Clinical and Genetic Studies in Paediatric Mitochondrial Disease Yehani N.G.G. Wedatilake Genetics and Genomic Medicine Programme UCL Great Ormond Street Institute of Child Health University College London January 2017 A thesis submitted for the degree of Doctor of Philosophy awarded by University College London 1 Declaration I, Yehani N.G.G Wedatilake, 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. Signed ______________________________________________________________ 2 Abstract Paediatric mitochondrial disease is a clinically and genetically heterogeneous group of disorders. Prior to the advent of next generation sequencing, many patients did not receive a genetic diagnosis. Genetic diagnosis is important for prognostication and prenatal diagnosis. The aim of this thesis was to study the genetic aetiology of paediatric mitochondrial disease. Paediatric patients with suspected mitochondrial disease were grouped to facilitate studying the genetic aetiology. This included grouping patients by respiratory chain enzyme deficiency, by affected end-organ type (cardiomyopathy) or by collectively investigating patients presenting with a similar, genetically undefined clinical syndrome. Whole exome sequencing (WES) was used to investigate genetic causes in patients with suspected mitochondrial disease associated with cytochrome c oxidase (COX) deficiency. In another paediatric cohort with mitochondrial cardiomyopathy, mitochondrial DNA sequencing, candidate gene sequencing or WES were used to identify genetic causes. Two families with a unique syndrome (febrile episodes, sideroblastic anaemia and immunodeficiency) were investigated using WES or homozygosity mapping. Candidate genes were functionally evaluated including the use of animal models. Furthermore a natural history study was performed in a monogenic mitochondrial disease (SURF1 deficiency) using clinical data from 12 centres. WES in COX-deficient patients revealed candidate genes for 14/30, including genes not targeted to the mitochondrion. In a mitochondrial cardiomyopathy (n=30) cohort, 18 genetic causes were identified. ATAD3A and MDH2 were identified as novel causes of human disease. Novel AARS2 and VARS2 mutations were found in patients with new clinical presentations. Tubular aggregates were identified in SERAC1 deficiency, as a novel histological finding in this disease. TRNT1 mutations were identified as causing a unique clinical syndrome. Two candidate genes (IGHMBP2 and SCYL1) were investigated as the cause for a mitochondrial DNA depletion syndrome. WES is a powerful tool for gene identification in mitochondrial disease leading to discovery of previously unrecognised, novel genetic causes. 3 Acknowledgements I am grateful to my supervisors Professor Shamima Rahman and Dr Jan-Willem Taanman for their support, enthusiasm and guidance. In addition I would like to thank Dr Elisa Fassone and Dr Rojeen Shahni postdoctoral researchers at the UCL Institute of Child Health for their time. I would like to express my thanks to Dr Iain Hargreaves who performs respiratory chain enzyme assays at the diagnostic lab at Neurometabolic Unit, Queen Square, London. In addition I am grateful to Mary Sweeney and Cathy Woodward for mitochondrial DNA sequencing, Suzie Drury for her supervision during the targeted sequencing project, Vincent Plagnol and Warren Emmett for their help with bioinformatics and Latifa Chentouf, clinical research nurse for her assistance with recruitment. I wish to thank all collaborators including Dr Peter Krawtiz, Dr Stephane Pelletier and Dr Michal Minczuk. I am especially grateful to the Wellcome Trust for awarding me a research training fellowship to pursue this exciting project. I am also grateful to CLIMB for providing funding for the targeted next generation sequencing panel. I am grateful to Kari and Magnus for their help and support during this time, especially with childcare. I also wish take this opportunity to express my gratitude to my parents who have always put us before themselves, and still continuously support me in everything I do. I thank my dearest E, who always encourages and supports me with my work; I thank him for his unwavering belief in me and for sharing his scientific point of view. Last but not least - thank you to our daughter who was born during this period of study, for all the joy she brings into our lives. 4 Publications arising from this work Wedatilake Y, Niazi R, Fassone E, Powell CA, Pearce S, Plagnol V, Saldanha JW, Kleta R, Chong WK, Footitt E, Mills PB, Taanman JW, Minczuk M, Clayton PT, Rahman S. TRNT1 deficiency: clinical, biochemical and molecular genetic features. Orphanet J Rare Dis. 2016 Jul 2;11(1):90. Wedatilake Y, Plagnol V, Anderson G, Paine SM, Clayton PT, Jacques TS, Rahman S. Tubular aggregates caused by serine active site containing 1 (SERAC1) mutations in a patient with a mitochondrial encephalopathy. Neuropathol Appl Neurobiol. 2015 Apr;41(3):399-402 Shahni R, Wedatilake Y, Cleary MA, Lindley KJ, Sibson KR, Rahman S. A distinct mitochondrial myopathy, lactic acidosis and sideroblastic anemia (MLASA) phenotype associates with YARS2 mutations. Am J Med Genet A. 2013 Sep;161A(9):2334-8. Wedatilake Y, Brown RM, McFarland R, Yaplito-Lee J, Morris AA, Champion M, Jardine PE, Clarke A, Thorburn DR, Taylor RW, Land JM, Forrest K, Dobbie A, Simmons L, Aasheim ET, Ketteridge D, Hanrahan D, Chakrapani A, Brown GK, Rahman S. SURF1 deficiency: a multi- centre natural history study. Orphanet J Rare Dis. 2013 Jul 5;8:96. 5 Table of contents Declaration ................................................................................................................................... 2 Abstract ........................................................................................................................................ 3 Acknowledgements ..................................................................................................................... 4 Publications arising from this work ........................................................................................... 5 List of figures ............................................................................................................................. 13 List of tables ............................................................................................................................... 15 List of abbreviations .................................................................................................................. 16 1. Introduction ........................................................................................................................ 20 1.1 The discovery of mitochondria ...................................................................... 20 1.2 The basic structure of mitochondria ............................................................. 20 1.3 Mitochondria are dynamic organelles ........................................................... 22 1.4 The mitochondrial genome ............................................................................. 22 1.5 Mitochondrial DNA replication ....................................................................... 25 1.6 Mitochondrial DNA transcription ................................................................... 26 1.7 Post- transcriptional modification of mitochondrial tRNAs ......................... 27 1.8 Mitochondrial protein translation ................................................................... 28 1.8.1 Initiation ...................................................................................................... 28 1.8.2 Elongation .................................................................................................. 28 1.8.3 Termination ................................................................................................ 29 1.8.4 Aminoacyl tRNA synthetases ..................................................................... 32 1.9 Mitochondrial oxidative phosphorylation ...................................................... 33 1.9.1 Complex I ................................................................................................... 35 1.9.2 Complex II .................................................................................................. 35 1.9.3 Complex III ................................................................................................. 35 1.9.4 Complex IV ................................................................................................. 36 1.9.4.1 The structure of cytochrome c oxidase .................................................... 36 1.9.4.2 COX assembly ........................................................................................ 37 1.9.5 Complex V .................................................................................................. 38 1.10 Mitophagy ...................................................................................................... 39 1.11 Mitochondria and human disease ................................................................ 39 1.11.1 Disorders due to Mitochondrial DNA mutations ........................................ 41 1.11.2 Disorders due to nuclear gene mutations ................................................. 41 1.11.3 The clinical syndromes of mitochondrial disease ...................................... 41 1.11.4 Cytochrome c oxidase deficient mitochondrial disease ............................