Gene discovery and mechanism of disease in the myopathies Heather Best Primary supervisor: A/Prof Sandra Cooper Associate supervisors: The Late A/Prof Nigel Clarke, Dr Michaela Yuen and Dr Frances Lemckert A thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy The Institute for Neuroscience and Muscle Research The Children’s Hospital at Westmead, Sydney, NSW, Australia Discipline of Child and Adolescent Health, Faculty of Medicine The University of Sydney, NSW, Australia February 2018 STATEMENT OF ORIGINALITY The contents of this thesis have not been presented for the award of a degree or diploma at this or any other university. The data presented are the original work of the author except where specifically indicated in the text. i AUTHOR CONTRIBUTION Chapter 2 Part A of this thesis is published as: O'Grady, G. L., Best, H.A., Oates, E.C., Kaur, S., Charlton, A., Brammah, S., Punetha, J., Kesari, A., North, K.N., Ilkovsli, B., Hoffman, E.P., Clarke, N.F. (2015). "Recessive ACTA1 variant causes congenital muscular dystrophy with rigid spine." Eur J Hum Genet 23, 883-886 Heather Best carried out the functional characterisation of the ACTA1 variant which became Figure 2 E-L of our publication. Heather contributed to drafting/revising the manuscript. NB: Dr Gina O’Grady’s PhD thesis included this publication. Dr O’Grady collated the clinical data for Patient 1 and 2, interpreted NGS results, drafted the manuscript, constructed the figures, and managed all aspects of paper submission. Chapter 4 Part A of this thesis is published as: O'Grady, G. L., α H. A. Best, α T. E. Sztal, α V. Schartner, # M. Sanjuan-Vazquez, # S. Donkervoort, # O. Abath Neto, R. B. Sutton, B. Ilkovski, N. B. Romero, T. Stojkovic, J. Dastgir, L. B. Waddell, A. Boland, Y. Hu, C. Williams, A. A. Ruparelia, T. Maisonobe, A. J. Peduto, S. W. Reddel, M. Lek, T. Tukiainen, B. B. Cummings, H. Joshi, J. Nectoux, S. Brammah, J. F. Deleuze, V. O. Ing, G. Ramm, D. Ardicli, K. J. Nowak, B. Talim, H. Topaloglu, N. G. Laing, K. N. North, D. G. MacArthur, S. Friant, N. F. Clarke, R. J. Bryson-Richardson, C. G. Bonnemann, J. Laporte§ and S. T. Cooper§ (2016). "Variants in the Oxidoreductase PYROXD1 Cause Early-Onset Myopathy with Internalized Nuclei and Myofibrillar Disorganization." Am J Hum Genet 99(5): 1086-1105. α These authors jointly share first authorship # These authors jointly share second authorship § These authors jointly share senior authorship This publication was the result of collaboration between multiple international translational genomics programs establishing variants within PYROXD1 as a new genetic cause of early onset myopathy. Heather Best was involved in the functional characterisation of PYROXD1. Heather developed and optimised lab tools to study PYROXD1 and subsequently performed characterisation of patient variants at the molecular genetic and protein level. As PYROXD1 was previously uncharacterised Heather carried out localisation studies of PYROXD1 in an overexpression system as well as primary cell lines and skeletal muscle. These experiments became four and a half out of nine figures in our paper. Heather played a major role in manuscript preparation, submission and production of Figures 1, 3-6. NB: Dr Gina O’Grady’s PhD thesis included this publication. Dr O’Grady was involved in analysis of WES data for family A, reviewed the affected siblings, helped coordinate international collaboration, collated clinical data on all families (Table1) and made a significant contribution to drafting of the manuscript and preparation of Figures 1 and 2 ii ACKNOWLEDGEMENTS I began my PhD journey under the primary supervision of the Late A/Prof Nigel Clarke. Late A/Prof Clarke’s outstanding clinical skill and passion for pushing medical science forward has left a huge hole within the INMR and the field of gene discovery. A/Prof Sandra Cooper was instrumental in carrying on The Late A/Prof Clarke’s legacy, and whom I was fortunate to come under primary supervision of. A/Prof Cooper’s drive and scientific skill are inspirational. A/Prof Cooper was a brilliant mentor to me and her support and guidance has shaped me into the scientist I am today. I appreciate the time A/Prof Cooper invested in me and am grateful for the opportunities granted. Dr Michaela Yuen is an outstanding scientist, immensely loyal friend and whom I feel privileged to have worked alongside. Dr Yuen has been a huge support to me during my time at the INMR, guiding me through my junior scientific training with kindness and dedication. I would like to thank Dr Yuen for believing in me and supporting me through the good and bad times of my PhD. I would like to thank Dr Biljana Ilkovski for her valuable guidance through the first year of my PhD and past and present members of the gene discovery team for their support and enabling enjoyable collaborative projects. I would also like to thank Adam Bournazos for assisting with experiments within the LMOD project. I am grateful to Dr Frances Lemckert who helped me navigate the world of mouse work with patience and enthusiasm. Prof Patrick Tam, Dr Nicolas Fossat and Vanessa Jones at the Children’s Medical Research Institute (CMRI) have been fantastic collaborators and I am very grateful for their help and expertise in mouse embryology. The transgenic facility at the Kids Research Institute (KRI) have been instrumental in the establishment of the Pyroxd1 mouse iii project. In particular I would like to express my gratitude to Kim Hewitt and Karen Knight for their hard work in a rapidly expanding project. I would like to thank our wonderful collaborators scattered all over the globe and the willing participation of patients and their families to the projects within this thesis. Without their support gene discovery would not be possible. I have been very fortunate to work in a lab of talented and friendly scientists. I would like to thank past and present INMR members for creating a friendly work environment and for their support during my PhD. In particular I would like to mention Greg Redpath, Fiona Lee, Natalie Woolger and Matthew Summers. I am grateful for the Australian Post-graduate award (APA) and Muscular Dystrophy NSW PhD Scholarship (MDNSW) for supporting me during my PhD project. I would also like to thank the Research Operations team at the KRI who have enabled an efficient working environment. My biggest thank-you is to my family. Who have supported me through my university years and made the undertaking of this PhD possible. In particular my Mum, Christine Best, who has always encouraged me to shoot for the stars and supported me in whatever I chose to undertake. And also, my step-sister, Katherine Barnsley, who has been a huge support to me during my time in Sydney. iv ABSTRACT Congenital myopathy and muscular dystrophy are two groups of inherited muscle diseases characterised by muscle weakness, and sub-classified by hallmark pathological features within a skeletal muscle biopsy. In order to understand the pathogenesis of inherited muscle disorders, and develop or apply therapies based on mechanistic insight, one must elucidate deep knowledge of the associated gene, genetic variant and the function of the encoded protein. This thesis focuses on three aspects of gene discovery in the inherited myopathies: (1) Identification of a novel variant and phenotype for a known disease gene; (2) understanding the functional role of a recently identified disease gene in skeletal muscle biology and disease; and (3) discovering a novel disease gene for congenital myopathy. We identified the first recessive variant within ACTA1 (encoding α-skeletal actin) as the genetic cause of congenital muscular dystrophy with rigid spine. This case uniquely describes recessive ACTA1 variants where α-skeletal actin protein is expressed. The unique clinical and histological presentation expands the spectrum of ACTA1 disease, and will help guide clinical care and future genetic diagnoses. Our team identified LMOD3 (leiomodin-3) as a novel disease gene for severe nemaline myopathy (NM). KLHL40 (encoding kelch-like family member 40) is another disease gene for severe NM. A recent study suggests mouse Klhl40 protects mouse Lmod3 protein from proteasome-mediated degradation, with the mechanistic basis of KLHL40-NM resulting from secondary loss of LMOD3. We investigated the regulation of human LMODs by human KLHL40, and unexpectedly found evidence that disputes the central paradigm that KLHL40 protects LMOD3 from proteasome-mediated degradation. v We identified PYROXD1 as a new genetic cause of early-onset congenital myopathy. We provide the first characterisation of PYROXD1 as a nuclear-cytoplasmic oxidoreductase and our discovery highlights oxidative distress as a core mechanistic pathway in the myopathies. We derived a mouse model of Pyroxd1 deficiency, determining that global loss of mouse Pyroxd1 is embryonic lethal. We subsequently developed a mouse model with skeletal muscle knock-out of Pyroxd1 – as a means to elucidate the role of PYROXD1 in biology and disease. vi CONFERENCE PROCEEDINGS SUPPORTING THIS THESIS Invited oral presentations 2016, Gage Muscle Conference, Canberra, ACT, Australia Mutations in an oxidoreductase cause early-onset myopathy Best HA, O’Grady GL and Cooper ST 2018, MDNSW meeting, Sydney, NSW, Australia Identifying a new genetic mechanism of muscle disease Best HAα, O’Grady GLα, Sztal TEα, Schartner V#, Sanjuan-Vazquez M#, Donkervoort S#, Neto OA, Sutton RB, Ilkovski B, Romero NB, Stojkovic T, Dastgir