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A Molecular and Genetic Analysis of Otosclerosis

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A Molecular and Genetic Analysis of Otosclerosis A molecular and genetic analysis of otosclerosis Joanna Lauren Ziff Submitted for the degree of PhD University College London January 2014 1 Declaration I, Joanna Ziff, confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis. Where work has been conducted by other members of our laboratory, this has been indicated by an appropriate reference. 2 Abstract Otosclerosis is a common form of conductive hearing loss. It is characterised by abnormal bone remodelling within the otic capsule, leading to formation of sclerotic lesions of the temporal bone. Encroachment of these lesions on to the footplate of the stapes in the middle ear leads to stapes fixation and subsequent conductive hearing loss. The hereditary nature of otosclerosis has long been recognised due to its recurrence within families, but its genetic aetiology is yet to be characterised. Although many familial linkage studies and candidate gene association studies to investigate the genetic nature of otosclerosis have been performed in recent years, progress in identifying disease causing genes has been slow. This is largely due to the highly heterogeneous nature of this condition. The research presented in this thesis examines the molecular and genetic basis of otosclerosis using two next generation sequencing technologies; RNA-sequencing and Whole Exome Sequencing. RNA–sequencing has provided human stapes transcriptomes for healthy and diseased stapes, and in combination with pathway analysis has helped identify genes and molecular processes dysregulated in otosclerotic tissue. Whole Exome Sequencing has been employed to investigate rare variants that segregate with otosclerosis in affected families, and has been followed by a variant filtering strategy, which has prioritised genes found to be dysregulated during RNA-sequencing. This has identified multiple variants predicted to be involved in splicing within genes involved in the bone disorder Osteogenesis Imperfecta, indicating a shared genetic aetiology for this condition and otosclerosis and a possible disease mechanism involving alternative splicing in the stapes. Whilst the hereditability of otosclerosis remains elusive, the identification of new candidate genes will make a significant contribution to the current literature. It is hoped that long term, this research will help reveal disease mechanisms and thereby improve treatment options for otosclerosis patients. 3 Dedication In loving memory of Henry Perlow 1928-2009 4 Acknowledgements Firstly, I thank my PhD supervisor Dr Sally Dawson for the opportunity to carry out this project and for her guidance and patience during the last three years. I appreciate the many hours of teaching and advice I have received from her throughout my PhD and during the preparation of this thesis. I also thank my friends and colleagues at UCL Ear Institute who have made my time here so memorable. In particular I thank Emily, Greg, Kiran, Lisa, Miriam, Naila, Paromita and Steph for sharing their knowledge and expertise with me and for all their assistance in the lab as well as their friendship. I also express my gratitude to my second supervisor Professor Shakeel Saeed for his enthusiasm for this project and for his valuable input into all clinical aspects of the study. I also thank him, along with Harry, Jeremy and Sherif, for their vital assistance with patient recruitment and sample collection which was crucial for the success of the project. I would also like to acknowledge Deafness Research UK/ Action on Hearing Loss for funding this PhD studentship and also to the UCL graduate school for funding my trip to the American Society of Human Genetics Annual conference in San Francisco. Most importantly, I thank my family and friends for their tireless support and encouragement throughout the past three years. They have helped me through all the problems I have encountered on this journey and enabled me to keep these in perspective. Finally, I would like to express my gratitude to the otosclerosis patients and their families who participated in this research study, without whom this project would not have been possible. 5 Abbreviations dNTPs Dideoxynucleotide triphosphates EDTA Ethylenediamine tetra-acetic acid FDR False discovery rate GWAS Genome wide association study LD Linkage disequilibrium NGS Next generation sequencing PCR Polymerse chain reaction qPCR Quantitative polymerase chain reaction RFLP Restriction fragment length polymorphism RNA-seq RNA sequencing RPKM Reads per kilobase per million mapped reads SNP Single nucleotide polymorphism TAE Tris-acetate-EDTA electrophoresis buffer UV Ultraviolet WES Whole exome sequencing WGS Whole genome sequencing 6 Table of Contents DECLARATION.......................................................................................................................... 2 ABSTRACT ................................................................................................................................ 3 DEDICATION ............................................................................................................................ 4 ACKNOWLEDGEMENTS ............................................................................................................ 5 ABBREVIATIONS ..................................................................................................................... 6 TABLE OF CONTENTS ............................................................................................................... 7 LIST OF FIGURES .....................................................................................................................10 LIST OF TABLES .......................................................................................................................13 1 INTRODUCTION ..............................................................................................................15 1.1 Deafness and hearing loss .................................................................................15 1.2 Structure and function of the ear ......................................................................16 1.3 Otosclerosis ......................................................................................................20 1.4 Development of the stapes ...............................................................................28 1.5 Bone remodelling in the otic capsule.................................................................29 1.6 Animal models for otosclerosis .........................................................................32 1.7 The disease aetiology of otosclerosis ................................................................33 1.8 Aims and objectives of this thesis ......................................................................58 2 MATERIALS AND METHODS ............................................................................................59 Materials .............................................................................................................................59 2.1 Chemicals and Equipment ................................................................................59 2.2 Abbreviations ...................................................................................................59 2.3 Stock Solutions .................................................................................................59 Methods .............................................................................................................................60 2.4 Tissue collection ...............................................................................................60 2.5 cDNA preparation ............................................................................................62 2.6 Gene Expression Assays ...................................................................................64 2.7 Genomic DNA preparation from blood and saliva samples ...............................67 2.8 Detection of genomic variation ........................................................................67 7 2.9 Agarose gel Electrophoresis .............................................................................72 2.10 Purification of PCR products .............................................................................72 3 COHORT CHARACTERISTICS ............................................................................................74 3.1 Overview .................................................................................................................74 3.2 Results .....................................................................................................................74 3.2.1 Collection of DNA samples from study participants ..........................................74 3.2.2 Characteristics of the otosclerosis cohort .........................................................75 3.2.3 Environmental factors to which cohort members have been exposed ..............79 3.2.4 Incidence of disease in cohort members...........................................................83 3.3 Discussion of cohort characteristics .........................................................................84 4 WHOLE EXOME SEQUENCING IDENTIFIES NOVEL GENETIC VARIANTS THAT SEGREGATE WITH OTOSCLEROSIS IN FAMILIES ......................................................................................92 4.1 Introduction.............................................................................................................92
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