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Whole Exome Sequencing to Investigate Genetic Variants of Non-Syndromic Hearing Impairment in a Population of African Ancestry

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Whole Exome Sequencing to Investigate Genetic Variants of Non-Syndromic Hearing Impairment in a Population of African Ancestry Whole exome sequencing to investigate genetic variants of non-syndromic hearing impairment in a population of African Ancestry By Noluthando Manyisa MNYNOL006 Submitted to the University of Cape Town in fulfilment of requirements of the degree MSc Med in Human UniversityGenetics of Cape Town University of Cape Town, Faculty of Health Sciences, Department of Pathology, Faculty of Human Genetics Supervisor: Professor Ambroise Wonkam Co-Supervisors: Professor Collet Dandara and Dr Emile Chimusa Word Count: 18 772 Date of Submission: February 2018 1 The copyright of this thesis vests in the author. No quotation from it or information derived from it is to be published without full acknowledgement of the source. The thesis is to be used for private study or non- commercial research purposes only. Published by the University of Cape Town (UCT) in terms of the non-exclusive license granted to UCT by the author. University of Cape Town Table of Contents Plagiarism declaration ................................................................................................................ I Acknowledgments..................................................................................................................... II List of Figures and Tables........................................................................................................ III Figures.................................................................................................................................. III Tables ................................................................................................................................... IV List of Abbreviations ................................................................................................................ V Abstract ...................................................................................................................................... 1 Chapter 1: Introduction and literature review ............................................................................ 6 Chapter 1.1: Overview ........................................................................................................... 6 Chapter 1.2: Definition and epidemiology of hearing impairment ........................................ 7 Chapter 1.3: Normal hearing.................................................................................................. 8 Chapter 1.4: Aetiology of hearing impairment .................................................................... 11 Chapter 1.5: Genetics of hearing impairment ...................................................................... 11 Chapter 1.5.1: Syndromic hearing impairment ................................................................ 12 Chapter 1.6: Prevalent genes associated with Non- Syndromic hearing impairment .......... 15 Chapter 1.6.1: Non-syndromic hearing impairment: prevalence of GJB2 and GJB6 ..... 15 Chapter 1.6.2: GJB2 and GJB6 mutations are rare among Africans ............................... 16 Chapter 1.7: Next generation sequencing and hearing impairment ..................................... 17 Chapter 1.7.1: Resolution of hearing impairment in African patients using targeted exome sequencing........................................................................................................................ 20 Chapter 1.8: Rationale of the study...................................................................................... 22 Chapter 1.10: Thesis outline .................................................................................................... 23 Chapter 1.11: Aim and objectives ............................................................................................ 25 Objective 1 ....................................................................................................................... 25 Objective 2 ....................................................................................................................... 25 Objective 3 ....................................................................................................................... 25 Chapter 2: Patients and methods .............................................................................................. 26 2 Chapter 2.1: Ethical consideration ....................................................................................... 26 Chapter 2.2: Patient recruitment and inclusion .................................................................... 26 Chapter 2.2.1: Patient recruitment ................................................................................... 26 Chapter 2.2.2: Inclusion and exclusion criteria ............................................................... 26 Chapter 2.2.3: Control population ................................................................................... 27 Chapter 2.3: DNA extractions and quality checks ............................................................... 27 Chapter 2.4: Whole exome sequencing ............................................................................... 27 Chapter 2.5: Bioinformatics analysis ................................................................................... 28 Chapter 2.5.1: Reads alignment and post-alignment quality checks ............................... 28 Chapter 2.5.2: Variant discovery ..................................................................................... 28 Chapter 2.5.3: Variant annotation and mutation prioritization ........................................ 29 Chapter 2.5.4: Genetic differentiation and population structure ...................................... 30 Chapter 2.5.5: Pathway enrichment analysis and protein-protein interactions ................ 30 Chapter 2.6: Evolutionary adaptation of human hearing ..................................................... 31 Chapter 2.7: Validation by Sanger Sequencing ................................................................... 34 Chapter 2.7.1: Primer Design........................................................................................... 34 Chapter 2.7.2: Primer Optimization ................................................................................. 37 Chapter 2.7.3: Polymerase Chain Reaction and product detection .................................. 37 Chapter 2.7.4: PCR clean-up ........................................................................................... 38 Chapter 2.7.5: Direct Cycle Sequencing and Capillary Electrophoresis ......................... 38 Chapter 2.7.7: Sequencing Results Analysis ................................................................... 38 Chapter 2.7.8: BAM file analysis .................................................................................... 39 Summary of bioinformatics methodology ........................................................................... 39 Summary of molecular methodology................................................................................... 40 Chapter 3: Genomics architecture of non-syndromic hearing impairment results .................. 42 Chapter 3.1: Patients clinical information ........................................................................... 42 Chapter 3.2: Variant filtering ............................................................................................... 43 3 Chapter 3.3: Population structure ........................................................................................ 48 Chapter 3.4: Genetic differentiation .................................................................................... 49 Chapter 3.5: PPI analysis and enrichment analysis ............................................................. 51 Chapter 3.6: Community network analysis .......................................................................... 52 Chapter 3.7: Evolutionary adaptation of human hearing ..................................................... 55 Chapter 3.8: Validation ........................................................................................................ 56 Summary of results .............................................................................................................. 62 Chapter 4: Discussion and perspectives ................................................................................... 64 Chapter 4.1: Originality of study and general comments .................................................... 64 Chapter 4.2: Candidate clinical variants .............................................................................. 65 Chapter 4.2.1: Collagen type IX alpha 3 chain. ............................................................... 66 Chapter 4.2.2: Myosin 3A ................................................................................................ 67 Chapter 4.2.3: Myosin 15A .............................................................................................. 68 Chapter 4.2.4: Resolution of hearing impairment ............................................................ 71 Chapter 4.3: Candidate population specific variants ........................................................... 71 Chapter 4.3.1: Forkhead Box D4 Like ............................................................................. 72 Chapter 4.3.2: Short chain dehydrogenase/reductase member 4 like 2 ........................... 72 Chapter 4.3.3: Ribosomal Protein L3 Like ...................................................................... 73 Chapter4.3.4: Vitronectin................................................................................................
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