MOLECULAR GENETICS OF VON WILLEBRAND DISEASE SUMITHA E Ph.D THESIS 2016 DEPARTMENT OF HAEMATOLOGY & CENTRE FOR STEM CELL RESEARCH (A UNIT OF INSTEM BENGALURU) CHRISTIAN MEDICAL COLLEGE, VELLORE SREE CHITRA TIRUNAL INSTITUTE FOR MEDICAL SCIENCES AND TECHNOLOGY, TRIVANDRUM Thiruvananthapuram MOLECULAR GENETICS OF VON WILLEBRAND DISEASE A THESIS PRESENTED BY SUMITHA E Department of Hematology & Centre for Stem Cell Research (A unit of inStem, Bengaluru) Christian Medical College, Vellore TO THE SREE CHITRA TIRUNAL INSTITUTE FOR MEDICAL SCIENCES AND TECHNOLOGY, TRIVANDRUM Thiruvananthapuram IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE AWARD OF DOCTOR OF PHILOSOPHY 2016 DECLARATION BY STUDENT I, Sumitha E hereby certify that I had personally carried out the work depicted in the thesis, entitled, “Molecular Genetics of Von Willebrand Disease”. No part of the thesis has been submitted for the award of any degree or diploma prior to this date. Signature: Name of the Candidate: Sumitha E Date: i CERTIFICATE BY GUIDE Dr. Alok Srivastava, MD, FRACP, FRCPA, FRCP Professor of Medicine, Department of Hematology, Head, Centre for Stem Cell Research, Christian Medical College, Vellore Tamil Nadu 632002. This is to certify that Ms Sumitha. E in the Department of Hematology of Christian Medical College, Vellore has fulfilled the requirements prescribed for the Ph.D degree of the Sree Chitra Tirunal Institute for Medical Sciences and Technology, Trivandrum. The thesis entitled "Molecular Genetics of Von Willebrand Disease" was carried out under my direct supervision. No part of the thesis was submitted for the award of any degree or diploma prior to this date. * Clearance was obtained from Institutional Ethics Committee for carrying out the study. Signature: Date: ii The Thesis entitled Molecular Genetics of Von Willebrand Disease Submitted by Sumitha.E for the degree of Doctor of Philosophy of SREE CHITRA TIRUNAL INSTITUTE FOR MEDICAL SCIENCES AND TECHNOLOGY, TRIVANDRUM Thiruvananthapuram Is evaluated and approved by --------------------------------- ---------------------- Dr. Alok Srivastava (Name of the guide) (Name of the thesis examiner) iii ACKNOWLEDGEMENTS First and foremost I thank God for the successful completion of the thesis. I express my sincere gratitude to my guide and mentor Dr. Alok Srivastava, for giving me the unique opportunity to work on coagulation disorders. I thank him for the systematic guidance and great effort he put into training me in the scientific field through various presentations and discussions. His insightful comments and constructive criticisms have helped me to remain focus and move forward. Without his advice, this thesis would not have come into being. I am truly grateful to Dr. Eunice Sindhuvi for her immense patience in reading the thesis and commenting on revision of the thesis. I am indebted for her invaluable insights and guidance throughout my thesis. I could like to express my pragmatic thanks to Dr. Jayandharan GR. He has taught me how to question thoughts and express ideas. The weekly discussions helped me to design various experiments with a time frame. My special thanks to Dr. Shaji RV and Dr. Poonkuzhali B for their insightful comments and encouragement that helped me to widen my research from different perspectives which helped me to complete my experiments with ease. I am thankful to Dr. Fouzia N.A, for helping me to finalize the bleeding scores. My humble gratitude to Dr. Auro Viswabandya my Doctoral Advisory Committee member for his devoted instructions to improve my research in various context. iv I would also especially like to thank Dr. Vikram Mathews, Dr. Biju George, Dr. Aby Abraham, and all other clinicians and nurses for their support throughout my study. I owe my sincere thanks to Mr. Abraham Sundar Singh without whom the samples for this study would not have been collected. I thank Dr. Sukesh Nair, and Dr. Joy Mammen, Department of Immunohematology and Transfusion Medicine for their help in providing details of hemostasis assessment and for the fruitful discussions. Many thanks to Ms. Ramya, Mr. Suresh, Mr. Surender, Mrs. Shenbagapriya, and all other technical staff for their patience in helping me to understand the methodology involved the hematological diagnosis of the study patients despite their busy schedule and answering all my queries patiently. I could like to express my profound gratitude to all my teachers whose blessings and enouragement deeply empowered me to move forward. I thank Dr. Jeyanthi for her support and motivation during my college days. I am especially grateful to all the patients included in this study, for their cooperation. Without them, this study would not have been possible. I also owe a debt of gratitude to Dr.Johannes Oldenburg and Dr. Arijit Biswas, Institute of Experimental Hematology and Transfusion Medicine, Bonn, Germany for their support rendered in molecular modelling. I am so thankful to Dr. Anne Goodeve and Dr. David Hampshire, University of Sheffield, Sheffield, United Kingdom for their valuable suggestions and guidance in the study. v I thank Mrs. Sankari Devi and Mr. Senthil Kumar, for facilitating my sequencing and gene scan experiments. I am appreciative of the help and support rendered by the core-laboratory supervisor at the Centre for Stem Cell Research, Mr. Vaidhyanathan. I am thankful to Dr. Sumitha Saluja, Dr. Arun and Dr. Madavi for immense guidance and suggestions in writing this thesis. I thank the following in the laboratory and office, Mr. Neelakandan, Mrs. Eswari, Mrs. Ruth, Mr. Raj Kumar, Mrs. Sumithra, Mrs. Monica, Mr. Gopinath, Ms. Sukanya, Mrs. Dhava priya, Mrs. Kotteeswari, Mr. Hari, Mrs. Selvi, Mrs. Sathya, Mrs. Vinodhini, Mr. Sam prabhakar, Mr. Mohan, Mrs. Rekha Singh and Mr. Joel Dennis for their tremendous assistance during the research. Many friends have helped me to stay calm through some difficult times. Their support and care helped me overcome setbacks and stay focused throughout my research work: Mrs. Divya, Mrs. Ansu, Mrs. Sangeetha, Ms. Sreeja, Mrs. Ezhil, Mrs. Bharathi, Mrs. Nancy, Mr. Nishanth, Mr. Sachin, Mr. Ajay, Mr. Abbas, Mr. Saravanan and Mr. Hemanth. I am thankful to Dr. Dwaipayan Sen, Dr. Ruchita Selot, Dr. Sabna and Mr. Balaji, for their immense guidance and motivation. Besides this, several people have knowingly and unknowingly helped me in the successful completion of this thesis. Furthermore I deeply thank my parents Mr S. Elayaperumal and Mrs. K. Suseela for the unconditional trust, timely encouragement all these years. I greatly value the support and encouragement from my brother and sister. I appreciate the help extended from my in-laws. vi I could like to express my immense accolade to my husband Mr S. Yuvaraj for his valuable inputs in thesis writing and supporting me morally. Finally I thank my son Y. Suraj for being my strength to complete this thesis. Sumitha E vii TABLE OF CONTENTS Page Declaration by student i Certificate by guide ii Approval of thesis iii Acknowledgements iv Table of Contents viii List of Figures xii List of Tables xv Abbreviations xvii Synopsis xx 1. Introduction 1 1.1 Objectives 3 1.2 Brief overview of thesis chapters 3 1.2.1 Literature review and Materials and methods 3 1.2.2 Results 4 1.2.3 Discussion 6 1.2.4 Summary and conclusion 6 2. Literature review 7 2.1 Overview of Blood Coagulation 7 2.1.1 Evolution of Blood Coagulation 7 2.2 Molecular Basis of Blood Coagulation 14 2.2.1 Endothelium 15 2.2.2 Primary hemostasis 16 2.2.3 Secondary hemostasis 20 2.2.4 Regulation of Blood Coagulation 23 2.3 Biology of Von Willebrand Factor 28 2.3.1 Von Willebrand Factor 29 2.3.2 Domain structure 30 2.3.3 Synthesis of Von Willebrand Factor 34 viii 2.3.4 Functions of VWF 41 2.3.5 Clearance of Von Willebrand Factor 43 2.4 Molecular Basis of Von Willebrand Disease 47 2.4.1 Inheritance pattern of Von Willebrand Disease 47 2.4.2 Genetic background and Molecular basis of VWF subtypes 49 2.5 Clinical Features Diagnosis Classification and Treatment of VWD 64 2.5.1 Historical aspects 64 2.5.2 Diagnostic approaches to VWD 66 2.5.3 Alloantibodies to VWF 72 2.5.4 Classification of VWD subtypes 73 2.5.5 Treatment of Von Willebrand Disease 79 2.6 Von Willebrand Diseases in India 84 2.6.1 Potential and relevance of VWD 84 2.6.2 Diagnosis of von Willebrand Disease in India 86 2.6.3 Molecular diagnosis of VWD in India 90 2.6.4 Management of VWD in India 91 3. Patients and methods 95 3.1 Patients 95 3.1.1 Inclusion criteria 95 3.1.2 Definitions 95 3.1.3 Clinical evaluation 96 3.1.4 Frequency and site of Bleeding 96 3.2 Laboratory evaluation-Sample Processing 96 3.3 Coagulation studies 97 3.3.1 Baseline Investigations 97 3.3.2 Screening test for Von Willebrand Disease 99 3.3.3 Tests for Von Willebrand Factor 100 3.4 Genotypic analysis 102 ix 3.4.1 DNA extraction 102 3.4.2 RNA extraction 103 3.4.3 DNA and RNA quantification 103 3.5 Uniplex PCR and CSGE for VWF gene 103 3.5.1 PCR amplification of VWF gene 103 3.6 Screening for mutations using CSGE 111 3.6.1 Principle 111 3.6.2 Method 112 3.7 DNA sequencing 116 3.7.1 Principle of the method 116 3.8 Confirmation of deletions 118 3.9 Gene dosage analysis 120 3.9.1 Principle of the method 120 3.10 Restriction fragment length polymorphism (RFLP) 123 3.10.1 Principle 123 3.11 In silico analysis 125 3.11.1 Evolutionary conservation studies 125 3.11.2 Splice site prediction 127 3.11.3 Molecular modelling 130 3.12 Haplotype analysis 130 3.13 In vitro cell expression studies 131 3.13.1 Patients 131 3.13.2 VWF expression plasmid 132 3.13.3 Site-Directed Mutagenesis 133 3.13.4 Expression of recombinant VWF in eukaryotic cell 137 3.14 Antibodies 137 3.14.1 Immunofluorescence Analysis 138 4.