The identification and characterisation of novel genes associated with cardiomyopathy Dean Graeme Phelan Submitted in total fulfilment of the requirements of the degree of Doctor of Philosophy March 2017 Department of Paediatrics The University of Melbourne The Bruce Lefroy Centre for Genetic Health Research The Murdoch Childrens Research Institute Abstract Cardiomyopathies are heart muscle disorders that result in inadequate pumping of the heart and thus poor cardiac blood flow. Cardiomyopathies are a common cause of heart failure, they affect all ages, are a significant contributor to morbidity and mortality and often require multidisciplinary care from health and community services. These disorders have a diverse clinical presentation, spectrum of severity and underlying aetiology. Hypertrophic cardiomyopathy (HCM) is a disease characterised by hypertrophy of the left ventricle of the heart. HCM has a prevalence of ~1 in 500 individuals and is the leading cause of sudden cardiac death among young individuals. Although mutations in more than 20 genes have been shown to cause HCM, over 20% of cases remain of unknown aetiology. Linkage analysis and whole exome sequencing identified a mutation in the gene encoding alpha protein kinase 3 ( ALPK3 ) in a family with two affected individuals presenting with hypertrophic cardiomyopathy and amyoplasia. Sanger sequencing analysis of ALPK3 in a cohort of 52 individuals with HCM excluded for known aetiologies, did not identify any predicted pathogenic variants. Targeted next generation sequencing (NGS) analysis of >500 unrelated individuals with heart disease of unknown cause was performed but no disease causing alterations were identified. These observations suggest that alterations in ALPK3 are not a common cause of heart disease. To investigate the function of ALPK3 we utilised induced pluripotent stem cells (iPSCs) generated from the skin cells of the affected individuals and human ESC-derived stem cells genetically edited to lack ALPK3 . Ultra-structural analysis of stem cell derived cardiomyocytes revealed disordered sarcomeres and intercalated discs. Multi- electrode array analysis and calcium imaging demonstrated extended field potential duration and abnormal calcium handling in mutant contractile cultures. Tissue engineering also revealed a reduction in contractile force in ALPK3 deficient cells. Identification of novel genes associated with cardiomyopathy increases diagnostic yield, permits early diagnosis and the provision of cascade testing. Early diagnosis and i identification of at risk individuals can vary clinical implications and outcomes. This study, and recently published genetic evidence, demonstrate that mutations in ALPK3 can cause familial cardiomyopathy. It also extends the ALPK3 phenotype to include congenital pterygia. The results provide functional insights into the molecular pathogenesis underlying ALPK3-mediated cardiomyopathy and suggest affected individuals may be at increased risk of potentially fatal arrhythmias. We anticipate these findings will alter patient management and help identify novel therapies for cardiomyopathy. ii Declaration This is to certify that: I. The thesis comprises only my original work towards the PhD except where indicated in the Preface, II. Due acknowledgement has been made in the text to all other material used, III. The thesis is less than 100,000 words in length, exclusive of tables, maps, bibliographies and appendices Dean Phelan: Date: iii Preface Chapter 3: Identification of Novel Genes Associated with Cardiomyopathy Contribution to the project: I was involved in all aspects of this project. I conceptualised and designed the study with Paul Lockhart. I performed majority of the experimental work and the data analysis. NGS was performed fee for service at Macrogen, Korea. Dr Melanie Bahlo and Dr Peter Hickey contributed to the bioinformatics analysis of the SNP array and NGS data. Chapter 4: Characterisation of ALPK3 Contribution to the project: I was involved in all aspects of this project. I conceptualised and designed the study with Paul Lockhart. I performed all the experimental work and the data analysis. Sarah Stephenson contributed to the molecular evolution analysis and Joe Sim contributed to the protein modelling. Chapter 5: Functional Analysis of ALPK3 Contribution to the project: I was involved in all aspects of this project. I conceptualised and designed the study with Paul Lockhart and David Elliott. I generated the ALPK3 KO hESCs and performed majority of the experimental work and data analysis. Dr Raymond Wong and Dr Alice Pébay generated the patient derived iPSCs. Dr Sarah Howden generated the ALPK3 corrected iPSCs. TEM images were captured by Dr Eric Hanns fee for service at The Electron Microscope Unit, Bio21. Dr James Hudson performed the tissue engineering experiments. iv Publications Dean G. Phelan, David J. Anderson, Sara E. Howden, Raymond C.B.Wong, Peter F. Hickey, Kate Pope, Gabrielle R.Wilson, Alice Pébay, Andrew M. Davis, Steven Petrou, Andrew G. Elefanty, Edouard G. Stanley, Paul A. James, Ivan Macciocca, Melanie Bahlo, Michael M. Cheung, David J. Amor, David A. Elliott, and Paul J. Lockhart 2016. ALPK3- deficient cardiomyocytes generated from patient-derived induced pluripotent stem cells and mutant human embryonic stem cells display abnormal calcium handling and establish that ALPK3 deficiency underlies familial cardiomyopathy. European Heart Journal 2016 Sep 01; 37(33):2586-90 Impact factor 15 Corresponding chapter in the thesis: Appendix 13 Contribution to manuscript : I was involved in all aspects of this manuscript. I conceptualised and designed the study with PJL and DAE, performed the majority of experimental work, conducted the majority of data analysis and co-wrote the paper with DAE and PJL. Corresponding chapter in the thesis: Chapter 5 DG. Phelan, GR Wilson, PA James, PJ Lockhart 2013. The genetics of cardiomyopathy, new technologies and the path to personalised medicine . OA Genetics Aug 01; 1(1):9 Impact factor 2 Contribution to manuscript : I was involved in all aspects of this manuscript. I conceptualised and designed the investigation with PJL, conducted the majority of literature analysis and co-wrote the paper with PJL. Corresponding chapter in the thesis: Chapter 1 v Acknowledgements First and foremost I would like to thank my supervisors. Associate Professor Paul Lockhart, Dr Gabrielle Wilson, Dr Desiree du Sart and Associate Professor Paul James have been fantastic in providing support, encouragement and guidance throughout the entire project. The experience and expertise provided by collaborator Dr David Elliott was instrumental in the success of this project. I would like to thank everyone that has contributed in some way to the work of this project. In particular past and present staff of the Bruce Lefroy Centre, Cardiac development, and Stem Cell laboratories at MCRI. I would also like to thank collaborators Dr Melanie Bahlo and Dr Peter Hickey, Walter and Eliza Hall Institute, Dr Raymond Wong, Dr Alice Pébay, Centre for Eye and Ear research, Dr James Hudson, The University of Queensland, Dr Eric Hanns of The Electron Microscope Unit, Bio21 for their contribution. Finally, but no less importantly, I would like to thank my family and friends for their support and understanding. vi Table of Contents Abstract ................................................................................................................. i Declaration ........................................................................................................... iii Preface ................................................................................................................. iv Publications ........................................................................................................... v Acknowledgements .............................................................................................. vi Abbreviations ..................................................................................................... xiii List of Tables ........................................................................................................ xv List of Figures ...................................................................................................... xvi Chapter 1 - Introduction ..................................................................................... 1 1.1 Introduction .......................................................................................................... 2 1.2 Embryology of the human heart .......................................................................... 3 1.2.1 Transcriptional Control of Cardiac Development .................................. 4 1.2.2 Other important regulators of cardiac development ............................ 7 1.2.3 Wnt/β-Catenin pathway ........................................................................ 7 1.2.4 Epigenetic factors................................................................................... 7 1.2.5 miRNAs involved in cardiac development ............................................. 8 1.3 The human heart .................................................................................................. 9 1.3.1 Structure and function of the human heart .......................................... 9 1.3.2 Cardiac Cycle ........................................................................................ 10 1.3.3 Cardiomyocyte structure ....................................................................