GENETIC AND FUNCTIONAL INVESTIGATION OF INHERITED NEUROPATHIES Ellen Cottenie MRC Centre for Neuromuscular Diseases, UCL Institute of Neurology Supervisors: Professor Mary M. Reilly, Professor Henry Houlden and Professor Mike Hanna Thesis submitted for the degree of Doctor of Philosophy University College London 2015 1 Declaration I, Ellen Cottenie, confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis. 2 Abstract With the discovery of next generation sequencing techniques the landscape of pathogenic gene discovery has shifted drastically over the last ten years. For the purpose of this thesis, focus was applied on finding genetic causes of inherited neuropathies, mainly Charcot-Marie-Tooth disease, by using both old and new genetic techniques and the accompanying functional investigations to prove the pathogenicity of these variants. Mutations in ATPase 6, the first mitochondrially encoded gene responsible for an isolated neuropathy, were found in five families with CMT2 by a traditional Sanger sequencing approach. The same approach was used to expand the phenotype associated with FIG4 mutations, known as CMT4J. Compound heterozygous mutations were found in a patient with a proximal and asymmetric weakness and rapid deterioration of strength in a single limb, mimicking CIDP. Several appropriate cohorts were screened for mutations in candidate genes with the traditional Sanger sequencing approach; however, no new pathogenic genes were found. In the case of the HINT1 gene, the originally stated frequency of 11% could not be replicated and a founder effect was suggested, underlying the importance of considering the ethnic background of a patient when screening for mutations in neuropathy-related genes. After the incorporation of exome sequencing, five CMT families were provided with a genetic diagnosis due to mutations in three novel genes and two previously known pathogenic genes. Many more families are currently under investigation and candidate genes have been found in some. Lastly, a series of divergent functional techniques was used to investigate the pathogenicity of IGHMBP2 mutations in 11 families with CMT2. IGHMBP2 mutations normally lead to SMARD1 and fibroblast and lymphoblast studies indicate that the IGHMBP2 protein levels are significantly higher in CMT2 than SMARD1, but lower than controls, suggesting that the clinical phenotype differences correlate to the IGHMBP2 protein levels. 3 Acknowledgments First of all, I would like to express my gratefulness and gratitude to my supervisors Professor Mary Reilly, Professor Henry Houlden and Professor Mike Hanna, for giving me the chance to work on this thesis under their guidance. I immensely appreciate the opportunities they have given me to grow as a person, both professionally and personally. None of this would have been possible without the endless support from my family and friends, old and new. I would especially like to thank my mum for always being there, for her encouragement and belief in me, and her never-ending care. My dad, who made moving countries so much easier and was always there to help, and my grandma for her continuous supply of cakes and stews from home. A special thanks to Julia, for keeping me sane and crazy at the same time; Elodie, the most amazing colleague I am so pleased to call my friend and Alice and Siobhan, who made me want to go sit in the office every day and provided entertaining distractions during long days in the lab and were so kind to welcome me amongst their friends. Marta and Ellie, for all the lovely coffees and dinners we had together and their tremendous support and help. All the other people from the lab that helped me along the way, especially Qiang, Amelie, Kira, Sybille, Alex H, Alex R, James, Mary, Cathy, Ese, Jason and Jacky, Chris and Matilde at the centre, and everyone from the ION Department of Molecular Neuroscience and the MRC centre for their help, advice, and friendship during my PhD. I also would like to thank all of my friends in London and my lovely housemates, Nicol and Ramona, for making me feel at home and being there in the best and worst times. For sharing their wisdom, cooking recipes and nail polish and putting up with some of my weird habits. My friends in Belgium, for always welcoming me back and not forgetting about me. Especially Karolien and Mireille, two of the most wonderful women I know. Annemie, Mairo, Laura and Willeke, girls I’ve known since first grade and I know will always be there for me. 4 Table of Contents Declaration .................................................................................................................. 2 Abstract ....................................................................................................................... 3 Acknowledgments ...................................................................................................... 4 Figure list .................................................................................................................. 11 Table list .................................................................................................................... 15 List of abbreviations ................................................................................................ 17 List of publications ................................................................................................... 23 Chapter 1: Introduction .......................................................................................... 25 1.1 Phenotypic spectrum of peripheral neuropathies ............................................. 25 1.1.1 The peripheral nervous system .................................................................. 25 1.1.2 Peripheral neuropathies ............................................................................. 26 1.1.3 Charcot-Marie-Tooth disease .................................................................... 27 1.2 Genetic diversity and classification of CMT .................................................... 27 1.2.1 Demyelinating CMT .................................................................................. 28 1.2.2 Axonal CMT .............................................................................................. 32 1.2.3 Intermediate CMT ..................................................................................... 37 1.2.4 Hereditary Sensory and Autonomic Neuropathy ....................................... 39 1.2.5 Distal Hereditary Motor Neuropathy ......................................................... 42 1.2.6 X-linked CMT ........................................................................................... 47 1.3 Pathogenic pathways involved in Charcot-Marie-Tooth .................................. 49 1.3.1 Myelin assembly and Schwann cell dynamics .......................................... 49 1.3.2 Altered protein synthesis, sorting and/or degradation ............................... 50 1.3.3 Axonal transport Altered Transport Processes .......................................... 53 1.3.4 Alterations of the Cytoskeleton ................................................................. 56 1.3.5 Mitochondrial network dynamics and ATP production ............................ 58 1.3.6 tRNA synthetase genes .............................................................................. 61 1.3.7 Unknown pathways ................................................................................... 62 1.4 Thesis aims ....................................................................................................... 63 Chapter 2: Material and methods .......................................................................... 64 2.1 Genetic studies ................................................................................................. 64 2.1.1 Ethical approval and patient selection ....................................................... 64 5 2.1.2 DNA extraction .......................................................................................... 64 2.1.3 DNA concentration and purity ................................................................... 65 2.1.4 Polymerase Chain Reaction (PCR) ............................................................ 65 2.1.5 Agarose gel electrophoresis of PCR products ........................................... 67 2.1.6 DNA purification ....................................................................................... 67 2.1.7 Sequencing ................................................................................................. 68 2.1.8 Clean-up reaction ....................................................................................... 68 2.1.9 Sequence analysis ...................................................................................... 69 2.1.10 Restriction endonuclease analysis ........................................................... 69 2.1.11 Fragment analysis to look at the C9orf72 expansion repeat .................... 69 2.1.12 Exome Sequencing .................................................................................. 70 2.1.13 Linkage analysis ...................................................................................... 72 2.1.14 Haplotyping ............................................................................................. 72 2.2 Cell Culture .....................................................................................................