb duplication of 17q21.33 associated with cognitive impairment and other anomalies‘ (American Journal of Medical Genetics Part A. 2009 Jun;149A(6):1257-62.), I was solely in charge of researching the genotype-phenotype correlation and preparing the manuscripts with input from all contributing authors. Chapters 5 and 6 are verbatim reproductions of published manuscripts for which I was a contributing author. Chapter 5, ‗Duplications of the critical Rubinstein-Taybi deletion region on chromosome 16p13.3 cause a novel recognizable syndrome‘ (Journal of Medical Genetics. 2010 Mar;47(3):155-61), contains a clinical comparison and discussion of candidate genes for a newly defined Intellectual Disability syndrome. I contributed clinical data on the patient from our center included in this study and also assisted with researching candidate genes for the syndrome. Chapter 6 ‗The duplication 8q12 Case: A characteristic syndrome associated with microduplication of 8q12, inclusive of CHD7‘ (European Journal of Medical Genetics. 2009 Nov-Dec;52(6):436-9.) contains a detailed clinical case report of a patient with a novel genetic defect detected in the same study iii headed by Dr. Jan M Friedman that led to the works appearing in chapters 2, 3 and 4. For this case I contributed an extensive genotype-phenotype correlation analysis, delineating candidate genes. The manuscript was prepared by Dr. Anna Lehman, who provided the clinical description of the patient. Chapters 1 and 8 contain large sections that are taken from two published review papers that I prepared. The discussion of microarrays appearing in chapter 1, the main technology underlying experiments detailed in this thesis, is taken from ‗The impact of array genomic hybridization on mental retardation research: a review of current technologies and their clinical utility‘ (Clinical Genetics. 2007 Oct;72(4):271-87) a review article published in 2007 written with Dr. Jan M. Friedman as my co-author. I have updated the text as required to reflect current knowledge for sections reproduced from this work. The sections introducing epigenetics and discussing the contribution of epigenetic regulation to neurodevelopment contained in chapter 1 and chapter 8 are taken from the review article, ‗Epigenetic impacts on neurodevelopment: pathophysiological mechanisms and genetic modes of action‘ (Pediatric Research. 2011 May;69(5 Pt 2):92R-100R) that I wrote in collaboration with Dr. Carolyn Brown. Chapter 7 is previously unpublished. The chapter details the main project of my doctoral research that I conducted in collaboration with Dr. Tracy Tucker who was in charge of a study to design and test a microarray probing selected genes. I was in charge of the selection of candidate genes to test my research hypothesis, which I contributed to the design (36% of the design). Dr. Jan Friedman, Dr. Sylvie Langlois and Dr. Patrice Eydoux provided the rest of the gene selection. Dr. Jacques Michaud (CHU Sainte-Justine, Montreal) provided patient samples and clinical reports. I was solely in charge of the entire validation component of the study that used an independent technology to assess the findings of the microarray. I have contributed this chapter as a joint primary author to a manuscript in preparation by Dr. Tracy Tucker detailing the results of this project. This study was approved by the University of British Columbia Clinical Research Ethics Board (certificate number H07-00392). iv Table of Contents Abstract .............................................................................................................................. ii Preface ................................................................................................................................iii Table of Contents ............................................................................................................... v List of Tables ......................................................................................................................xi List of Figures ...................................................................................................................xii List of Abbreviations ........................................................................................................ xiv Acknowledgements ...........................................................................................................xv Dedication ......................................................................................................................... xvi Chapter 1: Introduction .................................................................................................... 1 1.1 Definition of Intellectual Disability ............................................................................ 1 1.2 Etiology of Intellectual Disability .............................................................................. 1 1.2.1 The genetic etiology of Intellectual Disability ................................................ 2 1.2.1.1 Cytogenetically detectable genetic aberrations ............................................. 2 1.2.1.2 Genetic aberrations not detectable cytogenetically.................................... 2 1.2.1.3 Single gene mutations ............................................................................... 3 1.3 Epigenetics - definition and overview of epigenetic processes ................................ 4 1.4 Epigenetic perturbation in neurogenetic disorders .................................................. 7 1.4.1 Dosage change of genes encoding epigenetic factors as the mechanism of pathogenicity ...........................................................................................................11 1.5 Microarray technology ...........................................................................................12 1.5.1 Types of arrays and factors that influence their effective resolution ................14 1.5.2 The bioinformatics of AGH ..............................................................................17 1.5.3 Comparison to reference genome(s) ...............................................................18