Understanding the Genetic Basis of Rare Inherited Bleeding and Platelet Disorders: the Utility of Next-Generation Sequencing
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Understanding the genetic basis of rare inherited bleeding and platelet disorders: the utility of next-generation sequencing Claire Lentaigne A thesis submitted for the degree of Doctor of Philosophy Centre for Haematology Department of Medicine Imperial College London 2 Declaration of originality I hereby declare that the work presented in this thesis is my own. All collaborations and work performed by others are made clear and appropriately referenced. Copyright declaration The copyright of this thesis rests with the author and is made available under a Creative Commons Attribution Non-Commercial No Derivatives licence. Researchers are free to copy, distribute or transmit the thesis on the condition that they attribute it, that they do not use it for commercial purposes and that they do not alter, transform or build upon it. For any reuse or redistribution, researchers must make clear to others the licence terms of this work 3 Statement of collaboration The work presented in this thesis was all conducted as part of the BRIDGE-bleeding and platelet disorders international consortium study which was created by Prof Willem Ouwehand at the University of Cambridge in 2010-2011. During the course of my PhD, the BRIDGE study became incorporated into the NIHR BioResource for Rare Diseases. This study involved the enrolment of over one thousand patients with rare bleeding and platelet disorders with subsequent analysis and interpretation of their sequencing data. Collaboration between PhD fellows, clinicians, geneticists and bioinformaticians has been essential to the success of the study and the outcomes presented here. All the work presented herein was either performed by me or is work in which I had a major contribution. Work performed by others is clearly stated in the text. Some of my specific roles within the consortium and key collaborators are outlined below • Methodology design for recruitment and data collection alongside Clinical PhD fellow Dr Tadbir Bariana (Royal Free Hospital), Prof Kathleen Freson, Geneticist (University of Leuven, Belgium) and Dr Ernest Turro (Bioinformatician, University of Cambridge) • Optimisation and application of the Human Phenotype Ontology with Clinical PhD fellows Dr Tadbir Bariana, Dr Anne Kelly (University of Cambridge) and Dr Sarah Westbury (University of Bristol), Prof Kathleen Freson, Dr Ernest Turro and Dr Daniel Greene (Computational Scientist and PhD fellow, University of Cambridge). • Recruitment and phenotyping of all cases and relatives enrolled at Hammersmith Hospital. From 2014 to 2017 most recruitment was also performed by Dr Carolyn Millar and Prof Mike Laffan (Consultant haematologists, Imperial College NHS Healthcare Trust) and research assistants Alice Glaser and Nicola Window at Imperial College NHS Healthcare Trust. • Analysis of whole exome and whole genome sequencing data from all BRIDGE- BPD cases as part of the core analysis team. This comprised principally the study PI’s Prof Willem Ouwehand (University of Cambridge) and Prof Mike Laffan (Imperial College London), clinicians Prof Andrew Mumford (Bristol) and Dr Keith 4 Gomez (Royal Free Hospital), Geneticist Prof Kathleen Freson, Chief bioinformatician Dr Ernest Turro and PhD fellow Daniel Greene, study coordinator Dr Sofia Papadia and clinical PhD fellows Dr Sarah Westbury, Dr Tadbir Bariana and more recently Dr Suthesh Sivapalaratnam and Dr Janine Collins. Regular additional input was also provided by scientists from the University of Cambridge involved in co-segregation analysis, functional studies on genes under investigation and Thrombogenomics, particularly Dr Kate Downes and Dr Karyn Megy. The core analysis team would meet on a weekly basis to discuss novel analytical methods proposed by the bioinformaticians, candidate novel genes and strategies for follow-up co-segregation and functional studies. My specific roles included providing a clinical perspective to the analysis process, testing novel algorithms and ensuring that the methods applied were clinically relevant and coordinating follow-up studies on genes of interest. • Local analysis of Hammersmith cases and investigation of the candidate novel genes described in this thesis were performed by me. 5 Acknowledgements I would like to personally thank and acknowledge my supervisors, Dr Carolyn Millar and Prof Mike Laffan at Imperial College and Prof Willem Ouwehand at the University of Cambridge for their vision, support and guidance throughout my PhD. Being an integral member of the BRIDGE consortium has been an incredible and valuable experience and I thank all my supervisors for the opportunities this has provided. I also owe great thanks to the other core BRIDGE analysis team members, in particular Dr Ernest Turro, Prof Kathleen Freson, Dr Sofia Papadia, Dr Daniel Greene, Dr Suthesh Sivapalaratnam and clinical PhD fellows Dr Sarah Westbury and Dr Tadbir Bariana from whom I have learnt a great deal and they have also been wonderful colleagues. I would also like to acknowledge the help and support received from other colleagues in the Ouwehand group at the University of Cambridge, particularly Dr Kate Downes, Dr Karyn Megy, Dr Jonathan Stephens and Dr Stephanie Maiwald. I would also like to thank all my colleagues in the haemostasis lab at Hammersmith, particularly Dr Tom Mckinnon for his practical support and guidance on the bench. I am grateful to Prof Maddy Parsons at Kings College London for her knowledge and advice regarding ROCK1 experiments. I would also like to thank Haemophilia nurses Wendy Hutchinson and Sharon Alavian in the Haemophilia Centre for their help in patient recruitment and the research and admin teams, in particular Lisa Pape for all their administrative assistance with patient recruitment and recall. The work presented in this thesis has only been possible thanks to the support and dedication of all the patients and their families who are enrolled in the NIHR BioResource. I hope that all the hard work goes some way to improving the diagnosis and management of patients who suffer with bleeding and platelet disorders. Finally, I owe a huge debt of gratitude to my husband Julian who has supported me all the way through and my children who are my constant inspiration. 6 Abstract Inherited bleeding and platelet disorders (BPD) are rare, heterogeneous and rarely receive a specific genetic diagnosis. The BRIDGE-Bleeding and platelet disorders consortium was set up to address this using high-throughput sequencing (HTS). More than one thousand probands with inherited BPD of unknown aetiology were recruited to an international consortium study with two over-arching aims: to identify novel genetic loci involved in BPD and provide a better diagnosis for patients. Through applying comprehensive, standardised phenotyping to this large dataset combined with novel analytical methods, several novel candidate genes for BPD have been identified. HTS has also identified variants in known BPD genes, many of them pathogenic, thus providing a diagnosis to many patients with BPD. In this thesis I present the study design and methodology for recruitment and data collection for the BRIDGE-BPD study. I describe the optimisation and application of the Human Phenotype Ontology to phenotype rare BPD and show how this has facilitated gene discovery and improvements in patient diagnosis. In chapters 4 and 5 I show how HTS has identified many variants in known BPD genes and illustrate the challenges faced and methods required for the interpretation of these variants. Many novel candidate BPD genes have also been identified and in chapters 6 and 7 some examples are highlighted with methods for their characterisation. Overall, this thesis demonstrates the utility of HTS in the diagnosis of rare BPD and many of the challenges faced in the interpretation of data from large whole genome sequencing projects and application of this technology to rare diseases. 7 Table of Contents Declaration of originality...................................................................................................... 3 Copyright declaration ........................................................................................................... 3 Statement of collaboration .................................................................................................. 4 Acknowledgements .............................................................................................................. 6 Abstract ................................................................................................................................ 7 Table of Contents ................................................................................................................. 8 List of Figures ..................................................................................................................... 16 List of Tables ...................................................................................................................... 19 Introduction ................................................................................................ 21 1.1 Overview of haemostasis and bleeding ................................................................ 21 1.2 Megakaryopoiesis and platelet production .......................................................... 22 1.2.1 Megakaryopoiesis ........................................................................................ 22 1.2.1.1 Regulation of megakaryopoiesis..............................................................