Frequency, Phenotypic Expression, Hemostatic and Genetic Basis of Fibrinogen Based Inherited Coagulopathies in Pakistani Population A thesis submitted for the partial fulfilment of the requirement for the degree of Doctor of Philosophy in Haematology By Tehmina Nafees Sonia Khan (Ph.D.-30-2013) Supervised by Dr. Arshi Naz, PhD Assistant Professor National Institute of Blood Diseases & BMT, Karachi. Liaquat University of Medical and Health Sciences, Jamshoro. April 2019 Success is not final Failure is not fatal It is only a courage to continue that counts Winston Churchill. Dr. Arshi Naz Prof. Dr. Tahir S Shamsi Supervisor Dean Post Graduate Studies National Institute of Blood Disease and BMT _______________ _______________ Internal Examiner External Examiner i I Tehmina Nafees Sonia Khan hereby state that my Ph.D. thesis titled “Frequency, Phenotypic Expression, Hemostatic and Genetic Basis of Fibrinogen Based Inherited Coagulopathies in Pakistani Population” Author Name & Signature ________________________ Tehmina Nafees Sonia Khan ii iii iv v PLAGIARISM UNDERTAKING I solemnly declare that research work presented in the thesis titled “Frequency, Phenotypic Expression, Hemostatic and Genetic Basis of Fibrinogen Based Inherited Coagulopathies in Pakistani Population” is solely my research work with no significant contribution from any other person. Small contribution/help wherever taken has been duly acknowledged and that complete thesis has been written by me. I understand the zero tolerance policy of the HEC and National Institute of Blood Diseases and Bone Marrow Transplantation (NIBD) towards plagiarism. Therefore I as an Author of the above titled thesis declare that no portion of my thesis has been plagiarized and any material used as reference is properly referred/ cited. I undertake that if I am found guilty of any formal plagiarism in the above titled thesis even after award of PhD degree, the University reserves the rights to withdraw/revoke my PhD degree and that HEC and the University has the right to publish my name on the HEC/University Website on which names of students are placed who submitted plagiarized thesis. Author Name & Signature _________________________________ Tehmina Nafees Sonia Khan vi TABLE OF CONTENTS Pg. No Certificate of Approval i Author’s Declaration ii Dedication iii Preface iv Plagiarism Undertaking vi Table of Contents vii List of Tables xi List of Figures xii List of Abbreviations xiv Show Case of Published Work xxi Abstract 1 Chapter 1: Introduction 4 1 Introduction 4 1.1 Background 4 1.2 Disease Biology of Congenital Afibrinogenemia 4 1.3 Literature Review 5 1.4 Aim of the Study 22 1.5 Objectives 22 1.6 Rationale of the Study 23 1.7 Hemostasis 24 1.7.1 Hemostasis Development In neonates 24 1.7.2 Components of Hemostasis 25 1.7.3 Endothelium 25 1.7.4 Functions of Endothelium 28 1.7.5 Platelets 29 1.7.5.1 Structure of Platelets 29 1.7.5.2 Platelets Receptors 29 1.7.5.3 Platelet Activation and Adhesion 36 1.8 Clotting Factors 40 1.9 Phases of Hemostasis 42 1.9.1 Vascular Smooth Muscle Contraction 42 vii TABLE OF CONTENTS Pg. No 1.9.2 Loose Platelet Plug Formation 44 1.9.3 Secondary Platelet Plug Formation 44 1.9.4 Clot Resolution(Fibrinolysis) 44 1.10 Types of Hemostasis 44 1.10.1 Primary Hemostasis 46 1.10.2 Secondary Hemostasis 46 1.10.3 Tertiary Hemostasis 46 1.11 Mechanism of Hemostasis 47 1.11.1 Coagulation Pathway 47 1.11.2 Extrinsic Pathway 49 1.11.3 Intrinsic Pathway 51 1.12 Fibrinolysis 51 1.13 Discovery of Fibrinogen 54 1.14 What is Fibrinogen 54 1.14.1 Structure of Fibrinogen 55 1.14.2 Fibrinogen Gene Regulation 60 1.14.3 Fibrinogen conversion to Fibrin 61 1.14.4 Functions of Fibrinogen 65 1.14.5 Interactions of Fibrinogen 66 1.14.5.1 Platelet Aggregation 66 1.14.5.2 Platelet Aggregation Other Interactions 66 1.15 Plasma Fibrinogen Fluctuation 68 1.15.1 Low Fibrinogen Levels In Plasma 68 1.15.1.1 Acquired Causes 68 1.15.1.2 Genetic Causes 68 1.15.1.3 High Levels Of Fibrinogen 69 1.15.1.3.1 Acquired Causes 69 1.15.1.3.2 Genetic Causes of High Fibrinogen Plasma levels 70 1.16 Disorders of Fibrinogen 71 1.17 Types of Fibrinogen Deficiencies 71 viii TABLE OF CONTENTS Pg. No 1.18 Congenital Afibrinogenemia 74 1.19 Etiology of Congenital Afibrinogenemia 74 1.20 Diagnostic Criteria 75 1.21 Treatment 75 1.21.1 Mechanism of Action of Fibrinogen Concentrates 78 1.21.2 Complications 78 Chapter 2: Materials & Methods 82 2.1 Contemplation/Survey 82 2.2 Study Design 84 2.2.1 Ethical Guidelines 84 2.2.3 Study Coalitions 84 2.3 Patient’s Selection 87 2.3.1 Exclusion Criteria 87 2.3.2 Sample Size 88 2.3.3 Sampling Technique 88 2.3.4 Data Inference 88 2.3.5 Pedigree Mapping 90 2.3.6 Sample Collection and Storage 90 2.4 Sample Preparations 91 2.5 Blood Parameters 92 2.6 Assessment of Coagulation Parameters 92 2.6.1 Prothrombin Time (PT) 92 2.6.2 Activated Partial Thromboplastin Time (APTT) 94 2.6.3 Fibrinogen Assay by Clauss Method 94 2.7 DNA Extraction 96 2.8 PCR and Gene Sequencing 96 2.9 Primers for Fibrinogen Alpha, Beta and Gamma Genes 101 2.10 Primers Reconstitution 104 2.11 Biocomputing Analysis (Bioinformatics) 109 2.12 Pathogenicity Scoring 109 ix TABLE OF CONTENTS Pg. No 2.12.1 MUpro (predictions of protein stability changes upon mutations) 109 2.12.2 PROVEAN (Protein Variation Effect Analyzer) 109 2.12.3 PolyPhen-2(polymorphism phenotyping v2) 110 2.12.4 SIFT (Sorting Intolerant from Tolerant) 111 2.12.5 SNP&GO (Single nucleotide polymorphism and GO) 111 2.13 Molecular Remodeling 111 2.13.1. I-TASSER (Iterative Threading ASSEmbly Refinement) 111 Chapter 3: Results 115 3.1 Demographic Details 115 3.2 Patient’s Description 116 3.3 Baseline Screening Results and Phenotypic Manifestations 116 3.4 Genetic Analysis Results 121 3.4.1 FGA Genetic Variants 124 3.4.2 FGB genetic Variants 125 3.4.3 FGG genetic Variants 125 3.5 Pathogenicity Scoring Results 127 3.6 Molecular Modeling Analysis 129 3.6.1 Alpha chain missense mutations 130 3.6.2 Beta chain missense mutations 133 Chapter 4: Discussion 135 Discussion 136 Conclusion 145 Future Prospects 147 Limitations of the study 150 Bibliography 152 Annexure 181 x LIST OF TABLES Table 1.1 Data records of published variants of CAF 16 Table 1.2 Classification of Quantitative and Qualitative deficiencies of 73 Fibrinogen disorder Table 2.1 Principal of Detection Methods for Different Cellular 93 Components of CBC. Table 2.2 The components of PCR reaction mix 99 Table 2.3 Composition of master mix 100 Table 2.4 Quantitation of 1X PCR buffer 100 Table 3.1 Summarization the findings of Baseline screening tests in all 118 selected study patients Table 3.2 Genotypic expression of mutations in fibrinogen gene (FGA, 123 FGB & FGG) Table 3.3 Pathogenicity Score of Missense Mutations. 128 xi LIST OF FIGURES Figure 1.1 ---------------------------------------------------------------------------------- 14 Figure 1.2 ---------------------------------------------------------------------------------- 26 Figure 1.3---------------------------------------------------------------------------------- 27 Figure 1.4---------------------------------------------------------------------------------- 30 Figure 1.5---------------------------------------------------------------------------------- 37 Figure 1.6 ---------------------------------------------------------------------------------- 38 Figure 1.7---------------------------------------------------------------------------------- 39 Figure 1.8---------------------------------------------------------------------------------- 41 Figure 1.9---------------------------------------------------------------------------------- 43 Figure1.10---------------------------------------------------------------------------------- 45 Figure 1.11---------------------------------------------------------------------------------- 47 Figure 1.12---------------------------------------------------------------------------------- 50 Figure 1.13---------------------------------------------------------------------------------- 53 Figure 1.14---------------------------------------------------------------------------------- 53 Figure 1.15---------------------------------------------------------------------------------- 55 Figure 1.16---------------------------------------------------------------------------------- 56 Figure 1.17---------------------------------------------------------------------------------- 56 Figure 1.18---------------------------------------------------------------------------------- 57 Figure 1.19---------------------------------------------------------------------------------- 59 Figure 1.20---------------------------------------------------------------------------------- 62 Figure 1.21---------------------------------------------------------------------------------- 64 Figure 1.22---------------------------------------------------------------------------------- 67 xii Figure 1.23---------------------------------------------------------------------------------- 72 Figure 1.24---------------------------------------------------------------------------------- 76 Figure 1.25 ---------------------------------------------------------------------------------- 77 Figure 2.1 ---------------------------------------------------------------------------------- 81 Figure 2.2 ---------------------------------------------------------------------------------- 83 Figure 2.3 ---------------------------------------------------------------------------------- 86 Figure 2.4 ----------------------------------------------------------------------------------