Using Massively Parallel Sequencing to determine the genetic basis of Leigh Syndrome, the most common mitochondrial disorder affecting children Nicole Janet Lake ORCID ID 0000-0003-4103-6387 Doctor of Philosophy January 2018 Department of Paediatrics Faculty of Medicine, Dentistry and Health Sciences University of Melbourne Submitted in total fulfilment of the requirements of the degree of Doctor of Philosophy Abstract Mitochondrial diseases are debilitating illnesses caused by mutations that impair mitochondrial energy generation. The most common clinical presentation of mitochondrial disease in children is Leigh syndrome. This neurodegenerative disorder can be caused by mutations in more than 85 genes, encoded by both nuclear and mitochondrial DNA (mtDNA). When this PhD commenced, massively parallel sequencing for genetic diagnosis of Leigh syndrome was transitioning into the clinic, however its diagnostic utility in a clinical setting was unknown. Furthermore, a significant number of Leigh syndrome patients remained without a genetic diagnosis, indicating that further research was required to expand our understanding of the genetic basis of disease. To identify the maximum diagnostic yield of massively parallel sequencing in patients with Leigh syndrome, and to provide insight into the genetic basis of disease, unsolved patients from a historical Leigh syndrome cohort were studied. This cohort is comprised of 67 clinically- ascertained patients diagnosed with Leigh or Leigh-like syndrome according to stringent criteria. DNA from all 33 patients lacking a genetic diagnosis underwent whole exome sequencing, with parallel sequencing of the mtDNA. A targeted analysis of 2273 genes was performed, which included known and candidate mitochondrial disease genes, and differential diagnosis genes underlying distinct disorders with phenotypic overlap. This study provided a genetic diagnosis for 11 of the 12 unsolved Leigh syndrome patients and 7 of the 21 unsolved Leigh-like patients investigated. Eight reported and eight novel pathogenic variants were identified in twelve disease genes; ALDH18A1, MT-ATP6, MT-ND3, MT-ND5, MT-ND6, MTFMT, NARS2, SCO2, SERAC1, SLC19A3, PDHA1, and PDHX. A genetic diagnosis has now been established in 78% of the total cohort, including in 34 of 35 Leigh syndrome patients and 18 of 32 Leigh-like patients. Candidate genetic diagnoses, including in differential diagnosis genes, were identified in an additional 7 Leigh-like patients, where investigation of synonymous variants, complex CNVs and heterozygous X-linked variants highlight challenges associated with variant follow-up in a diagnostic context. This thesis also describes the study of an additional patient with a firm diagnosis of Leigh syndrome. Whole exome sequencing of this patient with targeted analysis identified a homozygous splice site mutation in MRPS34, which was shown to cause abnormal splicing and loss of wild-type MRPS34 protein. MRPS34 encodes a mitochondrial ribosomal protein, and variants in this gene had not previously been described to cause disease. The data presented in this thesis demonstrate that mutation of MRPS34 causes a disorder of mitochondrial energy generation by destabilising the small mitochondrial ribosomal subunit, and therefore reducing the synthesis of mtDNA-encoded proteins. The rescue of cellular defects by lentiviral-mediated expression of wild-type MRPS34 establishes it as a bona fide disease gene. In conclusion, this thesis describes the identification of novel mutations and a new disease gene, thereby expanding our understanding of the genetic basis of Leigh syndrome. The characterisation of the aetiological basis of disease in the cohort, as well as evaluation of the outcomes of massively parallel sequencing of cohort patients, provides key insight into the diagnostic utility of this testing approach in a clinical setting. i Declaration This is to certify that: (i) The thesis comprises my original work towards the PhD, except where indicated in the Preface, (ii) Due acknowledgement has been made in the text to all materials used, (iii) The thesis is fewer than 100,000 words in length, exclusive of tables, maps, bibliographies and appendices. ii Preface (i) Several colleagues and collaborators have contributed to the research described in this thesis. Except where indicated below, the thesis is my original work towards this PhD. The following contributions were made to the content of Chapters 3, 4 and 5: Patients were referred to the Mitochondrial Diagnostic Service, part of the Mitochondrial Research Laboratory at the Murdoch Children’s Research Institute and Victorian Clinical Genetics Service in Melbourne, Australia, and diagnostic enzymology assays were performed by staff members. Simone Tregoning assisted with collation of enzymology results. Massively parallel sequencing was performed at the Broad Institute in Boston, USA in collaboration with Prof. Vamsi Mootha and Dr Sarah Calvo. Variant call files were generated from the sequencing data by Dr Sarah Calvo. The following contributions were made to the content of Chapter 3: Sections 3.2.1.4, 3.2.1.8, and 3.2.3: The SNP array was performed on DNA from LS7, LS18 and LS20a by the Victorian Clinical Genetic Service, and genotype data from LS7 and LS18 was analysed by staff member Ralph Oertel. Section 3.2.3: Comparative genomic hybridisation arrays were performed previously on DNA from LS20a and LS20b in the Mitochondrial Research Laboratory by Drs Alison Compton, Hayley Mountford and Elena Tucker. The following contributions were made to the content of Chapter 4: Section 4.2.1.3: Blue-Native (BN)-PAGE and SDS-PAGE immunoblots examining complex I in fibroblasts from LL45, and complementation studies in HEK293T cells, were performed at Monash University in Melbourne by Dr Luke Formosa. Data for Figure 4-6(a-c) were generated solely by Dr Luke Formosa. Section 4.2.2.1: The SNP array on DNA from LL43a and LL43b was performed by the Victorian Clinical Genetic Service, and genotype data was analysed by staff member Ralph Oertel. Section 4.2.2.2: Sanger sequencing of the DOK7 mutations in genomic DNA from DT110 was performed in the Mitochondrial Research Laboratory by Shalini Thirukeswaran. Section 4.2.2.5: The mitochondrial translation assay for LL42 was performed at Monash University with the assistance of Dr David Stroud and Elliot Surgenor. Section 4.2.3: The sequencing data from LL40 was analysed by Shalini Thirukeswaran. The following contributions were made to the content of Chapter 5: Section 5.1.1.1: The clinical summary was provided by Prof. John Christodoulou. Section 5.2.1: Analysis of the sequencing data, and Sanger sequencing of the mutation in genomic DNA, was performed by Dr Hayley Mountford. Sections 5.2.3 and 5.2.4: BN-PAGE immunoblotting and the mitochondrial translation assay was performed with the assistance of Dr David Stroud and Elliot Surgenor. Sections 5.2.3 and 5.2.6: Quantitative proteomics was performed by Dr David Stroud. Figure 5-5a, Figure 5-10a and Figure 5-10c were generated with the assistance of Dr David Stroud. iii Section 5.2.5: Lentiviral-mediated correction of cell lines was performed by Dr Alison Compton. Section 5.2.6: Sucrose gradient centrifugation and SDS-PAGE immunoblots examining the mitoribosome were performed at the Henry Perkins Institute in Perth, Australia by Dr Tara Richman. (ii) Several colleagues and collaborators have contributed to publications included in this thesis. The following contributions were made to the content of the article “Leigh syndrome: One disease, more than 75 monogenic causes” which is included in Chapter 1: Professor David Thorburn, Dr Alison Compton and Professor Shamima Rahman contributed to reviewing publications on the genetic, clinical, and pathological features of Leigh syndrome, and to the drafting and review of the manuscript. (iii) The publication status of all chapters presented (in part) in article format are as follows: Chapter 1 includes the article “Leigh syndrome: One disorder, more than 75 monogenic causes”. This was published by Annals of Neurology on 15th December 2015. (iv) Acknowledgement of all sources of funding, including grant identification numbers where applicable: The following organisations provided funding to support this PhD project: the University of Melbourne (Australian Postgraduate Award, Ackman Travelling Scholarship, Department of Paediatrics Travelling Scholarship), the National Health and Medical Research Council (Project Grant GNT 1068409 to D Thorburn, A Compton, D Bruno and M Ryan), the Australian Mitochondrial Disease Foundation (PhD Top-up and Travel Scholarship), the Australian National University (Gowrie-Patrick Hore-Ruthven Memorial Scholarship), the Murdoch Children’s Research Institute (Student Conference Scheme Scholarship), and the Australasian Society for Inborn Errors of Metabolism (Travel Grant). iv Acknowledgments Firstly, I thank my primary supervisor Prof. David Thorburn. It’s a unique opportunity to learn from someone who is an international leader of their field, and I am grateful for the opportunity to do a PhD in his lab. I have greatly appreciated the various opportunities he provided, research and beyond, supporting presentations at local and international conferences, committee involvement, media engagement and peer-review of articles. These activities helped me to mature as an early-career researcher. Thank you for your support and mentorship throughout this PhD. I am also very thankful to my co-supervisor