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Molecular and Genetic Basis of Inherited Optic Neuropathies Padraig James Flannery BA MSc Thesis submitted to Newcastle University in candidature for the degree of Doctor of Philosophy Institute of Genetic Medicine Faculty of Medical Sciences Newcastle University To Mum, Dad and Ciara i Abstract Inherited optic neuropathies represent an important cause of chronic visual morbidity among children and young adults. This group of disorders is genetically heterogeneous and it can be caused by pathogenic mutations within both the mitochondrial and nuclear genomes. The unifying pathophysiological theme is mitochondrial dysfunction, but the specific disease mechanisms that ultimately precipitate neuronal loss, particularly retinal ganglion cell (RGC) degeneration, still remain unclear. The work presented in this thesis provides further insight into the molecular and genetic basis of two classical forms of inherited optic neuropathy, namely autosomal dominant optic atrophy (DOA) and Wolfram syndrome. Dominant optic atrophy (DOA) secondary to pathogenic OPA1 mutations is the most common inherited optic neuropathy diagnosed in clinical practice. The pathology is characterised by the preferential loss of RGCs within the inner retina and optic nerve degeneration. Although most OPA1 mutation carriers will only develop isolated optic atrophy, a subgroup of patients, referred to as DOA plus (DOA+), will develop more severe neuromuscular complications in addition to visual failure. The complexity of these clinical presentations may be due in part, to the various roles of OPA1 in the mitochondrial compartment such as regulating mitochondrial fusion and cristae structure,sequesterisation of pro-apoptotic molecules, mitochondrial DNA (mtDNA) maintenance,proper functioning of the oxidative phosphorylation system and calcium homoeostasis. To investigate the disease mechanisms that could explain the varying clinical manifestations and severity of OPA1 mutations, I made use of a cohort of eight fibroblast cell lines established from four patients with pure optic atrophy (OA) and four patients with DOA+ phenotypes. OPA1 expression and mitochondrial fragmentation patterns were compared between these two groups. There was no significant disruption in OPA1 transcription, mitochondrial OXPHOS and mtDNA maintenance. DOA primary fibroblasts showed increased fragmentation of the mitochondrial network and cell lines established from patients with DOA+ phenotypes were found to be particularly susceptible to fragmentation under basal conditions, which had not been reported previously. ii To further explore the findings obtained in OPA1 mutant fibroblasts, I made use of a cohort of nine myoblast cell lines that had previously been established from patient muscle biopsies. Interestingly, a similar mitochondrial fragmentation pattern was observed in OPA1 mutant primary myotubes and this was associated with decreased mitochondrial DNA molecule number in DOA+ myotubes. I also investigated two sisters from a consanguineous Arab Muslim family who developed a fatal form of juvenile-encephalopathy complicated by optic atrophy and cardiomyopathy. Exome sequencing identified a putative homozygous OPA1 mutation, which was confirmed by both functional studies and in silico modelling. Whole-exome analysis was carried out on a cohort of fourteen patients with optic atrophy that had previously been found to be OPA1-negative. Pathogenic mutations in the Wolframin (WFS1) gene, which is known to cause Wolfram syndrome, were identified in 3/14 (21%) patients. Based on our results, WFS1 mutations are an important cause of inherited optic atrophy and genetic testing should be considered in OPA1-negative patients. In conclusion, the body of work presented in my PhD thesis has provided further insight into the expanding genotypic and phenotypic spectrum of inherited optic neuropathies, which is highly relevant for clinical diagnosis and patient management. iii Author’s Declaration This thesis is submitted to Newcastle University for the degree of Doctor of Philosophy. Research described within was conducted between the years 2012-2016 and was supervised by Dr. Patrick Yu-Wai-Man and Professor Patrick Chinnery. I certify that none of the material contained within this thesis has been previously submitted by me for a degree or other qualification at this or any other university. iv Acknowledgements First and foremost, I would like to thank my supervisors Dr. Patrick Yu-Wai-Man and Professor Patrick Chinnery for giving me the opportunity to work on an excellent project. Their outstanding mentorship and guidance over the past few years were invaluable and it was a privilege to work with them. I would also like to offer my special thanks to both Kamil Sitarz and Florence Burte who provided patience, continual support and guidance over the course of my PhD. They helped to mould the scientist I am today. Of course, I must also mention Dr. Aurora Gomez-Duran who provided valuable insight and expertise and was always available anytime I had a ‘quick question’. Her scientific guidance will serve me in years to come over the course of my scientific career. A special mention is also deserved for Drs. Gavin Hudson, Angela Pyle and Glyn Nelson who were more than willing to share their technical knowledge and experience which helped shape the course of my PhD project. Moreover, I must also thank my other colleagues within the PFC lab group for providing a friendly and enjoyable work environment. They supported my scientific endeavours and I had a great experience, both working and socialising, with a fantastic group of scientists these past few years. Finally, I would like to thank the UK Medical Research Council and Newcastle University for providing me with a PhD studentship to advance my scientific career and making all of this possible. v List of Contents CHAPTER 1 INTRODUCTION ............................................................................................ 1 CHAPTER 2 RESEARCH AIMS ........................................................................................ 75 CHAPTER 3 MATERIALS AND METHODS ................................................................... 77 CHAPTER 4 GENOTYPE-PHENOTYPE CHARACTERISATION IN AUTOSOMAL DOMINANT OPTIC ATROPHY ........................................................................................ 98 CHAPTER 5 MITOCHONDRIAL NETWORK AND NUCLEOID DISTRIBUTION IN OPA1-MUTANT MYOTUBES ........................................................................................... 134 CHAPTER 6 NOVEL HOMOZYGOUS OPA1 MUTATION IN A CONSANGUINEOUS ISRAELI FAMILY ....................................................................... 169 CHAPTER 7 WHOLE-EXOME ANALYSIS OF A PATIENT COHORT WITH INHERITED OPTIC ATROPHY ...................................................................................... 191 CHAPTER 8 WFS1 MUTATIONS IN PATIENTS WITH INHERITED OPTIC ATROPHY ............................................................................................................................ 223 CHAPTER 9 GENERAL DISCUSSION ........................................................................... 244 APPENDIX A OPA1 PROTEIN LEVELS ........................................................................ 252 APPENDIX B OPA1 GENE EXPRESSION ..................................................................... 254 APPENDIX C MITOCHONDRIAL NETWORK ANALYSIS ...................................... 255 vi APPENDIX D MITOCHONDRIAL MAINTENANCE ANALYSIS .............................. 257 APPENDIX E OXPHOS DISTURBANCES ...................................................................... 258 APPENDIX F MITOCHONDRIAL NETWORK ANALYSIS IN MYOTUBES .......... 263 APPENDIX G MITOCHONDRIAL NUCLEOID DISTRIBUTION ............................. 267 APPENDIX H BENIGN VARIANTS FOUND IN WFS1 SCREEN ............................... 270 BIBLIOGRAPHY ................................................................................................................. 271 vii Table of Contents CHAPTER 1 INTRODUCTION ............................................................................................ 1 1.1 INHERITED OPTIC NEUROPATHIES ..................................................................................... 2 1.1.1 Epidemiology ............................................................................................................ 2 1.1.2 Classification ............................................................................................................ 4 1.2 LEBER HEREDITARY OPTIC NEUROPATHY (LHON) .......................................................... 6 1.2.1 Epidemiology ............................................................................................................ 6 1.2.2 Molecular genetics .................................................................................................... 6 1.2.3 Clinical manifestations ............................................................................................. 8 1.2.4 LHON plus phenotypes ............................................................................................. 9 1.2.5 Pathophysiology ........................................................................................................ 9 1.2.6 Other mtDNA mutations causing optic neuropathies ............................................. 10 1.3 AUTOSOMAL DOMINANT OPTIC ATROPHY ...................................................................... 12 1.3.1 Epidemiology .........................................................................................................

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