Clinicogenetic and functional studies in rare hereditary neurodegenerative movement disorders by Dr. Sarah Wiethoff A thesis submitted to University College London for the degree of Doctor of Philosophy Department of Molecular Neuroscience Institute of Neurology University College London (UCL) May 2016 1 I, Sarah Wiethoff, 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. Collaborative work is also indicated in this thesis. Signature: Date: 2 Abstract Neurodegenerative diseases are equally fascinating as they are devastating. They illustrate both function and pathology of neurons, the most complex cells in the human body. In the past, technological progress has allowed the identification of genetic variation that can lead to neurodegenerative processes. However, for many patients with different neurodegenerative diseases to date, no genetic diagnosis is obtained despite thorough investigation. For another significant proportion of neurodegenerative diseases the genetic defect and the resulting clinical phenotype/spectrum is known, but exact pathomechanisms remain elusive. This delays successful translational research and eventual clinical treatment. The objective of this thesis is to combine both aspects and employ two main techniques to further advance the search for better pathophysiologic understanding of neurodegenerative diseases: whole exome sequencing (WES) and induced pluripotent stem cell (iPSC) technology. Firstly, the thesis aims to improve clinical characterisation and genetic analysis of neurodegenerative patients to identify genetic causes and genetic modifiers of disease. Secondly, it aims to establish functional models in search of pathogenic and potentially druggable mechanisms using iPSCs in clinically and genetically characterised groups of patients. This thesis thereby investigated the clinicogenetic and pathophysiological bases of rare hereditary movement disorders with focus on hereditary ataxias, trinucleotide repeat and complex parkinsonism disorders, including neurodegeneration with brain iron accumulation (NBIA). Clinical characterisation, WES, targeted sequencing, homozygosity mapping and traditional Sanger sequencing were used to yield clinical description, identify underlying genetic causes and to investigate disease modifiers in different trinucleotide repeat disorders. To finally establish functional readouts of pathogenic mutations in a well characterised patient cohort, iPSC technology and directed differentiation was used to create a human neuronal model of spinocerebellar ataxia type 15 and to derive non-forebrain neuronal precursors to facilitate the study of cerebellar diseases using iPSCs in the future. 3 Table of contents Abstract .................................................................................................................................... 3 Table of contents ................................................................................................................... 4 Acknowledgements .............................................................................................................. 6 Abbreviations ......................................................................................................................... 8 List of tables ......................................................................................................................... 13 List of figures ....................................................................................................................... 15 Publications/Book chapters ........................................................................................... 18 1 Chapter 1: General introduction ............................................................................ 20 1.1 General introduction and aims of this thesis .......................................................... 20 1.2 Genetics in neurodegenerative disease .................................................................... 22 1.3 Clinicogenetics of neurodegeneration with brain iron accumulation ............ 35 1.4 Polyglutamine disorders and inherited cerebellar ataxias ............................... 68 1.5 Induced pluripotent stem cell technology and its usefulness for cerebellar disease modelling ........................................................................................................................ 74 2 Chapter 2: Methods ..................................................................................................... 91 2.1 Methods Chapter 3 ............................................................................................................ 91 2.2 Methods Chapter 4 .......................................................................................................... 105 2.3 Methods Chapter 5 .......................................................................................................... 109 2.4 Methods Chapter 6 .......................................................................................................... 115 3 Chapter 3: Dissecting neurodegeneration with brain iron accumulation clinically, genetically and cellularly ................................................................ .......... 127 3.1 Introduction ..................................................................................................................... 127 3.2 Results ................................................................................................................................ 129 3.3 Discussion ......................................................................................................................... 152 4 Chapter 4: Genetics in families with rare neurodegenerative movement disorders: Examples from McLeod syndrome and hereditary ataxias .......... 168 4.1 McLeod syndrome: Identification of a novel single base-pair deletion ....... 168 4.2 SYNE1: Novel mutations and extension of the clinicogenetic spectrum ...... 175 4.3 PNPLA6-associated disorders: Novel homozygous variants in a large consanguineous Parsi kindred with pure cerebellar ataxia ....................................... 184 4 5 Chapter 5: Trinucleotide disorders: The conundrum of modifiers of age at onset, somatic and repeat instability and repeat interruptions ...................... 193 5.1 DNA repair pathways underlie a common genetic mechanism modulating onset in polyglutamine diseases .......................................................................................... 193 5.2 The role of repeat interruptions in SCA6 ............................................................... 203 6 Chapter 6: Modelling cerebellar disease with human iPSCs ....................... 211 6.1 Generating a human iPSC model to study spinocerebellar ataxia type 15 . 211 6.2 Generating non-cortical neuronal derivatives to improve the accuracy in modelling cerebellar disease ................................................................................................ 241 7 Conclusions and future directions ...................................................................... 250 References .......................................................................................................................... 258 8 APPENDIX ..................................................................................................................... 282 5 Acknowledgements I would like to thank my supervisors Professor Henry Houlden and Professor John Hardy for their excellent supervision and support, laboratory environment and mentorship on exciting projects. I would also like to express my thankfulness towards my subsidiary supervisors Dr. Conceicao Bettencourt and Dr. Rickie Patani for advising, guiding and supporting me throughout my different projects. Thanks to our patients and their families in providing their patience, time and material as well as to our numerous clinical collaborators who contribute with excellent clinical cases. A warm thanks to the members of the iPSC-dream-team rota who made my life in the second part of the PhD so much more wonderful. Special thanks to Prof. Andrew Singleton, Monica Federoff, Dr. Monia Ben-Hamad Hammer, Dr. Jinhui Ding, Steve Clipman, Dr. Raphael Gibbs and Dr. Dena Hernandez for their collaboration, guidance and support during my stay at the NIH and beyond as well as to Hallgeir Jonvick, Dr. Boniface Mok and Dr. Alan Pittman for helping with data transfer and analysis strategies at the IoN. Thanks to all members of the diagnostic lab, to Chris Steele, Dr. Lee Stanyer, June Smalley, Mark Gaskin, Deborah Hughes, Stephanie Efthymiou, Alexandra Zirra, Chris Lovejoy, Dr. Qiang Gang, Dr. Lucia Schottlaender, Dr. Niccolo Mencacci, Dr. Claudia Manzoni, Dr. Joshua Hersheson, Dr. Lasse Pihlstrom, Dr. Reema Paudel, Dr. Arianna Tucci, Dr. Alice Gardiner, Dr. Ellen Cottenie, Dr. David Lynch, Dr. Steven Lubbe, Claire Hall, Dr. Martina Hallegger, Dr. Chris Sibley, Dr. Zhi Yao, Dr. Sonia Gandhi, Dr. Rosella Abeti, Dr. Sarah Crisp, Dr. Monika Madej, Dr. Raffaele Ferrari, Dr. Davina Hensman, Dr. Enrico Bugiardini, Dr. Roman Praschberger, Dr. Mike Flower, Dr. Chelban, Andreea Manole and Dr. James Polke for their help and guidance at IoN as well as special thanks to Dr. Abi Li, Dr. Giulia Tyzack, Dr. Selina Wray, Professor Tom Warner, Dr. Rohan de Silva, Dr. Nuria Seto-Salvia, Dr. Charles Arber, Elisavet Preza, Luke Hill, Daniel Cotfas, Teresa Sposito, Dr. Rubika Balendra, Lee