The identification and characterisation of novel 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 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 in the encoding alpha 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.

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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:

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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