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The Genetic Aetiology of Otosclerosis in the Population of Newfoundland and Labrador

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The Genetic Aetiology of Otosclerosis in the Population of Newfoundland and Labrador The Genetic Aetiology of Otosclerosis in the Population of Newfoundland and Labrador By © Nelly Abdelfatah A thesis submitted to the School of Graduate Studies in partial fulfillment of the requirements for the degree of Doctor of Philosophy Discipline of Genetics, Faculty of Medicine Memorial University of Newfoundland Oct, 2014 St. John’s Newfoundland and Labrador Abstract Background Otosclerosis is a common form of conductive and mixed hearing loss in Caucasian populations, with an estimated prevalence of 0.3-0.4%. Since 1998, eight loci have been mapped to otosclerosis in families with apparent autosomal dominant (AD) otosclerosis but none of the causative genes have been identified. Objective As no otosclerosis gene has yet been identified, the main objective of this thesis was to identify otosclerosis-disease causing genes by studying Newfoundland (NL) families. Methods Families with familial otosclerosis were identified and chracterized clinically. Those which fit the diagnostic criteria for otosclerosis were recurited for this study. Molecular genetic analyses of these families were carried out by genotyping, haplotyping, Sanger sequencing of candidate genes in linked regions and exome sequencing. Results One Family (2081) was solved through identification of a pathogenic variant (FOXL1c.976_990hetdel) in the FOXL1 gene at chromosome (Chr) 16q that was present in all affected individuals. The 15 base pair (bp) deletion was also identified in a second family from Ontario (ON) and the possible pathways involving FOXL1 in the pathogenesis of otosclerosis were suggested. In the second otosclerosis family, three II candidate variants were identified through exome sequencing of the candidate regions under a dominant model. Conclusion I have identified the first otosclerosis gene, FOXL1, a transcription factor involved in the disease pathogenicity. I also identified three possible candidate mutations for a second otosclerosis family. This finding will have a major impact on molecular genetic studies of other otosclerosis families and it will allows for genetic counselling and the possibility for gene therapies in the future. III Acknowledgments First, I would like to take this opportunity to thank my supervisor Dr. Terry Lynn Young for her generosity, support, guidance, teaching and patience. Words are not enough to thank her. When I came to her lab, I had a little experience in experimental design, critical thinking and scientific writing. Dr. Terry Young taught me how to be a great scientist. Thank you for believing in me and giving me the opportunity of my life. Thanks to my supervisory committee, Drs Kathy Hodgkinson, Jane Green and Ban Younghusband for their encouragement, guidance and helpful advice throughout the completion of my project. Thanks to The Young Lab, for being great, supportive and amazing co-workers. Special thanks to Dante Galutira and Jim Houston for your help and assistance, and for your patience and generosity. Thank you Tammy Benteau and Catherine Street for your support. Thank you Nancy Merner and Annika Haywood for being valuable members of the lab, and for your help and support. I spent a great and enjoyable time working with Lance Doucette and I wish that we could continue working with each other. Thank you David McComiskey, Jessica Squires, Cindy Penney and Andrew Bullen for being excellent peers and making learning fun. Special thanks for Jim Houston, Dante Galutira and Tammy Benteau for editing my thesis. I would like to thank Genome Canada (Atlantic Medical Genetics and Genomics Initiative), CIHR (Canadian Institutes of Health Research), RDC NL (Research & IV Development Corporation of Newfoundland and Labrador) and Memorial University Graduate Studies for funding. Also I would like to thank CIHR, RDC NL for my fellowship. I would like to thank the Journals: European Journal of Human Genetics and Human Mutation for publishing my two papers. I dedicate this work to my lovely Mom for her support, love and concern from abroad and in memory of my beloved Dad. Thank you to my husband Ahmed Mostafa for his continued support, help and encouragement. Without your support, I would not be able to finish my PhD. Thank you to my lovely adorable kids, Marwan and Merna, for your understanding that Mom is busy; for your laughter, love, support and encouragement which gave me the energy to continue. V Table of Contents The Genetic Aetiology of Otosclerosis in the Population of Newfoundland and Labrador ............................................................................................................................. I Abstract .............................................................................................................................. II Acknowledgments ........................................................................................................... IV List of Tables ..................................................................................................................... X List of Figures ......................................................................................................................................... XI Chapter 1: General introduction...................................................................................................... 1 1.1 Purpose of the study and significance .............................................................................. 1 1.2 Anatomy and function of the ear ........................................................................................ 2 1.3 How sound is transmitted..................................................................................................... 4 1.4 Hearing loss… ............................................................................................................................ 5 1.4.1 Classification of hearing loss ............................................................................................... 5 1.5 Gene discovery in non-syndromic hearing loss using NL families ..................... 12 1.5.1 Ascertainment of families ................................................................................................... 12 1.5.2 Complications of family ascertainment......................................................................... 15 1.5.2.1 Penetrance………. ................................................................................................................... 15 1.5.2.2 Variable expression .............................................................................................................. 16 1.5.2.3 Phenocopy……….. ................................................................................................................... 17 1.5.3 Genetic analysis of families with hearing loss ........................................................... 17 1.5.3.1 Exclusion of known loci or genes .................................................................................... 18 1.5.3.2 Candidate gene and genome-wide association approaches ................................. 18 1.5.3.3 Genome-wide linkage analysis ......................................................................................... 20 1.5.3.4 Positional candidate gene sequencing .......................................................................... 25 1.5.3.5 Mutation identification and validation ......................................................................... 26 1.5.3.6 Next generation sequencing.............................................................................................. 30 1.6 Otosclerosis…. .......................................................................................................................... 32 1.6.1 Overview……….. ...................................................................................................................... 32 1.6.2 Bone remodeling and otic capsule .................................................................................. 35 1.6.3 History and stages of otosclerosis ................................................................................... 38 1.6.4 Diagnosis of otosclerosis..................................................................................................... 40 1.6.5 Genetic causes of otosclerosis........................................................................................... 44 1.7 Founder population ............................................................................................................... 51 1.8 The NL population ................................................................................................................. 51 1.8.1 Gene discovery in the NL population ............................................................................. 52 VI 1.9 Hypothesis and objectives .................................................................................................. 53 1.10 Co-authorship statement .................................................................................................... 54 Chapter 2: Test for linkage of NL Families to published OTSC loci and associated candidate genes…………. ........................................................................................ 55 2.1 Introduction… .......................................................................................................................... 56 2.2 Subjects and methods ........................................................................................................... 56 2.2.1 Working definition of clinical
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