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Molecular Genetic Investigation Into Inherited Thrombocytopenia

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Molecular Genetic Investigation Into Inherited Thrombocytopenia Molecular Genetic Investigation into Inherited Thrombocytopenia by Ben David Johnson A thesis submitted to the University of Birmingham for the degree of DOCTOR OF PHILOSOPHY Birmingham Platelet Group Institute of Cardiovascular Sciences College of Medical and Dental Sciences University of Birmingham October 2016 University of Birmingham Research Archive e-theses repository This unpublished thesis/dissertation is copyright of the author and/or third parties. The intellectual property rights of the author or third parties in respect of this work are as defined by The Copyright Designs and Patents Act 1988 or as modified by any successor legislation. Any use made of information contained in this thesis/dissertation must be in accordance with that legislation and must be properly acknowledged. Further distribution or reproduction in any format is prohibited without the permission of the copyright holder. Abstract Inherited thrombocytopenias are a heterogeneous group of disorders characterised by abnormally low platelet counts, often with secondary qualitative defects in platelet function, which can be associated with abnormal bleeding. Next generation sequencing has only previously been employed in small-scale studies and for the confirmation of suspected variants. This study presents the first large-scale approach to fully categorise inherited thrombocytopenia. Ninety-five patients were recruited to the UK-Genotyping and Phenotyping (GAPP) study with inherited thrombocytopenia of unknown genetic aetiology. An average platelet count of 88x109/L was observed across all individuals. Platelet function testing revealed a secondary phenotypic defect in addition to the reduction in platelet count in 71% (61/86). Of the 95 patients, 69 patients, encompassing 47 index cases, were analysed by whole exome sequencing. A variant with a positive prediction of pathogenicity was identified in 40% (19/47) of patients and overall plausible candidate variants of disease were identified in 69% of index cases. Subsequently an inherited thrombocytopenia specific gene panel was developed to serve as a pre-screen for patients prior to whole exome sequencing. In total, candidate variants in genes previously known to be implicated with disease were identified in 77% (20/26) of patients analysed. In conclusion, the application of a combined phenotyping and genotyping approach to patients with inherited thrombocytopenia is an effective and efficient means of complete diagnosis. It also has the added benefit of identifying novel candidate variants in genes not previously known to cause disease that may further our understanding of the processes surrounding haemostasis through subsequent functional studies. Acknowledgements Firstly, I would like to thank all collaborators (in particular members of the West Midlands Regional Genetics Service), consultants and referring physicians, providers of equipment and resources, and staff that have helped me over the past three years. To everyone in the Watson (Platelet) group, thank you for all of your continued help and expertise throughout. Whether you have directly helped me or whether you have just been around to share a cup of tea and distract me from my work with office projectiles, lunges or pictures of cats, it has definitely helped. Thank you to everyone in particular within the Morgan Group. Abs, Annabel (Annie Mac), Jane, Sarah and Sian. You have all been amazing and I really appreciate everything you have done for me during my thesis. A special thank you to Neil. I’m sorry for being a pain over the past three years, for suggesting strange ideas that may not have fully worked and for repeating sequencing on patients that was completely unnecessary (I’ll pay you back that £800 at some point). Thank you so much though, you have been a great supervisor and a good friend, and although I have moved to the dark side of industry I hope it can stay that way. Lastly to my friends, family and my girlfriend Grace. You have kept me sane, supplied me with sugar and warm beverages, and have ultimately made sure that I have submitted this one piece of work that I have been slaving over for three years. Thank you. Contents 1. Introduction ........................................................................................................... 4 1.1. Megakaryopoiesis and thrombopoiesis ......................................................... 4 1.2. Functional role and lifespan of platelets ...................................................... 12 1.3. Platelet function testing ............................................................................... 16 1.4. Inherited thrombocytopenia – Symptoms, treatment and misdiagnosis ...... 18 1.5. Genetic causes of inherited thrombocytopenia ........................................... 22 1.5.1. Disorders of megakaryopoiesis.............................................................. 27 1.5.2. Disorders affecting proplatelet formation/platelet release ...................... 36 1.5.3. Disorders of platelet clearance/unknown functional disorders ............... 47 1.6. Genetic studies into inherited thrombocytopenia ........................................ 50 1.6.1. Pre-genomic era .................................................................................... 50 1.6.2. Post-genomic era ................................................................................... 51 1.7. Thesis Hypothesis and Aims ....................................................................... 54 2. Materials and Methods ........................................................................................ 55 2.1. Materials ..................................................................................................... 55 2.2. Study approval ............................................................................................ 57 2.3. Patient recruitment ...................................................................................... 57 2.4. Healthy controls .......................................................................................... 59 2.5. Sample collection ........................................................................................ 59 2.6. Preparation of whole citrated blood ............................................................. 60 2.7. Preparation of wash platelets ...................................................................... 61 2.8. Platelet counts in whole EDTA-anticoagulated blood .................................. 61 2.9. Platelet function testing ............................................................................... 62 2.9.1. Light transmission aggregometry with lumiaggregometry ...................... 63 2.9.2. Flow cytometry ....................................................................................... 66 2.10. Sample storage – Protein and buffy coat .................................................. 68 2.11. Preparation of platelet lysates from washed platelets ............................... 69 2.12. Preparation of lysates for protein analysis from cell lines ......................... 70 2.13. DNA extraction from buffy coat and whole blood ...................................... 70 2.14. DNA quantification .................................................................................... 72 2.15. Next generation sequencing ..................................................................... 73 2.15.1. Whole exome sequencing .................................................................. 73 2.15.2. Thrombocytopenia specific panel sequencing .................................... 77 2.16. Polymerase chain reaction ........................................................................ 83 2.17. Sanger sequencing ................................................................................... 84 2.18. PCR based X-inactivation assay ............................................................... 85 2.19. Western blot analysis ................................................................................ 86 2.20. Platelet spreading and immunohistochemistry .......................................... 88 2.21. TTF2 guide RNA in silico guide design and cloning .................................. 89 .................................................................................................................................. 92 3. Patient phenotypical presentations and platelet function testing .................. 93 3.1. Summary of background to this research .................................................... 93 3.2. Aims ............................................................................................................ 94 3.3. Patients with inherited thrombocytopenia recruited to the UK-GAPP study 94 3.4. Patient characteristics – Age, gender and ethnicity .................................... 98 3.5. Patient bleeding symptoms and treatment ................................................ 100 3.6. Platelet counts and morphology ................................................................ 109 3.7. Whole blood white cell counts ................................................................... 112 3.8. Platelet function testing ............................................................................. 112 3.8.1. Light transmission aggregometry ......................................................... 112 3.8.2. Flow cytometry ..................................................................................... 119 3.9. Discussion ................................................................................................
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