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The Genetic Architecture of Familial Hypercholesterolaemia

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The Genetic Architecture of Familial Hypercholesterolaemia The Genetic Architecture of Familial Hypercholesterolaemia Marta Futema University College London A thesis submitted in accordance with the regulations of the University College London for the degree of Doctor of Philosophy Centre for Cardiovascular Genetics UCL Genetics Institute 1 “Nothing in life is to be feared, it is only to be understood. Now is the time to understand more, so that we may fear less.” Maria Skłodowska-Curie 2 Declaration I, Marta Futema confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis. Regarding the Oxford FH cohort, I would like to acknowledge Prof. Andrew Neil and Dr. Mary Seed on behalf of the Simon Broome Familial Hypercholesterolaemia Register Group for providing the patients samples, the clinical information and expertise. Regarding the cohort of FH patients of South-Asian origin, samples were provided by Mr Darren Harvey, Dr. Anjly Jain and Dr. Devi Nair (Royal Free Hospital, London), Sister Emma McGowan and Dr. Michael Khan (University Hospitals Coventry and Warwickshire NHS Trust), Dr. Julian Barth (Leeds General Infirmary), and Dr. Manjeet Mehta (Kokilaben Dhirubhai Ambani Hospital and Medical Research Institute). Dr. Emma Ashton and Dr. Nicholas Lench from the North East Thames Regional Genetics Service (Great Ormond Street Hospital) kindly provided their advice and equipment for MLPA experiments. The replication cohorts for LDL-C gene score analysis were provided by Albert Wiegman and Joep Defesche (Academic Medical Center, Amsterdam, the Netherlands), Rob Hegele and Mary Aderayo Bamimore (University of Western Ontario, London, Canada), and Dr. Euridiki Drogari and Dr. Vasiliki Mollaki (University of Athens Medical School, “Aghia Sophia” Children's Hospital, Athens, Greece). The UK10K consortium, including the project production team, was responsible for the library preparation, sequence capture and bioinformatics of the whole exome sequencing FH cohort and the controls. The full list of the members can be found at: http://www.uk10k.org/consortium.html. Dr. Vincent Plagnol was involved in the whole exome sequencing data processing and analysis. Ms KaWah Li did some of the 12 SNPs genotyping for the LDL-C gene score analysis. 3 Collaborators who provided FH samples for whole exome sequencing include: Dr. Stefano Bertolini (University of Genoa, Italy), Dr. Sebastiano Calandra (University of Modena and Reggio Emilia, Modena, Italy), Dr. Olivier S Descamps (Centre de Recherche Médicale de Jolimont, Haine St-Paul, Belgium), Dr. Colin Graham (Belfast Health and Social Care Trust / City Hospital, Belfast, Northern Ireland, UK), Dr. Fredrik Karpe (University of Oxford, Churchill Hospital, Oxford, UK), Dr. Eran Leitersdorf (Hadassah Hebrew University Medical Centre, Jerusalem, Israel), Dr. Handrean Soran (Central Manchester University Hospitals Foundation Trust, Manchester, UK), Dr. Frank Van Bockxmeer (Royal Perth Hospital, Wellington St, Perth, Australia). 4 Acknowledgements Firstly, I would like to acknowledge all the patients who participated in this study. It is through their willingness to donate their samples for research that new medical and scientific knowledge has been gained. I would like to thank my teachers, whose courage, commitment and passion have inspired me over the years. I have been extremely fortunate to learn from you. Especially I would like to express my sincere thanks to my supervisor Professor Steve Humphries, whose guidance throughout my project helped me to bring out the best in myself. I would like to also convey my gratitude to Vincent Plagnol and Ros Whittall, who generously shared their time and expertise. I extend my thanks to all members of the Cardiovascular Genetics lab, who make a friendly and exciting team of scientists always keen to share their knowledge. I am extremely grateful to the Medical Research Council and Gen-Probe Incorporated, who have supported me financially during the project. The research carried out in this thesis was also partially founded by the British Heart Foundation and the Wellcome Trust (the UK10K project). I thank my family and friends for their constant support. Special thanks to my parents who managed to provide stability and support throughout my life. Your love and care gave me strength and courage in fulfilling my ambitions. Many thanks to Artus, my husband, who was always there for me and whose positive attitude was extremely motivating in difficult moments. 5 Publications resulting from the work in this thesis: 1. Futema M, Whittall RA, Kiley A, Steel LK, Cooper JA, Badmus E, Leigh SE, Karpe F, Neil HAon behalf of the Simon Broome Register Group, and Humphries SE. Analysis of the frequency and spectrum of mutations recognised to cause familial hypercholesterolaemia in routine clinical practice in a UK specialist hospital lipid clinic. Atherosclerosis. 2013;229(1):161-8. 2. Futema M, Plagnol V, Whittall RA, Neil HA on behalf of the Simon Broome Register Group, Humphries SE and the UK10K Consortium. Use of targeted exome sequencing as a diagnostic tool for Familial Hypercholesterolaemia. J Med Genet. 2012 Oct;49(10):644-9. Other publications produced during the thesis: 1. Talmud PJ, Shah S, Whittall R, Futema M, Howard P, Cooper JA, Harrison SC, Li K, Drenos F, Karpe F, Neil HA, Descamps OS, Langenberg C, Lench N, Kivimaki M, Whittaker J, Hingorani AD, Kumari M, Humphries SE. Use of low-density lipoprotein cholesterol gene score to distinguish patients with polygenic and monogenic familial hypercholesterolaemia: a case-control study. Lancet. 2013 Apr 13;381(9874):1293-301. 2. Pećin I, Whittall R, Futema M, Sertić J, Reiner Z, Leigh SE, Humphries SE. Mutation detection in Croatian patients with familial hypercholesterolemia. Ann Hum Genet. 2013 Jan;77(1):22-30. Manuscripts can be found at the back of the thesis. 6 Abstract Familial Hypercholesterolaemia (FH) is a common autosomal dominant disorder of the defective plasma clearance of LDL-cholesterol (LDL-C). Mutations in three genes, LDLR/APOB/PCSK9, can be detected in 60-90% of definite FH patients. DNA-based testing for FH mutations has important clinical utility and is recommended by the UK and European guidelines to identify affected relatives. This thesis aimed to determine the frequency and spectrum of FH mutations in two independent cohorts of FH patients (from one Oxford lipid clinic, and of Indian background). The FH mutation spectrum was shown to be highly heterogeneous and the mutation detection rate was significantly dependent on the pre-treatment total cholesterol and triglyceride levels. This project also validated the findings that a proportion of clinically diagnosed FH patients have a polygenic cause of hypercholesterolaemia due to an accumulation of common mild LDL-C-raising alleles by analysing LDL-C gene score in 88 mutation negative and 21 mutation positive FH patients, and by replicating the results in further 231 FH patients. A high-throughput DNA sequencing method was assessed as a novel diagnostic tool for detection of FH mutations, and compared it with the currently used methods. This highlighted the need for updating the current FH mutation screening methods as well as the need for more efficient bioinformatics for the next generation sequencing data analysis. Lastly, whole exome sequencing of 125 definite FH patients with no mutations detected in known genes was performed to identify novel monogenic causes of FH. Variants in two genes, CH25H and INSIG2, were identified as potential novel FH mutations. Overall, the results of this thesis demonstrate the heterogeneous FH aetiology and help to understand the genetic architecture of the disease. 7 Contents List of Figures .................................................................................................................... 12 List of Tables ..................................................................................................................... 15 Abbreviations .................................................................................................................... 18 I. CHAPTER ONE - General Introduction ............................................................. 21 1.1. Lipid metabolism ................................................................................................ 22 1.1.1. Lipids and Lipoproteins ............................................................................... 22 1.1.2. Exogenous pathway ....................................................................................... 25 1.1.3. Endogenous pathway .................................................................................... 25 1.1.4. Reverse cholesterol transport ................................................................... 26 1.1.5. Cholesterol homeostasis .............................................................................. 27 1.2. Atherosclerosis.................................................................................................... 28 1.3. Overview of the molecular pathogenesis of FH ....................................... 33 1.4. Clinical diagnosis of FH ..................................................................................... 34 1.5. Genetic diagnosis of FH..................................................................................... 41 1.6. Clinical utility of DNA testing.......................................................................... 44 1.7. Cascade testing to identify FH patients ......................................................
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