Investigating Gene Mutations in a South African Paediatric Cohort Diagnosed with Mitochondrial Disease
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Investigating gene mutations in a South African paediatric cohort diagnosed with mitochondrial disease M Schoonen orcid.org 0000-0001-6762-2232 Thesis submitted in fulfilment of the requirements for the degree Doctor of Philosophy in Biochemistry at the North-West University Promoter: Prof FH van der Westhuizen Co-promoter: Prof I Smuts Graduation May 2019 20574495 “It is paradoxical, yet true, to say, that the more we know, the more ignorant we become in the absolute sense, for it is only through enlightenment that we become conscious of our limitations. Precisely one of the most gratifying results of intellectual evolution is the continuous opening up of new and greater prospects.” Nikola Tesla “Persistence is very important. You should not give up unless you are forced to give up.” Elon Musk iii iv ACKNOWLEDGEMENTS I would like to thank the following individuals and institutions, whom all played a pivotal part in completing this thesis: First, I would like to express my utmost thanks to my promotor, Prof. Francois H. van der Westhuizen, and co-promoter, Prof. Izelle Smuts, for their kindness, support, guidance and expert knowledge throughout this study. I will always be grateful for your invaluable time. Dr. Marianne Pretorius, who showed me unconditional love and support throughout this study. Thank you for all the “uppers and downers”, the care packages when times were tough, and the smile of encouragement every day. Dr. Laneke Luies for the comprehensive and timely formatting of my thesis and your endless and unconditional support throughout this study. Thank you for being the most wonderful best friend a girl could ask for. Prof. Eugene Engelbrecht, for comprehensive and prompt language editing. My Mitochondrial Research Laboratory friends, in particular John-Drew Bosshoff, Jaundrie Fourie, Michelle Mereis, and Liesel Mienie, all of whom helped me, encouraged me, and made my life easier over the past five years. Thank you for all the laughs and craziness that kept me sane. My dearest family-friends, Ds. Leon Geel, Oom Nielen and Tannie Rita Conradie. Thank you for the constant belief in me, your endless hugs and warm words of encouragements and above all, your sincere interest in my work. I would like to express my deepest appreciation to my family, in particular my parents, Johan and Joanie Schoonen. Thank you for accepting nothing less than excellence from me. You are my pillars and my strength. I love you. Bertie Seyffert, I would never have been able to complete this huge undertaking without your constant support, love and encouragements. I love you. v Finally, the financial assistance of the National Research Foundation (NRF), the South African Medical Research Council (MRC), and the North-West University (NWU) towards this research is hereby acknowledged. Opinions expressed and conclusions arrived at, are those of the author and are not necessarily to be attributed to the NRF, MRC, and NWU. vi SUMMARY Mitochondrial diseases (MD) are a clinically heterogenous group of genetic disorders that affect the neuromuscular system, the central nervous system (collectively known as encephalomyopathies), and other high-energy demanding organs, often with multi-system involvement. This involvement, whether single- or multi-system, can have extensive phenotypical features which could make the diagnosis of MD a daunting task. Over recent years, substantial progress has been made towards a better understanding of the most common mitochondrial encephalomyopathies including their clinical manifestation and underlying genetic cause. This progress has been made in mainly non-African patient populations. For these well-studied populations, routine clinical, biochemical, and genetic diagnostic approaches have consequently been established, improving diagnosis of MD in paediatric and adult patients alike. The limited diagnostic capacity in developing countries such as South Africa with its ethnically diverse populations, has hindered the advancement of diagnostic procedures for MDs in these countries. As a result, there is limited information available on clinical manifestations and genetic causation for the most common MDs in these understudied patient populations. However, over the last decade the following progress has been made towards an understanding of the underlying genetic cause of MD in South African patients: Firstly, since 1998 paediatric clinical referrals and assessments have been performed on paediatric patients with mitochondrial-disease-like signs and symptoms at the Steve Biko Academic Hospital, Pretoria (forming the cohort for this investigation). Secondly, biochemical analyses also commenced shortly afterwards and were performed for every patient who has been clinically diagnosed with an MD, which at the time of this investigation was 212. Thirdly, genetic investigations followed, starting with a small number of patients (n = 71) for whom whole mitochondrial genome next-generation sequencing was performed. This number has been expanded throughout recent years, and to date there are whole mitochondrial DNA sequencing data for 123 patients. Results from this initial mtDNA sequencing approach revealed relatively few pathogenic mutations. For the majority of these cases, a clear MD aetiology could not be established. It was collaboratively decided to perform targeted gene panel sequencing on nuclear-encoded genes associated with MD. Three panels were designed and used in this study, each consisting of genes directly involved with the mitochondrion. An in- house bioinformatics pipeline was developed during this study to analyse the sequence data. The results for the nuclear gene investigations revealed that a clear genotype-phenotype correlation could be established in only two of the 85 selected cases. One of these patients was vii extensively investigated and later published as a case-report. Due to the disappointingly low diagnostic yield of panel sequencing, whole exome sequencing was performed on a small number of African patients to probe the outcome of this approach. These initial results were promising as six of the eight sequenced cases presented with a pathogenic or likely pathogenic variant. Additional screening for two putative African-population-specific gene mutations was also performed on all of the African cases. Combined, from the results, the aetiology could be determined in ten cases; eight cases using next-generation sequencing, and two cases using selected gene mutation screening. In conclusion, this study was the first in size and scope to investigate the molecular genetics of MD in an understudied ethnically diverse population, providing new knowledge on these patients and insight into future strategies. Diagnosis of MD remains a daunting task in South Africa and will only improve once diagnostic capacity has been significantly enhanced. Key words: African paediatric cohort; mitochondrial disease; bioinformatics; panel sequencing; whole exome sequencing; mitochondrial DNA; nuclear DNA viii TABLE OF CONTENTS ACKNOWLEDGEMENTS...................................................................................................... v SUMMARY .......................................................................................................................... vii LIST OF FIGURES .............................................................................................................. xv LIST OF TABLES ............................................................................................................. xxiii LIST OF ABBREVIATIONS AND SYMBOLS .................................................................. xxvii CHAPTER 1: INTRODUCTION ............................................................................................. 1 1.1 BACKGROUND AND MOTIVATION ...................................................................... 1 1.2 RESEARCH AIM AND OBJECTIVES .................................................................... 3 1.3 STRUCTURE OF THESIS AND RESEARCH OUTPUTS ...................................... 6 1.4 AUTHOR CONTRIBUTIONS ................................................................................. 8 CHAPTER 2: LITERATURE REVIEW ................................................................................. 11 2.1 MITOCHONDRIAL BIOLOGY .............................................................................. 11 Structure and function of mitochondria .............................................................. 11 Electron transport chain and oxidative phosphorylation ..................................... 12 Oxidative phosphorylation dysfunction ............................................................... 13 2.1.3.1 Complex I ..................................................................................................... 14 2.1.3.2 Complex II .................................................................................................... 15 2.1.3.3 Complex III ................................................................................................... 16 2.1.3.4 Complex IV .................................................................................................. 17 2.1.3.5 Complex V ................................................................................................... 18 2.1.3.6 Electron transfer proteins ............................................................................. 19 2.2 MITOCHONDRIAL GENETICS ............................................................................ 20 Mitochondrial genome ......................................................................................