Genetic Analysis of Hereditary Sensory, Motor and Autonomic
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Genetic analysis of hereditary sensory, motor and autonomic neuropathies, including a rat model Ming-Jen Lee A thesis submitted to the University of London for the degree of Doctor of Philosophy August 2002 Institute of Neurology University of London ProQuest Number: 10016056 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 10016056 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 To my wife and my parents Who supported me throughout my studies. Acknowledgements I would like to thank Dr. Mike Groves and Professor Francesco Scaravilli, for their help in the animal breeding and phenotype characterization. I am also indebted to Dr. Dennis Stephenson, MacLaughlin Institute, Grate Fall, Montana, USA. for advice on positional cloning and on the construction of the rat/mouse/human comparative genetic map. His idea on BLAST searches between Celera mouse genome database and rat markers, made a breakthrough in selecting candidate genes, which lead me to identify the m f gene. I am grateful to Dr. Pete Dixon and Dr. Mary Davis for their advice on genetic linkage and physical mapping. I would also like to thank Ms. Mary Sweeney, for her expert advice on genotyping. Thanks to Dr. Henry Houlden for his help in the initial stage of this project. I am thankful to both of my supervisors. Dr. Mary M. Reilly and Prof. Nicholas W. Wood. They initiated the entire project and I am truly grateful that I have the chance to be involved from the very beginning. I appreciated the support and guidance that both provided. I gratefully acknowledge the financial support from Brain Research Trust and from the Ministry of Education, Taipei, Taiwan. Lastly, I thank God for his blessing on my work and my family living in London. Abstract The subject of this thesis is genetic research in two inherited neuropathies, the hereditary sensory and autonomic neuropathies (HSAN) and Charcot-Marie- Tooth diseases (CMTs). Two sections are included; the first is dedicated to identifying the causative gene of a hereditary sensory neuropathy in a spontaneous mutant rat {mf rat). The second involves the sequencing of two known genes in patients with CMTs. The locus for the m f disease was linked to the distal end of rat chromosome 14. Using the sequences of the closely linked rat microsatellite markers, database BLAST searches identified a three Megabase region on mouse chromosome 11 whose gene content was co-linear with that on human 2pl5-pl6. Sequencing the cDNA of genes in this interval revealed a c.l349G>A substitution in subunit 4 of the chaperonin containing TCP-1 protein {Cct4) in the mutant rat. This change replaced a highly conserved cysteine450 with tyrosine. This result demonstrates that a mutation in the Cct4 protein causes an inherited sensory neuropathy in rats. The genes, myelin protein zero {MPZ) and gap junction protein betal {GJBl), were screened for mutations in patients with CMT. Most forms of CMT are either clearly demyelinating or axonal. Mutations in MPZ and GJBl can cause both types of CMT. Two novel MPZ mutations were identified in five Taiwanese patients with a demyelinating neuropathy. Forty-six British patients with axonal CMT were also screened but only one mutation was identified. The frequency of MPZ mutations in axonal CMT is low. GJBl is the gene responsible for X-linked CMT (CMTX). The clinical features of CMTX patients are variable and the neurophysiological findings can suggest a demyelinating or an axonal neuropathy. 24 mutations (6 novel ones) were identified in 139 patients with CMT. Detailed clinical and electrophysiological studies in these patients are presented. In conclusion, this thesis identified the genetic defect of the m f rat, an animal model for HSAN, and identified mutations in MPZ and GJBl in CMT patients. Contents TITLE PAGE 1 DEDICATION 2 ACKNOWLEDGEMENTS 3 ABSTRACT 4 CONTENTS 6 LIST OF FIGURES 10 LIST OF TABLES 11 ABBREVIATIONS 12 CHAPTER 1 INTRODUCTION 14 1.1 Hereditary Sensory and Autonomic Neuropathies (HSANs) 14 1.1.1 Classification and molecular genetics of the HSANs 16 1.1.1.1 HSAN I 16 1.1.1.2 HSAN II 18 1.1.1.3 HSAN III 19 1.1.1.4 HSAN IV 21 1.1.1.5 HSAN V 23 1.2 Charcot-Marie-Tooth disease 26 1.2.1 Historical reyiew 26 1.2.2 Classification of Charcot-Marie-Tooth disease 27 1.2.3 Clinical and Pathological features of Charcot-Marie-Tooth disease 30 1.2.3.1 CMTl 30 1.2.3.2 X-linked CMT 31 1.2.3.3 HNPP 32 1.2.3.4 CMT2 32 1.2.3.5 Dejerine-Sottas Disease 34 1.2.3.6 CHN 35 1.2.3.7 DI-CMT(dominant intermediate-CMT) 36 1.2.3.8 Autosomal recessiye CMT (ARCMT) 36 1.2.4 Molecular Genetics of CMT 38 1.2.4.1 CMTl, HNPP, DSD and CHN 38 1.2.4.2 HNPP 43 1.2.4.3 X-linked CMT 44 1.2.4.4 DI-CMT 45 1.2.4.5 CMT2 45 1.2.4.6 ARCMT 48 1.2.4.7 X-linked CMT2 52 1.2.5 Conclusion 53 1.3 Rat genetic study 54 1.3.1 History 54 1.3.2 Mutilated foot rat 55 1.3.3 Pathological findings of the m f rat 56 1.3.4 Animal model for HSAN 60 1.3.5 Genetic analysis of the m f rat 62 1.3.5.1 Strategies for disease gene identification 62 1.3.5.2 Resources 65 1.3.5.2.1 Marker loci 66 1.3.5.2.2Bioinformatic resources 67 1.3.5.2.3 Homologous/syntenic mapping 68 1.3.5.2.4Clone resources 70 CHAPTER 2 MATERIAL AND METHODS 72 2.1 The rat project 72 2.1.1 Materials 72 2.1.1.1 Breeding protocol 72 2.1.1.2 Phenotype characterization and tissue collection 73 2.1.2 Methods 74 2.1.2.1 Genetic linkage analysis 74 2.1.2.1.1 Introduction 74 2.1.2.1.2 Genome-wide screening and linkage study 75 2.1.2.1.3 Selecting the BAG clones 79 2.1.2.1.4 Anchoring the m/locus on the mouse genome 80 2.1.2.1.5 Candidate gene screening 84 2.1.2.2 Nucleic acid extraction 84 2.1.2.2.1 Extraction of genomic DNA from rat soft tissue 84 2.1.2.2.2 Quantification of DNA 85 2.1.2.2.3 Extraction of total RNA from fresh rat tissue 86 2.1.2.2.4 Bacterial culture and DNA extraction from BAG clones 86 2.1.2.3 Polymerase chain reaction 88 2.1.2.3.1 Amplification of rat genomic DNA 89 2.1.2.3.2 Reverse transcription PCR (RT-PCR) 90 2.1.2.3.3 Degenerate PCR 90 2.1.2.3.4 Fluorescent incorporation PCR 91 2.1.2.3.5 Purification of PCR product 92 2.1.2.3.6 Sequencing reaction 93 2.1.2.4 Electrophoresis 95 2.1.2.4.1 Agarose gel electrophoresis 95 2.1.2.4.2 Single-strand conformation polymorphism (SSCP) gel electrophoresis 96 2.1.2.4.3 Photography of electrophoresis gel under UV light transilluminator 97 2.1.2.4.4 Polyacrylamide gel electrophoresis 97 2.1.2.4.4.1 Polyacrylamide gel preparation 97 2.1.2.4.4.2 Gel loading and conditions for electrophoresis 98 2.1.2.4.5 Data analysis 100 2.1.2.4.5.1 Genescan data analysis 100 2.1.2.4.5.2 DNA sequencing data analysis 100 2.1.2.5 Southern blot hybridization 101 2.1.2.6 Northern blot hybridization 103 2.1.2.6.1 Probe tailing and determination of labeling efficiency 104 2.1.2.6.2 Transferring and fixing the RNA onto a nylon membrane 105 2.1.2.6.3 Prehybridization and hybridization 105 2.1.2.6.4 Signal detection 106 2.2 Human CMT disease project 107 2.2.1 Patients 107 2.2.1.1. Patients for MPZ mutation screening 107 2.2.1.1.1 Taiwanese patients 107 2.2.1.1.2 British CMT2 patients 109 2.2.1.2 Patients for GJBl mutation screening 109 2.2.2 Methods 110 2.2.2.1 Extraction of genomic DNA from human whole blood 110 2.2.2.2 PCR 111 2.2.2.3 Restriction fragment length polymorphism (RFLP) analysis 112 2.2.2.4 Genotyping and sequencing 112 CHAPTER 3 RESULTS OF GENETIC ANALYSIS IN THE MUTILATED FOOT {mf) RAT 113 3. 1_____ Positional independent candidate gene study 113 3.2 Genetic linkage analysis 114 3.2.1 Choosing mating strain 114 3.2.2 Genome wide screening 117 3.2.3 Refinement of linkage region 119 3.3 Identification of the responsible gene 121 3.3.1 Selecting BAC clones 121 3.3.2 Anchoring linked rat segment to the mouse genome 124 3.3.3 Candidate gene screening 130 3.3.3.1 Otxl 130 3.3.3.2 Kiaa0127 131 3.3.3.3 B elli A 132 3.3.3.4 Identifying the gene 132 3.3.3.5 The expression of Cct4 138 CHAPTER 4 DISCUSSION: RAT GENETIC STUDY 142 4.1 Summary of the rat project 142 4.2 The function of Cct4 and its role in m f disease 143 4.2.1 mutation 143 4.2.2 Pathogenesis of m f disease 145 4.2.2.1 Actin and apoptosis 147 4.2.2.2 Independent role in neuronal apoptosis 149 4.3 Conclusion 150 CHAPTER 5 RESULTS OF SCREENING OF MPZ IN CMT PATIENTS 151 5.1 MPZ mutations in CMT 1 patients 151 5.1.1 Molecular genetic findings 151 5.1.2 Clinical features of patients with MPZ mutations 153 5.2 MPZ mutation in CMT2 patients 157 5.2.1 Molecular genetic findings 157 5.2.2 Clinical features of patient with MPZ mutation 157 CHAPTER 6 DISCUSSION; MPZ MUTATIONS IN CMT PATIENTS 161 6.1 Genetic findings 161 6.2 Phenotypes in patients with MPZ mutations 162 6.3 Functional implications for MPZ mutations 164 6.4 Conclusion 166 CHAPTER 7 RESULTS OF SCREENING GJBl IN CMT PATIENTS 168 7.