GENETICSOFDISTALHEREDITARY MOTOR NEUROPATHIES By alexander peter drew A thesis submitted for the Degree of Doctor of Philosophy Supervised by Professor Garth A. Nicholson Dr. Ian P. Blair Faculty of Medicine University of Sydney 2012 statement No part of the work described in this thesis has been submitted in fulfilment of the requirements for any other academic degree or qualification. Except where due acknowledgement has been made, all experimental work was performed by the author. Alexander Peter Drew CONTENTS acknowledgements ............................. i summary .................................... ii list of figures ................................ v list of tables ................................ viii acronyms and abbreviations ..................... xi publications ................................. xiv 1 literature review ........................... 1 1.1 Molecular genetics and mechanisms of disease in Distal Hereditary Motor Neuropathies . .1 1.1.1 Small heat shock protein family . .2 1.1.2 Dynactin 1 (DCTN1).....................9 1.1.3 Immunoglobulin mu binding protein 2 gene (IGHMBP2) 11 1.1.4 Senataxin (SETX)....................... 14 1.1.5 Glycyl-tRNA synthase (GARS)............... 16 1.1.6 Berardinelli-Seip congenital lipodystrophy 2 (SEIPIN) gene (BSCL2)......................... 18 1.1.7 ATPase, Cu2+-transporting, alpha polypeptide gene (ATP7A) 20 1.1.8 Pleckstrin homology domain-containing protein, G5 gene (PLEKHG5)........................... 21 1.1.9 Transient receptor potential cation channel, V4 gene (TRPV4) 22 1.1.10 DYNC1H1 ........................... 23 1.1.11 Clinical variability in dHMN . 24 1.1.12 Common disease mechanisms in dHMN . 29 2 general materials and methods ................. 32 2.1 General materials and reagents . 32 2.1.1 Reagents and Enzymes . 32 2.1.2 Equipment . 33 2.1.3 Plasticware . 33 2.2 Study participants . 34 2.3 DNA methods . 34 2.3.1 DNA extraction from blood . 34 2.3.2 DNA oligonucleotides . 35 2.3.3 PCR protocol . 35 2.3.4 Agarose gel electrophoresis . 36 2.3.5 DNA purification from PCR . 36 2.3.6 DNA sequencing . 37 3 genetic mapping ............................. 39 3.1 Introduction . 39 3.1.1 Overview . 39 3.1.2 Homologous recombination and linkage disequilibrium 39 3.1.3 Genetic linkage analysis . 41 3.1.4 Penetrance in dHMN . 44 3.1.5 Characterisation of dHMN1 in a large Australian family 44 3.1.6 Previous genetic studies in dHMN1 at 7q34-q36 . 44 3.1.7 Hypothesis and aims . 48 3.2 Materials and Methods . 50 3.2.1 Microsatellite analysis . 50 3.2.2 Microsatellite markers . 51 3.2.3 Haplotype analysis . 51 3.2.4 Genetic linkage analysis . 52 3.2.5 Association analysis . 53 3.3 Results . 54 3.3.1 Fine mapping of family CMT54 . 54 3.3.2 Linkage analysis using new markers in CMT54 . 57 3.3.3 Identification of additional dHMN families . 60 3.3.4 Linkage power analysis . 62 3.3.5 Genetic linkage analysis in additional dHMN families . 65 3.3.6 Suggestive linkage in family CMT44 . 65 3.3.7 Uninformative families . 66 3.3.8 dHMN families excluded from 7q34-q36 . 74 3.3.9 Haplotype association analysis . 75 3.4 Discussion . 76 3.4.1 Linkage analysis in CMT54 . 76 3.4.2 Linkage analysis in the extended dHMN cohort . 79 3.4.3 Proportion of dHMN caused by a pathogenic gene at 7q34-q36 . 82 4 analysis of candidate genes .................... 84 4.1 Introduction . 84 4.1.1 Chapter overview . 84 4.1.2 Strategy for gene identification . 84 4.1.3 Identifying genes within a disease interval . 85 4.1.4 Selection of candidate genes . 86 4.1.5 Gene screening methods . 88 4.2 Materials and Methods . 90 4.2.1 In-silico candidate gene prioritisation . 90 4.2.2 PCR and sequencing for gene screening . 90 4.2.3 Analysis of effect of variants on exon splicing . 91 4.2.4 RNA studies of splicing variants . 92 4.2.5 RNA folding prediction . 93 4.3 Results . 94 4.3.1 Positional candidate genes . 94 4.3.2 Functional candidate gene selection . 96 4.3.3 Screening candidate genes . 99 4.3.4 Analysis of the interval defined by significantly associ- ated haplotypes . 106 4.4 Discussion . 107 4.4.1 Gene screening summary . 107 4.4.2 Was the mutation missed? . 108 5 copy number and structural variation ............ 109 5.1 Introduction . 109 5.1.1 CNV and structural variation . 109 5.1.2 CNV in dHMN and related peripheral neuropathies . 112 5.1.3 Methods of detecting CNV . 113 5.1.4 Hypothesis and Aim . 115 5.2 Materials and Methods . 115 5.2.1 Custom CGH Microarray . 115 5.2.2 Array processing . 116 5.2.3 Software Nexus 5 . 117 5.2.4 PCR confirmation of deletions . 118 5.3 Results . 118 5.3.1 Cytogenetic analysis . 118 5.3.2 CNV analysis at 7q34-q36 using aCGH . 119 5.4 Discussion . 130 5.4.1 Summary . 130 5.4.2 Comparison of methods for CNV analysis . 130 5.4.3 Limitations of array-based CGH . 133 6 next generation sequencing of cmt54 ............. 134 6.1 Introduction . 134 6.1.1 NGS technology . 134 6.1.2 NGS of large genomes . 138 6.1.3 Applications of NGS to Mendelian disease . 139 6.2 Materials and methods . 141 6.2.1 NimbleGen array-based targeted sequence capture . 141 6.2.2 454 GS FLX sequencing . 142 6.2.3 NimbleGen array-based exome capture and Solexa se- quencing . 143 6.2.4 Confirmation of sequence variants using Sanger sequencing144 6.2.5 Sequence variant analysis using Galaxy Browser . 144 6.2.6 NGS assembly and annotation using Bowtie and SAMtools144 6.2.7 Unequal coverage comparison . 145 6.2.8 In silico prediction of functional impacts of sequence vari- ants . 145 6.3 Results . 146 6.3.1 Chromosome 7q34-q36 target region sequencing . 146 6.3.2 7q34-q36 sequence variant analysis . 147 6.3.3 Off-target and repeat mapping reads . 154 6.3.4 Exome sequencing . 155 6.3.5 Further sequencing analysis using unequal coverage anal- ysis . 158 6.3.6 Exome wide analysis . 163 6.4 Discussion . 164 6.4.1 Limitations of the NGS analysis . 166 7 exome sequencing of cmt44 .................... 169 7.1 Introduction . 169 7.2 Materials and methods . 170 7.2.1 Exome sequencing . 170 7.2.2 NGS variant analysis . 171 7.2.3 HRM analysis . 172 7.3 Results . 172 7.3.1 Exome sequencing in CMT44 . 172 7.3.2 Clinical detail of CMT44 . 173 7.3.3 MFN2 genotyping in CMT44 using high resolution melt analysis . 177 7.3.4 Exome wide sequence variant analysis in CMT44 . 177 7.3.5 Comparison of SNPs between CMT44 and CMT54 . 179 7.4 Discussion . 179 8 general discussion .......................... 185 8.1 Genetic studies in family CMT54 . 185 8.2 Genetic linkage analysis in additional dHMN families . 190 8.3 Genetic studies in family CMT44 . 191 8.4 Conclusion . 192 a custom gnu bash scripts ...................... 194 a.1 Linkage analysis . 194 a.2 NGS coverage visualisation . 197 a.3 NGS variant comparison with fold coverage analysis . 198 b pcr primers ................................ 205 c pedigrees for additional dhmn families ........... 216 d two point lod scores for additional dhmn families ............................... 230 e multi-point lod scores for additional dhmn families ............................... 240 f 7q34-q36 control haplotypes ................... 249 g 7q34-q36 candidate genes and reported gene expression .............................. 251 h next generation sequencing .................... 255 i cnv identified with array cgh .................. 257 bibliography ................................. 261 ACKNOWLEDGEMENTS It is a pleasure to thank the people who made this PhD project possible. My warmest thanks to my supervisors Professor Garth Nicholson and Dr Ian Blair for your support, encouragement and the opportunities presented to me throughout this project. To Garth, your expertise with the clinical aspects of peripheral neuropathies as well as your academic experience have proved invaluable. To Ian, thank you for your guidance in the lab, the opportunities and resources I was given and the assistance with preparation of my thesis. I am extremely grateful to all participating dHMN family members, without whom this research would not be possible. To my colleagues, in particular, Kelly Williams, for assistance with the targeted sequencing of candidate genes and Jenn Solski, for assistance with sequencing variants identified with NGS. To all the staff and students in the Northcott Neuroscience and Molecular Medicine laboratories, thank you for your friendly assistance over these years. The help of Annette Berryman in the Molecular Medicine office and the procedural guidance of Tracey Dent and Annet Doss in the ANZAC research institute office has been most helpful. The support and encouragement of my friends and family has been essential. My parents, Paul and Josephina Drew, have been a constant source of support, both emotionally and financially during my postgraduate years. Finally, my warmest thanks go to my girlfriend Victoria, without her loving support and patience this thesis would never have been possible. You always bring me joy. This work was supported in part by an Australian Postgraduate Award and funding from the National Health and Medical Research Council of Australia (511941). i SUMMARY The distal hereditary motor neuropathies (dHMN) are a clinically and geneti- cally heterogeneous group of disorders that primarily affect motor neurons, without significant sensory involvement. Using genome wide linkage analy- sis in a large Australian family (CMT54), a form of dHMN was previously mapped by this laboratory, to a 12.98 Mb interval on chromosome 7q34-q36. The axonal neuropathy seen.