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Gene Identification and Characterisation for Blinding

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Gene Identification and Characterisation for Blinding Gene identification and characterisation for blinding inherited eye diseases Shahrbanou Javadiyan (MSc) Thesis is submitted for the degree of Doctor of Philosophy Department of Ophthalmology School of Medicine Faculty of Medicine, Nursing and health Sciences Flinders University of South Australia July 2015 Table of contents Declaration ...................................................................................................................... vii Thesis Summary ............................................................................................................. viii Acknowledgment .............................................................................................................. x Publications and conference abstracts arising from this thesis ....................................... xii Chapter1: Introduction ...................................................................................................... 1 1.1 Overview ................................................................................................................. 2 1.2 Anatomy of the human eye ..................................................................................... 3 1.2.1 Lens .................................................................................................................. 5 1.2.2 Trabecular meshwork and circulation of aqueous humor ................................ 7 1.3 Cataract ................................................................................................................... 9 1.3.1 Paediatric cataract ............................................................................................ 9 1.3.2 Genetics of paediatric cataract ....................................................................... 12 1.4 Glaucoma .............................................................................................................. 19 1.4.1 Primary congenital glaucoma ......................................................................... 21 1.5 Mendelian disease gene discovery and candidate gene screening methods ......... 24 1.6 Aims ...................................................................................................................... 25 1.6.1 Aim 1: Screening known paediatric cataract and PCG genes ........................ 26 1.6.2 Aim 2: Undertaking gene discovery in paediatric cataract and PCG families ................................................................................................................................. 27 i Chapter 2: Materials and methods................................................................................... 29 2.1 Ethics statement .................................................................................................... 30 2.2 Participant recruitment .......................................................................................... 31 2.3 Control population ................................................................................................ 33 2.4 DNA extraction ..................................................................................................... 33 2.5 Primers design ....................................................................................................... 34 2.6 Validation of potential mutations by Sanger sequencing ...................................... 34 2.7 Genotyping methodology for control population screening ................................. 35 2.8 Variant Filtering strategy ...................................................................................... 36 2.9 Definition of some terms ....................................................................................... 37 Chapter 3: Investigating the genetics of primary congenital glaucoma in Australian cases ................................................................................................................................ 38 3.1 Introduction ........................................................................................................... 39 3.2 Aims ...................................................................................................................... 39 3.3 Methods ................................................................................................................. 40 3.3.1 Candidate gene screening ............................................................................... 40 3.3.2 Whole exome sequencing in an affected family ............................................ 41 3.4 Results ................................................................................................................... 42 3.4.1 TEK gene screening in Australian PCG cohort .............................................. 42 3.4.2 Novel gene identification in an affected family ............................................. 47 3.5 Discussion ............................................................................................................. 50 3.5.1. Candidate gene screening: TEK .................................................................... 50 ii 3.5.2 GREB1, a novel candidate gene: evaluation of its involvement in PCG ....... 53 3.6 Summary and conclusion ...................................................................................... 55 Chapter 4: Massively parallel sequencing of known paediatric cataract genes identifies causative mutations in Australian and Asian cohorts ..................................................... 56 4.1 Introduction ........................................................................................................... 57 4.2 Aim ........................................................................................................................ 58 4.3 Methods ................................................................................................................. 58 4.3.1 Known paediatric cataract genes selection for sequencing ............................ 58 4.3.2 Primer design for targeted massively parallel sequencing ............................. 61 4.3.3 Library preparation......................................................................................... 61 4.3.4 Sequencing and analysis ................................................................................ 62 4.3.5 Validation, segregation analysis and evaluating potential functional effects of mutations ................................................................................................................. 63 4.3.6 Screening novel variants in population controls ............................................ 63 4.3.7 Haplotype analysis of families CSA110 and CSA91 with a mutation in CRYAA .................................................................................................................... 64 4.3.8 Assessment of sequence conservation ........................................................... 65 4.4 Results ................................................................................................................... 65 4.4.1 Screening Australian paediatric cataract samples with family history of paediatric cataract.................................................................................................... 70 4.4.2 Screening sporadic paediatric cataract Australian cohort .............................. 97 4.4.3 Screening Asian paediatric cataract cases .................................................... 107 4.5 Discussion ........................................................................................................... 121 iii 4.5.1. Mutations detected in membrane or cytoskeleton proteins encoding genes ............................................................................................................................... 121 4.5.2 Mutations detected in crystallin genes ......................................................... 124 4.5.3 Mutations detected in transcription factors, signalling molecules or in genes associated with syndromic and enzymatic paediatric cataract .............................. 127 4.6 Summary and conclusion .................................................................................... 133 Chapter 5: Identification of novel genes and mechanisms underlying paediatric cataract using whole exome sequencing ..................................................................................... 135 5.1 Introduction ......................................................................................................... 136 5.2 Aim ...................................................................................................................... 136 5.3 Methods ............................................................................................................... 137 5.3.1 Exome sequencing ....................................................................................... 137 5.3.2 Variant filtering strategy .............................................................................. 137 5.3.3 Processing exome sequencing data for linkage analysis .............................. 138 5.3.4 Linkage analysis using exome sequence data .............................................. 139 5.3.5 Copy number variation (CNV) analysis ....................................................... 140 5.3.6 Quantitative PCR to confirm CRYBB1 partial duplication .......................... 140 5.3.7 Protein extraction from surgical lens specimen ........................................... 141 5.3.8 Denaturing protein gel electrophoresis and western
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