BIOMARKERS FOR ISCHAEMIC STROKE NAZEEHA HASAN A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY SUPERVISORS: DR PANKAJ SHARMA DR ROBERT EDWARDS DEPARTMENT OF MEDICINE IMPERIAL COLLEGE LONDON DECEMBER 2013 ABSTRACT Background: Current diagnostic, prognostic and risk stratification tools are inadequate for effective ischaemic stroke management. Hypothesising that –omics approaches can be used to detect novel candidate biomarkers for ischaemic stroke, this thesis aimed to evaluate the current status of biomarkers for ischaemic stroke and develop strategies for biomarker discovery. Methods: Systematic literature review and individual patient data meta-analyses were performed to assess current candidate biomarkers associated with ischaemic stroke. Proteomic SELDI-TOF MS profiling was undertaken to identify novel blood-based protein biomarkers for the diagnosis of acute ischaemic stroke, consisting of a pilot study and a subsequent well- powered discovery study of 104 patients. In an integrative genomics study, transcriptomics data from carotid endarterectomy samples was combined with a genome-wide association study meta-analysis and subjected to functional enrichment analysis to detect differential gene expression or alternative splicing profiles that may be under the control of a genetic variant. Results: Systematic review and meta-analysis concluded that no current candidate biomarkers could be recommended for routine clinical practice, supporting the pursuit of novel biomarkers for ischaemic stroke and informing the design of subsequent experimental studies. SELDI-TOF MS detected two plasma protein ions, m/z 3699 and m/z 6640, which could differentiate between acute cerebral ischaemia and stroke mimics. Protein ion m/z 6640, identified as ApoC-1, highlighted the role of lipid dysregulation and was postulated to be a novel candidate biomarker for acute cerebral ischaemia. Integrative genomics provided evidence for the genetic regulation of cytoskeletal organisation and extracellular matrix remodelling processes in carotid disease. The LTBP4 gene was found to be a candidate risk biomarker for ischaemic stroke by predicting plaque instability and rupture. Conclusions: This work provides workflows for successful biomarker discovery using innovative –omics approaches, highlights key pathogenic pathways and identifies novel candidate biomarkers for ischaemic stroke diagnosis and risk stratification. 3 The work presented in this thesis is original and my own. Information derived from other sources has been appropriately acknowledged and referenced. 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. 4 PUBLICATIONS AND PRESENTATIONS PUBLICATIONS *Hasan N, Altman DG, on behalf of the SU:GAR Group. Association of Admission Glucose Levels with Symptomatic Intracerebral Haemorrhage Following Acute Stroke Thrombolysis: Quantification of Risk Using Individual Patient Data. [Manuscript in Preparation]. Cotlarciuc I, Malik R, Holliday EG, Ahmadi KR, Pare G, Psaty BM, Fornage M, Hasan N, Rinne PE, Ikram MA, Markus HS, Rosand J, Mitchell BD, Kittner SJ, Meschia JF, van Meurs JBJ, Uitterlinden AG, Worrall BB, Dichgans M, Sharma P, on behalf of METASTROKE and the International Stroke Genetics Consortium. Effect of genetic variants associated with plasma homocysteine levels on stroke risk. Stroke, 2014 [In Publication]. Yadav S, Cotlarciuc I, Munroe PB, Khan MS, Nalls MA, Bevan S, Cheng YC, Chen WM, Malik R, McCarthy NS, Holliday EG, Speed D, Hasan N, Pucek M, Rinne PE, Sever P, Stanton A, Shields DC, Maguire JM, McEvoy M, Scott RJ, Ferrucci L, Macleod MJ, Attia J, Markus HS, Sale MM, Worrall BB, Mitchell BD, Dichgans M, Sudlow C, Meschia JF, Rothwell PM, Caulfield M, Sharma P; International Stroke Genetics Consortium. Genome- Wide Analysis of Blood Pressure Variability and Ischemic Stroke. Stroke. 2013;44(10):2703-2709. *Hasan N. Cardiovascular disease as a measure of sustainable development. Journal of the Royal Society of Medicine Cardiovascular Disease. 2013;2: 2048004013491731. Yadav S, Hasan N, Marjot T, Khan MS, Prasad K, Bentley P, Sharma P. Detailed analysis of gene polymorphisms associated with ischemic stroke in South Asians. PLoS One. 2013; 8(3):e57305. *Hasan N, McColgan P, Bentley P, Edwards RJ, Sharma P. Towards the identification of blood biomarkers for acute stroke in humans: a comprehensive systematic review. Br J Clin Pharmacol. 2012;74(2):230-40. Brooke J, Hasan N & Sharma P. Efficacy of information interventions in reducing transfer anxiety from a critical care setting to a general ward: a systematic review and meta-analysis. Journal of Critical Care. 2012;27(4): 425.e9-15. Shalhoub J, Davies KJ, Hasan N, Thapar A, Sharma P, Davies AH. The utility of collaborative biobanks for cardiovascular research. Angiology. 2012;63(5):367-77. Marjot T, Yadav S, Hasan N, Bentley P & Sharma P. Genes associated with adult cerebral thrombosis. Stroke 2011; 42: 913-918. ORAL AND POSTER PRESENTATIONS *Platform Presentation: Integrative Genomics for Symptomatic Carotid Disease. International Stroke Genetics Consortium meeting, Lund, Sweden. November 2013. *Poster Presentation: Whole Genome Exon Array of Human Carotid Plaques. Imperial College London Department of Medicine Young Scientist Day, London, UK. April 2012. *Platform presentation: Towards the Identification of Blood Biomarkers for Acute Stroke. London Cardiovascular Society, London Medical Society, London, UK. April 2012. *Poster Presentation: Whole Genome Exon Array of Human Carotid Plaques: Differential Gene Expression, Alternative Splicing and the Role of IRF5 in advanced lesion development. British Heart Foundation Centre of Research Excellence Symposium, Cambridge, UK. June 2011. *Platform Presentation: Too Sweet to Treat? Rethinking Current Glucose Guidelines for Thrombolytic Therapy. 5th UK Stroke Forum, Glasgow UK. December 2010. * Publications and presentations relevant to this thesis. 5 ACKNOWLEDGEMENTS First and foremost I would like to thank my supervisors: Dr Pankaj Sharma for his support and guidance throughout my PhD, and Dr Robert Edwards, with whom every conversation has been a humbling lesson in how to be a ‘good’ scientist. Special thanks go to the many people who have contributed their time and expertise to the completion of this thesis. I am particularly indebted to Dr Waheedah Abdul-Salam for dedicating her time and help to the proteomics study, as well as providing constant support throughout my PhD. To Dr Paul Bentley, Dr Peter McColgan, Muhammad Saleem-Khan, Professor Doug Altman and the collaborators of the SU:GAR group for their statistical guidance and contribution to the biomarker systematic review and individual patient data meta-analysis. I would like to thank Dr Joseph Shalhoub, Professor Alun Davies, Dr Daniah Trabzuni and Dr Mina Ryten for their involvement with gene expression profiling, as well as Dr Ioana Cotlarciuc, Dr Sunaina Yadav, and Dr Doug Speed for their advice throughout the integrative genomics study. Thank you to the members of the Imperial College Cerebrovascular Research Unit (ICCRU) for making the past four years so enjoyable. To my family and friends for their patience and support, especially Kartik, for being my rock, Madiha and Siddik for providing a home away from home, and little Samaya, for being my stressball and a much-needed source of sanity. And finally, to my parents. None of this would have been possible without their unwavering love and encouragement. This thesis is dedicated to them, and all those who have taught me. 6 CONTENTS ABSTRACT .............................................................................................................................................. 3 PUBLICATIONS AND PRESENTATIONS ................................................................................................... 5 ACKNOWLEDGEMENTS........................................................................................................................... 6 LIST OF FIGURES .................................................................................................................................. 12 LIST OF TABLES ................................................................................................................................... 14 ABBREVIATIONS .................................................................................................................................. 16 CHAPTER ONE: INTRODUCTION .................................................................................................................................... 19 1.1 The Burden of Vascular Disease and Stroke ................................................................................ 20 1.2 Stroke Aetiology and Classification ................................................................................................ 21 1.2.1 Ischaemic Stroke Subtypes .................................................................................................................. 21 1.2.2 Transient Ischaemic Attack ................................................................................................................. 24