MOLECULAR GENETIC STUDIES OF RECESSIVELY INHERITED EYE DISEASES by SARAH JOYCE A thesis submitted to The University of Birmingham for the degree of DOCTOR OF PHILOSOPHY School of Clinical and Experimental Medicine The Medical School University of Birmingham July 2011 University of Birmingham Research Archive e-theses repository This unpublished thesis/dissertation is copyright of the author and/or third parties. The intellectual property rights of the author or third parties in respect of this work are as defined by The Copyright Designs and Patents Act 1988 or as modified by any successor legislation. Any use made of information contained in this thesis/dissertation must be in accordance with that legislation and must be properly acknowledged. Further distribution or reproduction in any format is prohibited without the permission of the copyright holder. Abstract Cataract is the opacification of the crystalline lens of the eye. Both childhood and later-onset cataracts have been linked with complex genetic factors. Cataracts vary in phenotype and exhibit genetic heterogeneity. They can appear as isolated abnormalities, or as part of a syndrome. During this project, analysis of syndromic and non-syndromic cataract families using genetic linkage studies was undertaken in order to identify the genes involved, using an autozygosity mapping and positional candidate approach. Causative mutations were identified in families with syndromes involving cataracts. The finding of a mutation in CYP27A1 in a family with Cerebrotendinous Xanthomatosis permitted clinical intervention as this is a treatable disorder. A mutation that segregated with disease status in a family with Marinesco Sjogren Syndrome was identified in SIL1. In a family with Knobloch Syndrome, a frameshift mutation in COL18A1 was detected in all affected individuals. Analysis of families with non-syndromic autosomal recessive cataracts was also performed, using genome-wide linkage scans to identify homozygous candidate regions and to then sequence candidate genes within these regions. The identification of a potential putative mutation in one family in CDC25A illustrated the challenges of distinguishing between rare benign variants and pathogenic mutations. Identification of novel genes involved in cataractogenesis will increase understanding of the pathways involved in cataract formation, and benefit affected families through genetic counselling, and, potentially personalised management. Acknowledgements I would like to thank my supervisors Professor Eamonn Maher and Dr Paul Gissen for giving me the opportunity to carry out this research and for their support during the entire process. So many people helped me in the lab, but I would particularly like to thank Dr Esther Meyer for unwavering support, often beyond the call of duty. Thanks also to Uncaar, Manju, Louise, Ania, Neil, Derek, Jane, Holly, Chris, Blerida, Shanaz, Nick, Dean, Dewi and Mike for advice and for answering my questions. I would also like to thank my family, especially my mum and my sister Kathleen, and my friends for all helping me through the tougher moments. I‟m sure my dad would be proud. I am grateful for the families who agreed to take part in this project, and for Fight For Sight for funding.this work. Declaration In this project, some work was carried out by other people. The Affymetrix Genechip Human Mapping 10K Array was carried out by MRC Geneservice. The Genechip Human Mapping 250K SNP array was carried out by Louise Tee or Fatimah Rahman. Dr Michael Simpson conducted the exome sequencing in this project. Dr Esther Meyer performed linkage analysis for cataract families 1, 5 and 6. DNA extraction was carried out by West Midlands Regional Genetics Molecular Genetics Laboratory. Table of Contents TABLE OF CONTENTS Title Abstract Acknowledgements Declaration Table of Contents List of Illustrations List of Tables Abbreviations 1. INTRODUCTION 1 1.1 Overview 1 1.2 The Eye 1 1.2.1 Eye Development 3 1.2.2 Lens Development in Humans 3 1.3 Eye Diseases 4 1.3.1 Hereditary Eye Diseases 6 1.3.1.1 The Genetics of Eye Development 6 1.3.1.1.1 Paired Box Gene 6 (PAX6) 7 1.3.1.1.2 Sonic Hedgehog Gene (SHH) 8 1.3.1.1.3 Sine Oculis Homeobox 3 Gene (SIX3) 8 1.3.1.1.4 SRY-Box 2 Gene (SOX2) 9 1.3.1.1.5 Retina and Anterior Neural Fold Homeobox Gene (RX) 9 1.3.1.2 The Genetics of Lens Development 10 1.3.1.2.1 Forkhead Box E3 Gene (FOXE3) 10 1.3.1.2.2 V-MAF Avian Musculoaponeurotic Fibrosarcoma Oncogene Homolog Gene (MAF) 11 1.3.1.2.3 Paired-like Homeodomain Transcription Factor 3 Gene (PITX3) 11 1.3.1.2.4 Crystallins 12 1.3.1.2.5 Connexins 14 1.3.2 Cataract 15 1.3.2.1 Cataract Classification 15 1.3.2.2 Types of Cataracts 15 1.3.2.3 Genetics of Cataracts 20 1.3.2.3.1 Beaded Filament Structural Protein 1 Gene (BFSP1) 21 1.3.2.3.2 Crystallins 22 1.3.2.3.2.1 Crystallin, Alpha-A (CRYAA) 22 1.3.2.3.2.2 Crystallin, Alpha-B (CRYAB) 22 1.3.2.3.2.3 Crystallin, Beta-A1 (CRYBA1) 23 1.3.2.3.2.4 Crystallin, Beta-A4 (CRYBA4) 23 1.3.2.3.2.5 Crystallin, Beta-B1 (CRYBB1) 23 Table of Contents 1.3.2.3.2.6 Crystallin, Beta-B2 (CRYBB2) 24 1.3.2.3.2.7 Crystallin, Beta-B3 (CRYBB3) 24 1.3.2.3.2.8 Crystallin, Gamma-C (CRYGC) 24 1.3.2.3.2.9 Crystallin, Gamma-D (CRYGD) 25 1.3.2.3.2.10 Crystallin, Gamma-S (CRYGS) 25 1.3.2.3.3 Galactokinase 1 (GALK1) 27 1.3.2.3.4 Glucosaminyl (N-acetyl) Transferase 2 Gene (GCNT2) 28 1.3.2.3.5 Gap Junction Alpha-8 Protein Gene (GJA8) 29 1.3.2.3.6 Heat Shock Transcription Factor 4 Gene (HSF4) 29 1.3.2.3.7 Lens Intrinsic Membrane Protein 2 Gene (LIM2) 30 1.3.2.3.8 Visual System Homeobox 2 Gene (VSX2) 31 1.4 Disease Gene Identification 32 1.4.1 Functional Candidate Gene Approach 32 1.4.2 Positional Approach 33 1.4.2.1 Genetic Mapping 34 1.4.2.2 Linkage Analysis 37 1.4.2.3 LOD Scores and Critical Values 39 1.4.2.4 Two-Point and Multi-Point Linkage Analysis 40 1.4.2.5 Markers 40 1.4.2.5.1 Restriction Fragment Length Polymorphisms (RFLPs) 40 1.4.2.5.2 Microsatellites 41 1.4.2.5.3 Single Nucleotide Polymorphisms (SNPs) 42 1.4.2.6 Autozygosity Mapping 44 1.4.2.6.1 Consanguinity 44 1.4.2.6.2 Principles of Autozygosity Mapping 46 1.4.2.6.3 Mathematical Principles of Autozygosity Mapping 47 1.4.2.6.4 The Power of Autozygosity Mapping 48 1.4.2.6.5 Pitfalls of Autozygosity Mapping 49 1.4.3 Exome Sequencing 50 1.4.4 Progress in Gene Identification 51 2. MATERIAL AND METHODS 52 2.1 Materials 53 2.1.1 Chemicals 53 2.2 Methods 57 2.2.1 DNA 57 2.2.2 Polymerase Chain Reaction (PCR) 57 2.2.2.1 PCR Conditions 59 2.2.2.2 Primer Design 60 2.2.3 Agarose Gel Electrophoresis 60 2.2.4 DNA Purification 61 2.2.4.1 ExoSAP-IT 61 2.2.4.2 MicroCLEAN 62 2.2.5 Linkage Studies 62 2.2.5.1 Microsatellite Marker Analysis 62 Table of Contents 2.2.5.2 SNP Chip 64 2.2.6 Sequencing 65 2.2.7 Next Generation Sequencing 66 2.2.8 Cloning of PCR Products 67 2.2.8.1 IMAGE Clone 67 2.2.8.2 Vectors 68 2.2.8.3 Antibiotic Plates and LB Medium 69 2.2.8.4 Plasmid DNA Purification 70 2.2.8.4.1 Miniprep of Plasmids 70 2.2.8.4.2 Maxiprep of Plasmids 70 2.2.8.5 Gel Extraction 70 2.2.8.6 Restriction Enzyme Digests 71 2.2.8.7 Ligation 72 2.2.8.8 Site-Directed Mutagenesis 73 2.2.8.8.1 PCR of Site-Directed Mutagenesis 73 2.2.8.8.2 Digest and Transformation 74 2.2.9 Functional Work 75 2.2.9.1 Cell Culture 75 2.2.9.1.1 Cell Storage and Thawing 75 2.2.9.1.2 Transfection 76 2.2.9.1.3 Cell Lysis 77 2.2.9.2 Protein Determination 77 2.2.9.3 Western Blotting 78 2.2.9.4 Protein Densitometry 81 2.2.9.5 Immunofluorescent Staining 82 2.2.9.5.1 Confocal Microscopy 83 2.2.9.6 Fluorescence Activated Cell Sorting (FACS) 84 3. SYNDROMIC CATARACT FAMILIES 86 3.1 CEREBROTENDINOUS XANTHOMATOSIS (CTX) 87 3.1.1 Introduction 87 3.1.2 Patient DNA 88 3.1.3 Previous Work 90 3.1.4 Molecular Genetics Method 91 3.1.4.1 Prioritised Candidate Genes 91 3.1.4.1.1 Indian Hedgehog (IHH) 91 3.1.4.1.2 Inhibin Alpha (INHA) 91 3.1.4.1.3 Tubulin Alpha 4a (TUBA4A) 92 3.1.4.1.4 Wingless-type MMTV Integration Site Family Member 10A (WNT10A) 92 3.1.4.1.5 Cytochrome P450 Family 27 Subfamily A Polypeptide 1 (CYP27A1) 93 3.1.5 Results 93 3.1.6 Discussion 94 3.2 MARINESCO-SJÖGREN SYNDROME (MSS) 96 Table of Contents 3.2.1 Introduction 96 3.2.1.1 Marinesco-Sjögren Syndrome (MSS) 96 3.2.1.2 SIL1 98 3.2.1.3 Cataract, Congenital, with Facial Dysmorphism and Neuropathy (CCFDN) 99 3.2.2 Patient DNA 99 3.2.3 Molecular Genetics Method 101 3.2.4 Results 101 3.2.5 Discussion 104 3.3 KNOBLOCH SYNDROME 105 3.3.1 Introduction 105 3.3.2 Patient DNA 106 3.3.3 Previous Work 107 3.3.4 Molecular Genetics Methods 108 3.3.5 Results 108 3.3.5.1 Chromosome 17 Open Reading Frame 42 (C17orf42) 109 3.3.5.2 Oligodendrocyte Myelin Glycoprotein (OMG) 109 3.3.5.3 Ecotropic Viral Integration Site 2A (EVI2A) 110 3.3.5.4 Ecotropic Viral Integration Site 2B (EVI2B) 110 3.3.5.5 Ring Finger Protein 135 (RNF135) 111 3.3.6 Discussion 114 4.