COPYRIGHT AND USE OF THIS THESIS This thesis must be used in accordance with the provisions of the Copyright Act 1968. Reproduction of material protected by copyright may be an infringement of copyright and copyright owners may be entitled to take legal action against persons who infringe their copyright. Section 51 (2) of the Copyright Act permits an authorized officer of a university library or archives to provide a copy (by communication or otherwise) of an unpublished thesis kept in the library or archives, to a person who satisfies the authorized officer that he or she requires the reproduction for the purposes of research or study. The Copyright Act grants the creator of a work a number of moral rights, specifically the right of attribution, the right against false attribution and the right of integrity. You may infringe the author’s moral rights if you: - fail to acknowledge the author of this thesis if you quote sections from the work - attribute this thesis to another author - subject this thesis to derogatory treatment which may prejudice the author’s reputation For further information contact the University’s Copyright Service. sydney.edu.au/copyright Neuropathy in childhood mitochondrial disease, including riboflavin transporter deficiency: phenotype, neurophysiology and disease-modifying therapy in a recently described treatable disorder Dr Manoj Peter Menezes A thesis submitted for the degree of Doctor of Philosophy in the faculty of Medicine, The University of Sydney, 2015. Submitted October 2015 1 DECLARATION I hereby declare that this submission is my own work and that, to the best of my knowledge and belief, it contains no material previously published or written by another person or material which to a substantial extent has been accepted for the award of any other degree or diploma of the university or other institute of higher learning, except where due acknowledgement has been made in the text. Signed ________________________ Date__________________ Manoj Peter Menezes 2 ABSTRACT Introduction: Defining the neurophysiological features of the peripheral neuropathy associated with childhood mitochondrial disease, including the neuropathy/neuronopathy associated with Brown-Vialetto-Van Laere syndrome (BVVL), will aid in classifying the mitochondrial syndrome, directing genetic testing and instituting therapy. Methods: Nerve conduction studies and clinical assessments were performed on children with nuclear or mitochondrial genome mutations. The clinical and genetic profile, neurophysiology, histopathology, audiology and response to riboflavin therapy of nine children with BVVL due to RFVT2 deficiency were evaluated. Results: Peripheral neuropathy was more frequent with nuclear gene mutations. SURF1 was associated with a demyelinating neuropathy while PDHc deficiency caused an axonal neuropathy. POLG mutations caused a sensory axonal neuropathy. The neuropathy associated with mitochondrial disease was not length-dependent. Children with RFVT2 deficiency presented with an auditory neuropathy and sensory ataxia, and developed upper limb weakness. Nerve excitability studies showed an increase in myelin permeability. Riboflavin therapy caused partial reversal of these changes and improvement in strength, audiology and respiratory function. Conclusions: Nerve conduction studies should be part of the initial assessment of children with suspected mitochondrial disease. Clinicians need to be familiar with the unique phenotype of RFVT2 deficiency so that affected children are diagnosed early and commenced on appropriate therapy. 3 ACKNOWLEDGEMENTS The supervisory team for my PhD is a veritable Who’s Who of neuromuscular research in Australia, and I have benefitted greatly from their knowledge, experience and enthusiasm. I would like to thank Prof Kathryn North, for encouraging me to pursue my interest in neuromuscular research, for giving me my first neuromuscular training position, co- authoring my first peer-reviewed paper and suggesting I undertake a postgraduate research degree. She introduced me to Prof Ouvrier and suggested that I pursue peripheral neuropathy as a subspecialty interest, and that interest is now a major part of my clinical and research work. Her support was also invaluable when I took time off clinical work to train in neuromuscular medicine and work on this PhD. I would like to thank my primary supervisor Prof Ouvrier for teaching me both the science and the art of peripheral nerve medicine. This PhD has coincided with probably the most exciting period in peripheral nerve research, with rapid advances in our understanding of the genetics of inherited peripheral nerve disease, due to the availability of next-generation sequencing technologies. I have benefitted from not only the extremely well phenotyped cohort of patients accumulated by Prof Ouvrier but also the national and international collaborations he has established. I would like to thank A/Prof Monique Ryan for not only spending time to carefully edit each chapter of this PhD but also her insightful comments and questions whenever I presented portions of this PhD at conferences. I would also like to thank the many co- authors on the different studies that make up this PhD (some awaiting publication), especially Prof Joshua Burns and Dr Michelle Farrar for teaching me the techniques of CMTPedS evaluation and nerve excitability studies. I would like to thank my wife Riona and son Xavier for accepting without complaint the late evenings and many weekends spent working on this PhD and the missed holidays, swimming lessons and visits to the park. I would also like to thank my parents, Joseph and Muriel, for instilling a hard work ethic and continuing to delight in my every achievement, however minor. Finally, I would like to thank all the patients and their families who participated in the studies on this PhD, especially the families of children with BVVL, and shared the journey from identification of the causative mutations to evaluating the benefits of therapy with riboflavin. 4 DEDICATION To Riona, for her unwavering support, and Xavier, for his beautiful smile. 5 TABLE OF CONTENTS Declaration ............................................................................................................................... 2 Abstract .................................................................................................................................... 3 Acknowledgements .................................................................................................................. 4 Dedication ................................................................................................................................ 5 Table of contents ...................................................................................................................... 6 List of abbreviations ................................................................................................................. 8 Publications, presentations and awards................................................................................... 10 Literature Review ................................................................................................................... 11 Hypothesis………................................................................................................................... 29 Methodology........................................................................................................................... 32 Section I Peripheral neuropathy associated with mitochondrial disease in children Chapter 1................................................................................................................................. 39 Neurophysiological profile of peripheral neuropathy in childhood mitochondrial disease Section II Brown-Vialetto-Van Laere syndrome due to RFVT2 deficiency Chapter 2 ................................................................................................................................ 60 Phenotype of Brown-Vialetto-Van Laere syndrome due to RFVT2 deficiency Chapter 3 ................................................................................................................................ 90 Auditory neuropathy in Brown-Vialetto-Van Laere syndrome due to RFVT2 deficiency 6 Chapter 4 .............................................................................................................................. 107 Pathophysiology of motor nerve dysfunction in Brown-Vialetto-Van Laere syndrome due to RFVT2 deficiency Chapter 5 .............................................................................................................................. 127 Outcome after therapy with riboflavin in Brown-Vialetto-Van Laere syndrome due to RFVT2 deficiency Summary and recommendations........................................................................................... 143 References ............................................................................................................................ 152 Appendix .............................................................................................................................. 178 7 LIST OF COMMON ABBREVIATIONS ABR Automated brainstem responses ANS Ataxia neuropathy spectrum ANSD Auditory neuropathy spectrum disorder APB Abductor pollicis brevis BVVL Brown-Vialetto-Van Laere syndrome CM Cochlear microphonic CMAP Compound muscle action potential CMT Charcot-Marie-Tooth disease CMTPedS CMT paediatric scale FAD Flavin