GENOMIC REARRANGEMENTS IN HUMAN AND MOUSE AND THEIR CONTRIBUTION TO THE WILLIAMS-BEUREN SYNDROME PHENOTYPE. by Edwin James Young A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy, Institute of Medical Science University of Toronto © Copyright by Edwin James Young (2010) Genomic rearrangements in human and mouse and their contribution to the Williams- Beuren Syndrome phenotype. Doctor of Philosophy (2010) Edwin James Young Institute of Medical Science University of Toronto Abstract: Genomic rearrangements, particularly deletions and duplications, are known to cause many genetic disorders. The chromosome 7q11.23 region in humans is prone to recurrent chromosomal rearrangement, due to the presence of low copy repeats that promote non-allelic homologous recombination. The most well characterized rearrangement of 7q11.23 is a hemizygous 1.5 million base pair (Mb) deletion spanning more than 25 genes. This deletion causes Williams-Beuren Syndrome (WBS; OMIM 194050), a multisystem developmental disorder with distinctive physical and behavioural features. Other rearrangements of the region lead to phenotypes distinct from that of WBS. Here we describe the first individual identified with duplication of the same 1.5 Mb region, resulting in severe impairment of expressive language, in striking contrast to people with WBS who have relatively well preserved language skills. We also describe the identification of a new gene for a severe form of childhood epilepsy through the analysis of individuals with deletions on ii chromosome 7 that extend beyond the boundaries typical for WBS. This gene, MAGI2, is part of the large protein scaffold at the post-synaptic membrane and provides a new avenue of research into both the molecular basis of infantile spasms and the development of effective therapies. Individuals with smaller than typical deletions of 7q11.23 have delineated a minimal critical region for WBS and have implicated two members of the TFII-I transcription factor family. To better understand the contribution of these genes to WBS, I have generated animal models with these genes deleted singly and in combination. Disruption of the first gene, Gtf2ird1, resulted in phenotypes reminiscent of WBS including alterations in social behaviour, natural fear response and anxiety. An alteration in serotonin function was identified in the frontal cortex and may be linked to these behavioural phenotypes. Together with a model for the second gene, Gtf2i, and the double deletion model that was generated using Cre-loxP technology, these resources will permit the study of the individual and additive effects of hemizygosity for Gtf2i and Gtf2ird1 and will greatly expand our understanding of the role the TFII-I gene family in WBS. iii Acknowledgements I want to first start off by thanking my supervisor Dr. Lucy Osborne and my committee members Dr Steve Scherer and Dr Paul Franklin for all their technical assistance and especially their patience. A special thank you to Drs. Bernice Morrow, Sue Quaggin and Johanna Rommens, for agreeing to be members of my doctoral defense committee. I would also like to thank the members of the Osborne lab past and present, in particular Jen O‘Leary for the countless hours of ‗scientific discussion‘ over the last several years. Thank you to the many collaborators and their respective lab members that I have had the priveledge of working with including Dr John Roder, Dr Howard Mount, Dr Paul Fletcher, Dr Evelyn Lambe (especially Eliane Proulx), Dr Andras Nagy (especially Marina Gerstenstein), the Marsden Lab (especially Brent Steer), The Centre for Applied Genomics, Dr Carolyn Mervis, Dr Colleen Morris and the WBS family members. A special thanks my parents and family for although I do not think they always understood why I did it, they were always there with motivation and enthusiasm. I would also like to thank my inlaws Lino and Kathy Vrigini for all encouragement and support they have provide me and my family throughout my studies. Of course the greatest thanks have to go to my family; my wife Lisa, my son Owen and our dog Lily. Lisa, I knew from the beginning that the journey would not be an easy one but your unwavering love and support made the completion my studies possible. Although my son Owen will not remember this period in his life, his safe arrival in the last year of my studies provided not only a impetus to finish but also a great deal of perspective of what is truly important in my life. iv TABLE OF CONTENTS Abstract ii Acknowledgements iv Table of Contents v List of Figures x List of Tables xii List of Abbreviations xiv Internet Resources xxi Chapter I: Introduction to Williams-Beuren syndrome. 1 1.1 Literature Review. 2 1.1.1: Williams-Beuren syndrome history 2 1.1.2: Williams-Beuren syndrome clinical phenotype 3 1.1.3: Williams-Beuren syndrome cognitive and behavioral phenotype 7 1.1.4: Williams-Beuren Syndrome Cognitive Profile (WBSCP): 7 1.1.5: Genomic structure and molecular basis of Williams-Beuren Syndrome 11 1.1.6: WBS genotype-phenotype correlations 13 1.1.7: Identification of a WBS critical region 14 1.1.8: Transcription factors in human disease 15 1.1.9: GTF2I transcription factor gene family 17 1.1.10: The transcription factor GTF2I 19 1.1.11: The transcription factor GTF2IRD1 22 1.1.12: The transcription factor GTF2IRD2 23 1.2: Research Aims and Hypothesis 24 1.3: References 26 Chapter II: Genomic rearrangements of the human 7q11-q21 region 35 2.1: Literature Review. 36 2.1.1: Chromosomal rearrangements and the human genome 36 2.1.2: Chromosomal rearrangements and association with disease 39 2.1.3: Inversion of the WBS region 40 2.1.4: Duplication of the WBS region 41 2.1.5: Large deletions of the WBS region 42 2.2: Methods: Severe Expressive-Language Delay Related to Duplication of the 43 Williams–Beuren Locus 2.2.1: Participants 43 2.2.2: Clinical evaluation of language fundamentals and physical 44 manifestations 2.2.3: Fluorescence in situ hybridization 44 v 2.2.4: Single-copy microsatellite markers 45 2.2.5: Site Specific Nucleotide (SSN) dosage analysis 45 2.2.6: Genomic analysis using quantitative PCR 46 2.2.7: Expression analysis using quantitative PCR 46 2.3: Methods: The Common Inversion of the Williams-Beuren Syndrome Region at 47 7q11.23 Does Not Cause Clinical Symptoms 2.3.1: Participants 47 2.3.2: Developmental assessment 48 2.3.3: Inversion testing 49 2.3.4: Expression analysis using quantitative PCR 49 2.3.5: Copy Number Variation (CNV) analysis 51 2.3.6: Genomic analysis using quantitative PCR 52 2.4: Methods: Infantile Spasms Is Associated with Deletion of MAGI2 on 52 Chromosome 7q11.23-q21.11 2.4.1: Participants 52 2.4.2: Preparation of genomic DNA 53 2.4.3: Copy Number Variation (CNV) analysis 53 2.4.4: Genomic analysis using quantitative PCR 54 2.5: Results: Duplications and its Association with Speech Language Delay 56 2.5.1: Mild physical manifestation of 7q11.23 duplication 56 2.5.2: Severe expressive language delay is the most striking feature of 58 7q11.23 duplication 2.5.3: Duplication of the 1.5 Mb WBS region 60 2.5.4: Single-copy microsatellite markers 61 2.5.5: The duplication is the reciprocal of the WBS deletion 64 2.5.6: Genes within the duplication show altered expression 64 2.6: Results: Common Inversion Does Not Cause Clinical Symptoms 66 2.6.1: Clinical assessment 66 2.6.1.1: Medical and family history Participant 1 67 2.6.1.2: Medical and family history Participant 2 67 2.6.1.3: Physical examination Participant 1 68 2.6.1.4: Physical examination Participant 2 70 2.6.2: INV-1 participant 1 and 2 developmental assessment 72 2.6.3: Inversion testing using three-colour interphase FISH 76 2.6.4: INV expression analysis 76 2.6.5: Copy Number Variation (CNV) analysis 79 2.7: Results: Identification of MAGI2 Deletions and its Association with IS 81 2.8: Conclusion and Discussion: 86 2.8.1: Severe expressive language delay related to duplication of the 86 Williams–Beuren locus: 2.8.2: The common inversion of 7q11.23 does not cause clinical symptoms 94 2.8.3: Infantile spasms (IS) is associated with deletion of MAGI2 102 2.9: References: 107 Chapter III: Analysis of Gtf2ird1 Mouse Model: 121 vi 3.1: Literature Review: 122 3.1.1: Contribution of the genes telomeric to elastin to the Williams-Beuren 122 syndrome phenotype 3.1.2: The neurobiology of fear, emotion and social cognition 125 3.1.3: The unique social profile seen in Williams-Beuren syndrome 126 3.1.4: Increased levels of generalized anxiety and specific phobias in WBS 128 3.1.5: Role of serotonin in emotional behaviors 130 3.2: Material and Methods: 132 3.2.1: Generation of targeted Gtf2ird1 mouse model 132 3.2.2: Expression analysis 133 3.2.3: General morphological analysis 135 3.2.4: Resident intruder/Olfactory function test 136 3.2.5: Elevated plus maze 136 3.2.6: Cube exploration/novel object recognition test 137 3.2.7: Locomotor activity in the Open Field 138 3.2.8: Morris Water Maze Test 138 3.2.9: Context and cued fear conditioning 139 3.2.10: Neurochemical analyses 140 3.2.11: Rotorod analysis 141 3.2.12: Microarray analysis 141 3.2.13: Western blotting analysis 142 3.2.14: Immunohistochemistry 143 3.2.15 Golgi-Cox Staining 143 3.2.16: Brain slice preparation and electrophysiology 144 3.2.17: Statistical Analysis 146 3.3: Results: 146 3.3.1: Characterization of Gtf2ird1 mice: 147 3.3.2: Phenotypic analysis of Gtf2ird1 targeted mice 151 3.3.3: Analysis of body weight 151 3.3.4: Assessment in the Open Field 152 3.3.5: Gtf2ird1-/- mice are less anxious