A Genetic Investigation of the Muscle and Neuronal Channelopathies: From Sanger to Next – Generation Sequencing Alice Gardiner MRC Centre for Neuromuscular Diseases and UCL Institute of Neurology Supervised by Professor Henry Houlden and Professor Mike Hanna 1 Declaration I, Alice Gardiner, 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. Signature A~~~ . Date ~.'t..J.q~ l.?,.q.l.~ . 2 Abstract The neurological channelopathies are a group of hereditary, episodic and frequently debilitating diseases often caused by dysfunction of voltage-gated ion channels. This thesis reports genetic studies of carefully clinically characterised patient cohorts with different episodic neurological and neuromuscular disorders including paroxysmal dyskinesias, episodic ataxia, periodic paralysis and episodic rhabdomyolysis. Genetic and clinical heterogeneity has in the past, using traditional Sanger sequencing methods, made genetic diagnosis difficult and time consuming. This has led to many patients and families being undiagnosed. Here, different sequencing technologies were employed to define the genetic architecture in the paroxysmal disorders. Initially, Sanger sequencing was employed to screen the three known paroxysmal dyskinesia genes in a large cohort of paroxysmal movement disorder patients and smaller mixed episodic phenotype cohort. A genetic diagnosis was achieved in 39% and 13% of the cohorts respectively, and the genetic and phenotypic overlap was highlighted. Subsequently, next-generation sequencing panels were developed, for the first time in our laboratory. Small custom-designed amplicon-based panels were used for the skeletal muscle and neuronal channelopathies. They offered considerable clinical and practical benefit over traditional Sanger sequencing and revealed further phenotypic overlap, however there were still problems to overcome with incomplete coverage. Large custom and non-custom pull-down panels were used to investigate patients with recurrent rhabdomyolysis patients. The contribution of genetic abnormalities was determined, and it was concluded that while the contribution of the RYR1 was substantial, it was minimal for the classic voltage-gated ion channels SCN4A and CACNA1S Lastly, whole-exome sequencing was applied to two large undiagnosed possible channelopathy families. One family was found to indeed harbour a channelopathy mutation, whilst the other did not. Overall, next-generation sequencing proved to be a more thorough and efficient method for channelopathy genetic diagnosis and several novel findings throughout the thesis expanded the current knowledge within the field. 3 Acknowledgements I would first like to thank my supervisors Professor Henry Houlden and Professor Mike Hanna for their support, encouragement and scientific guidance of me and my project throughout the last four years. I am deeply grateful for the opportunity, time and supervision that they have given me, and without which I could not have succeeded. I am also hugely grateful to my friends and family; in particular to my parents for their unwavering belief in me, their constant assistance in all things, for providing a lovely place for me to write when I needed to escape London, and to my mum for taking the time to read through this thesis. I thank my sister, Jenny, for her encouragement, wisdom, and the benefit of her scientific experience and my twin, Robert, for his constant non-scientific support. I also thank all of my friends for keeping me sane and for making the move to London one that I have not for a second regretted. I am especially indebted to Andy and Elaine for opening their house to me, first, when I needed somewhere to live three years ago and more recently when I needed somewhere to write, away from the builders. This PhD would have not been possible without unending help and insight from everyone within Molecular Neurosciences, the MRC centre for Neuromuscular Diseases and the Neurogenetics team, in particular Renata, Amelie, Fatima, Josh, Mark, Klaus, Cath, Ros, Rob, Roope, Alan, Debbie and Karen, to whom I express my sincerest gratitude. Special thanks go to Ellen, Siobhan and Qiang, with whom sharing an office has been a pleasure, for their entertainment, advice and friendship. Lastly, I would like extend my thanks to the Muscular Dystrophy Campaign, who funded this project, and to all of the patients and their families who have taken part in this research. 4 Table of Contents Declaration ................................................................................................................... 2 Abstract ........................................................................................................................ 3 Acknowledgements ....................................................................................................... 4 Table of Contents ......................................................................................................... 5 List of Figures ............................................................................................................. 13 List of Tables .............................................................................................................. 16 List of Abbreviations ................................................................................................... 19 Publications Arising from this Thesis........................................................................... 23 Chapter 1: Introduction ............................................................................................... 26 1. Voltage-Gated Ion Channels ................................................................................ 26 1.1. Action Potentials ........................................................................................... 27 1.2. Structure of Voltage-Gated Ion Channels ..................................................... 27 1.3. Families of Voltage-Gated Ion Channels....................................................... 30 1.3.1. Voltage-Gated Sodium Channels (VGSCs) ........................................... 31 1.3.2. Voltage-Gated Calcium Channels (VGCCs) .......................................... 31 1.3.3. Voltage-Gated Chloride Channels ......................................................... 31 1.3.4. Voltage-Gated Potassium Channels (VGKCs) ....................................... 31 1.4. Ryanodine Receptors (RyRs) ....................................................................... 39 2. Channelopathies .................................................................................................. 39 2.1. The Skeletal Muscle Channelopathies .............................................................. 40 2.1.1. The Non-Dystrophic Myotonias .............................................................. 43 2.1.2. The Periodic Paralyses .......................................................................... 45 2.2. The Neuronal Channelopathies .................................................................... 48 2.2.1. Episodic Ataxia ...................................................................................... 50 2.2.2. Familial Hemiplegic Migraine ................................................................. 53 2.2.3. The Paroxysmal Dyskinesias ................................................................. 55 2.3. Non-Channel Channelopathies ..................................................................... 63 3. Rhabdomyolysis .................................................................................................. 64 5 3.1. Channelopathies ........................................................................................... 66 3.1.1. MHS1 – RYR1 ....................................................................................... 66 3.1.2. MHS5 – CACNA1S ................................................................................ 67 3.1.3. SCN4A .................................................................................................. 67 3.2. Metabolic Muscle Disorders .......................................................................... 67 3.2.1. Glycogen Storage Diseases .................................................................. 68 3.2.2. Fatty Acid Metabolism Disorders ........................................................... 75 3.3. Muscular Dystrophy Genes........................................................................... 78 3.3.1. ANO5 .................................................................................................... 78 3.3.2. FKRP ..................................................................................................... 78 3.3.3. DMD ...................................................................................................... 78 3.3.4. SGCA and SGCB .................................................................................. 79 3.3.5. DYSF ..................................................................................................... 79 3.4. Miscellaneous Genes ................................................................................... 79 3.5. Common Polymorphisms .............................................................................. 81 4. Mutation Detection ............................................................................................... 84 4.1. Methods of Mutation Detection ....................................................................