Molecular Genetics of Corneal Dystrophy A THESIS SUBMITTED FOR THE M.D. TO THE UNIVERSITY OF LONDON MOHAMED EL-ASHRY, MB CHB FRCS (Ed) CLINICAL RESEARCH FELLOW DEPARTMENT OF MOLECULAR GENETICS INSTITUTE OF OPHTHALMOLOGY UNIVERSITY COLLEGE LONDON BATH STREET LONDON AND MOORFIELDS EYE HOSPITAL CITY ROAD LONDON 2001 ProQuest Number: 10013866 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest. ProQuest 10013866 Published by ProQuest LLC(2016). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. Microform Edition © ProQuest LLC. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 Abstract Abstract Comeal dystrophies are inherited disorders characterised by progressive accumulation of deposits in the cornea causing visual impairment. They occur in either an autosomal dominant or recessive form and are usually manifested in the first few decades of life. The present classification is solely based on the layer or layers of the cornea involved. This study aimed at better understanding of the underlying molecular basis of such disorders via linkage to specific chromosomal loci and then mutation screening of the disease genes by means of amplification of the genomic DNA using polymerase chain reaction and then sequencing and restriction enzyme digest analysis. A family with a combined cornea plana and microphthalmia phenotype was mapped to the autosomal recessive cornea plana (CNA2) locus on 12q21. A novel mutation in KERA gene (CNA2) has also been identified. The phenotype described is wider than that previously reported for KERA mutations and represents the first description of a stmctural protein causing microphthalmia. Mutation screening of a new carbohydrate sulfotransferase 6 gene {CHST6) in 5 families with autosomal recessive macular comeal dystrophy (MCD) type I has revealed 6 novel missense mutations, 4 homozygous and 2 compound heterozygous. This study provides further evidence that CHST6 is the MCD gene and the loss of gene function by these mutations may result in production of abnormal keratan sulfate, which would account for the MCD phenotype. Analysis of a human transforming growth factor-6 -induced gene (BIGH3) in 36 patients with lattice comeal dystrophy type I (LCDI), Avellino comeal dystrophy (ACD), granular comeal dystrophy (GCD), and Reis-Bucklers’ comeal dystrophy (RBCD) has revealed 4 heterozygous missense mutations. In conclusion, molecular genetics may provide a more accurate basis for the diagnosis and classification of comeal dystrophies. This work contributes to our understanding of the role of specific genes in comeal transparency and may help to identify novel therapeutic approaches for these dystrophies that cause significant visual impairment. Table o f contents Table of Contents Page Abstract 2 Table of contents 3 List of tables 12 List of figures 14 Declaration 18 Acknowledgements 19 List of abbreviations 20 Key to pedigree symbols 22 Chapter 1 23 Introduction 23 1.1 General introduction 23 1.2 Cornea 24 1.2.1 Gross anatomy 24 1.2.2 Microscopic anatomy 24 1.2.2.1 Epithelium 26 1.2.2.2 Bowman’s membrane 27 1.2.2.3 Stroma 27 1.2.2.4 Descemet’s membrane 28 1.2.2.5 Endothelium 28 1.2.3 Comeal transparency 29 1.2.4 Development of the cornea 29 1.2.5 Metabolism and physiology of the cornea 33 1.3 Corneal dystrophy: A phenotypic review 35 Table o f contents 1.3.1 Epithelial dystrophies 36 1.3.1.1 Meesmann's dystrophy 36 1.3.1.2 Epithelial basement membrane dystrophies 37 1.3.1.3 Band-shaped dystrophy 38 1.3.2 Bowman's layer dystrophies 3 8 1.3.2.1 Reis-Bücklers' comeal dystrophy (type I 38 Bowman’s layer, CDBI dystrophy, RBCD) 1.3.2.2 Honeycomb dystrophy of Thiel and Behnke 40 (type II Bowman’s layer, CDBII dystrophy) 1.3.2.3 Grayson-Wilbrandt dystrophy 41 1.3.3 Stromal dystrophies 43 1.3.3.1 Granular comeal dystrophy (GCD) 43 1.3.3.2 Lattice comeal dystrophy (LCD) 44 1.3.3.2.1 Lattice comeal dystrophy type I (LCD type I) 44 1.3.3.2.2 Lattice comeal dystrophy type II (LCD type II) 45 1.3.3.2.3 Lattice comeal dystrophy type HI (LCD type HI) 45 1.3.3.2.4 Lattice comeal dystrophy type IIIA (LCD type 46 IIIA) 1.3.3.2.5 Lattice comeal dystrophy type IV (LCD type IV) 46 1.3.3.3 Avellino comeal dystrophy (ACD) 48 1.3.3.4 Macular comeal dystrophy (MCD) 48 1.3.3.5 Fleck comeal dystrophy (Mouchetée or speckled comeal dystrophy) 49 1.3.3.6 Central crystalline dystrophy of Schnyder's 50 Table o f contents 1.3.4 Endothelial dystrophy 52 1.3.4.1 Fuch's dystrophy 52 1.3.4.2 congenital hereditary endothelial dystrophy (CHED) 52 1.3.4.3 Posterior polymorphous dystrophy (PPD) 53 1.3.5 Ectatic dystrophies 56 1.3.5.1 Keratoconus 5 6 1.3.5.2 Keratoglobus 56 1.3.5.3 Macrocomea 57 1.4 Anomalous corneal development 57 1.4.1 Cornea plana 57 1.4.2 Microphthalmia 59 1.4.3 Sclerocomea 60 1.4.4 Genetics of cornea plana and microphthalmia 61 1.5 Genetics of comeal dystrophy 63 1.6 Mapping single gene disorders by linkage analysis 65 1.6.1 History of linkage analysis 65 1.6.2 Linkage analysis 65 1.6.3 Genetic and physical map distances 66 1.6.4 Genetic markers 67 1.6.4.1 Requirements for genetic mapping 67 1.6.4.2 Restriction fragment length polymorphisms (RFLPs) 68 1.6.4.3 Variable number of tandem repeats (VNTRs) or 68 minisatellites 1.6.4.4 Microsatellite markers 69 (short tandem repeat polymorphisms [STRPs]) Table o f contents 1.6.4.3 Single nucleotide polymorphisms 69 (biallelic markers) or SNPs 1.6.5 Linkage analysis for autosomal dominant diseases 70 1.6.6 Linkage analysis for autosomal recessive diseases 70 1.6.7 Two point mapping to locate a disease locus 71 1.6.8 Evidence of genetic heterogeneity 72 1.7 The human genome project 72 1.7.1 Microsatellite maps 73 1.7.2 Identification of genes 74 1.7.3 Mutation screening 75 1.7.3.1 Identifying disease-causing mutations 76 1.8 Aims of the study 76 Chapter 2 78 Materials and methods 78 2.1 Extraction of DNA 78 2.1.1 Extraction of DNA from peripheral blood lymphocytes 78 2.1.2 Extraction of DNA from buccal smears 79 2.2 DNA amplification by polymerase chain reaction (PCR) 80 2.2.1 The PCR reaction 80 2.2.2 Standard parameters for PCR 82 2.2.3. Primer design 82 2.3 Fractionation of DNA by gel electrophoresis 83 2.3.1 Agarose gel electrophoresis 83 2.3.2 Non-denaturing polyacrylamide gel electrophoresis 84 2.4 Purification of DNA 86 Table o f contents 2.4 Purification of DNA 86 2.4.1 Phenol chloroform extraction and ethanol precipitation 86 2.4.2 Use of Sephacryl microspin columns (Sephacryl-S400 HR 87 columns, Pharmacia, UK) 2.4.3 Use of Qiagen gel extraction/ PCR purification kits 88 2.5 Restriction enzyme digests of DNA 89 2.6 Mutation detection techniques 89 2.6.1 Heteroduplex analysis method 89 2.6.2 Single strand conformation polymorphism analysis (SSCP) 91 2.6.3 DNA sequencing 92 2.7 Assay of sulphated keratan sulfate (KS) in serum 94 2.7.1 Specimens 94 2.7.2 Antibodies 95 2.7.3 Inhibition—Enzyme-Linked Immunosorbent Assay 95 (ELISA) for keratan sulfate detection 2.8 Computer aided analysis 96 2.8.1 Computational analysis of DNA sequence 96 2.8.2 Linkage computer programs (Cyrillic and MLINK) 96 2.9 Buffers and solutions 97 2.10 Electronic database information 98 Chapter 3 99 A novel mutation in keratocan causes autosomal recessive cornea plana and 99 microphthalmia. 3.1 Introduction 99 3.1.1 Cornea plana 99 Table o f contents 3.1.2.1 Autosomal dominant inheritance 100 3.1.2.2 Autosomal recessive inheritance 101 3.1.2.3 Identification of the gene for cornea plana 101 3.1.3 Microphthalmia 102 3.1.3.1 Phenotype of microphthalmia 102 3.1.3.2 Genetics of microphthalmia 103 3.1.4 Small leucine rich proteoglycans (SLRPs) 103 3.1.4.1 Class I SLRPs 104 3.1.4.2 Class II SLRPs 105 3.1.4.3 Class III SLRPs 105 2iA.5 Kera 107 3.2 Aim of the study 109 3.3 Patients and methods 110 3.3.1 Family and clinical data 110 3.3.2 Methods 114 3.3.2.1 Genotyping 114 3.3.2.2 Linkage analysis 114 3.3.2.3 Mutation screening of KERA by sequencing 116 3.4 Results 117 3.4.1 Linkage of the family to the known locus for cornea plana 117 3.4.2 Exclusion of linkage to the known loci of microphthalmia 118 3.4.3 Mutation screening of KERA 124 3.4.4 Functional analysis of the T215K mutation by protein modelling 126 3.5 Discussion 129 Table o f contents Chapter 4 133 Spectrum of BIGH3 mutations in comeal dystrophies 133 4.1 Introduction 133 4.1.1 Chromosome 5q linked dystrophies 133 4A2BIG H 3 gene 134 4.1.3 Kerato-epithelin 134 4.1.4 Comeal dystrophies caused by BIGH3 gene mutations 136 4.1.4.1 Granular comeal dystrophy (GCD) or 136 (Groenouw type I) (CDGGI) (OMIM NO 121900) 4.1.4.2 Reis- Bucklers' comeal dystrophy (RBCD) 137 (OMIM N0121900) 4.1.4.3 Avellino comeal dystrophy (ACD) 138 (OMIM NO 121900) 4.1.4.4 Lattice comeal dystrophies (LCD) 139 4.2 Aim of the study 140 4.3 Patients and methods 140 4.3.1 Patients 140 4.3.1.1 RBCD patients 141 4.3.1.2 GCD patients 145 4.3.1.3 ACD patients 150 4.3.1.4 LCDI patients 151 4.3.1.5 A Bangladeshi family with RBCD and LCDI phenotyp 156 4.3.2 Methods 158 4.3.2.1 DNA extraction 158 4.3.2.2 PCR amplification of BIGH3 gene 158 Table o f contents 4.3.2.3 Mutation detection 158 4.4 Results 160 4.4.1 Identification of R555Q mutation in RBCD Patients 160 4.4.2 Identification