Fibrinogen Based Inherited Coagulopathies in Pakistani Population

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 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

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

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

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

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

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 ------89

Figure 2.5------95

Figure 2.6------96

Figure 2.7------105

Figure 2.8------106

Figure 2.9------107

Figure 2.10------108

Figure 3.1------114

Figure 3.2------115

Figure 3.3------119

Figure 3.4------122

Figure 3.5------126

Figure 3.6------132

Figure 3.7------134

xiii

LIST OF ABBREVIATIONS A Adenine

ADP Adenosine DiPhosphate

AE Any Elution Buffer

AE Buffer Any Elution Buffer

AL Buffer A Lysis Buffer

Ala Alanine

ALT Alanine Transaminase

AMP Adenosine Monophosphate

Approx. Approximately

APTT Activated Partial Thromboplastin Time

Asn Asparagine

AST Aspartate Transaminase

ATP Adenosine Triphosphate

AW 2 Buffer A Wash Buffer 2

AW1 Buffer A Wash Buffer 1

BioLiP Biologically relevant Ligand Protein

BMC Biomed Central journal

C Cytosine c Complementary

C terminal Carboxyl terminal

CaCl2 Calcium Chloride

CAF Congenital Afibrinogenemia

CBC Complete Blood Count.

CD Cluster of Differentiation

CHG Chughtai’s Lab

xiv

LIST OF ABBREVIATIONS CHL Children Hospital

CLSI Clinical Lab and Standard Institute

Cys Cystein

D domain Distal Domain dATP Deoxyadenosine triphosphate dCTP Deoxycytidine triphosphate

Del Deletion dGTP Deoxyguanosine triphosphate

DNA Deoxy Ribonucleic Acid dNTP Deoxyribo Nucleotide Triphosphate. dTTP Deoxythymidine triphosphate

EBP Endothelium Binding Protein

EDRF Endothelial Derived Relaxing Factor

EDTA Ethylene Diamine Tetra Acetic Acid

ENDRF Endothelial Derived Growth Factor

EN-RBD European Network of Rare Bleeding Disorders

ENSG Ensembl Gene Annotation

EQAS External quality assessment

F Forward

F1 Factor 1

FFP Fresh Frozen Plasma

FGA Fibrinogen Gene Alpha

FGB Fibrinogen Gene Beta

FGG Fibrinogen Gene Gamma

FII Factor II

LIST OF ABBREVIATIONS FIII Factor III

FIX Factor IX

FpA Fibrinopeptides Alpha

FpB Fibrinopeptides Beta

FpG Fibrinopeptides Gamma

Fs Frameshift

FVII Factor VII

FX Factor X

FXa Factor X activated

FXI Factor XI

FXIII Factor XIII

G Guanine

G Protein Guanine nucleotide binding Protein

G(g) Gravity g/dl Grams/deciliter g/l Grams/Liter

Gln Glutamine

Gly Glycine

GP Glycoprotein

Hb Hemoglobin

HBsAg Hepatitis B surface Antigen

HCV Hepatitis C Virus

HGMD Human Gene Mutation Databases

His Histidine

HMWK High Molecular Weight Kininogen

xvi

LIST OF ABBREVIATIONS HNF Hepatic Necrosis Factor

ICAM-2 Intra Cellular Adhesion Molecule-2

IL Interleukin

ISO International Standardization Committee

ISTH BAT International Society of Thrombosis and Hemostasis Bleeding Assessment Tool

I-TASSER Iterative Threading ASSEmbly Refinement

K2EDTA Dipotassium Ethylene Diamine Tetra Acetic Acid

Kb Kilo base

KCCT Kaolin Cephalin Clotting Time kDa Kilo Dalton

KPK Khyber Pakhtun Khuwa

LPL-R Lyso Phosphatidic acid receptors

Lys Lysine

MCHC Mean Corpuscular Hemoglobin Concentration

MCV Mean Corpuscular Volume

MgCl2 Magnesium Chloride ml Milli liter mM Milli Molar mRNA Messenger Ribonucleic Acid

NaCl Sodium Chloride

NEQAS National External Quality Assessment Scheme ng Nanogram

NIBD National Institute of Blood Diseases nm Nano Meter

xvii

LIST OF ABBREVIATIONS NMD Nonsense Mediated Decay

NMR Nuclear Magnetic Resonance

NO Nitric Oxide

NRBC Nucleated Red Blood Cell

OCS Open Canalicular System

OMIM Online Mendalian Inheritence In Man p Short Arm of Chromosome

P Protein p Protein change

PAF Platelet Activating Factor

PAI-1 Plasminogen Activator Inhibitor-1

PCR Polymerase Chain Reaction

PDB Protein Data Base

PGE2 Prostaglandin E2

PGI2 Prostaglandin I2 pM Pico Mole

PolyPhen Polymorphism Phenotyping

PPP Platelet Poor Plasma

Pro Proline

PROVEAN Protein Variation Effect Analyzer

PS Phosphotidyl Serine

PT Prothrombin Time q Long Arm of Chromosome

R Reverse

RBC Red Blood Cell

xviii

LIST OF ABBREVIATIONS RDW Red cell Distribution Width

RIBDs Rare Inherited Bleeding Disorders

RIQAS Randox International Quality Assessment Scheme

RPM Revolution per Minute

RPM Revolution Per Minute

RST Rapid Serum Test

SD Standard Deviation

Ser Serine

SIFT Scale-Invariant Feature Transform

SNP&GO Single Nucleotide polymorphism

SNPs Single Nucleotide Polymorphism

T Thiamine

Ta Annealing Temperature

TAX2 Thromboxane 2

TF Tissue factor

TFPI Tissue Factor Pathway Inhibitor

THBS-1 Thrombospondin-1

Thr Threonine tPA Tissue Plasminogen Activator

Trp Tryptophan

VWD Von Willebrand Disease

WBC White Blood Cell

WFH World Fedaration of Hemophilia

WHESS.db Whole Human Exome Sequence Space Database

WHO World Health Organization

xix

LIST OF ABBREVIATIONS YASARA Yet Another Scientific Artificial Reality Application

α Alpha

α2AP Alpha 2 Anti Plasmin

β Beta

γ Gamma

µL Micro Liter

µM Micro Mole

Showcase of Published Work

xxi

Publications

Journal of Thrombosis (BMC) Published online 2017 DOI: https://doi.org/10.1186/s12959-017-0143-3.

•Identification of Novel Mutations in Congenital Afibrinogenemia Patients and Molecular Modeling of Missense Mutations in Pakistani Population

Characterization of phenotypic expression of In Review congenital afibrinogenemia in Pakistani patients

Blood Advances DOI:10.1182/bloodadvances.2018GS110924

•Establishment of diagnostic facilities for autosomal recessive bleeding disorders in Pakistan.

xxii

Poster Presentations

xxiii

International

The Annual 80th meeting of Japanese society of hematology, 12 to 14th October2018, Osaka,Japan.

Poster presentation accepted for 64th Annual Scientific and Standardization Committee (SSC) meeting in Dublin, Ireland on July 18th -21st 2018.

Poster presentation accepted at 63rd XXVI Congress of the International Society on Thrombosis and Haemostasis and 63rd Annual Scientific congress Berlin, Germany, from July 8th - 13th 2017

Poster presentation accepted at 61st annual meeting of GTH 2017, Basel, Switzerland.

Poster presented accepted at XXIX International Symposium on Technical Innovations in Laboratory Hematology, Milano, Italy May 12-14, 2016

xxiv

Poster presentation accepted at 62nd International society of thrombosis and hemostasis, 25th -28th May 2016, Montpellier, France.

Poster presentation at 13th International congress of Human Genetics, 3rd -7th April 2016, Kyoto, Japan.

E-Poster presentation accepted in 20th Congress of European Association of hematology (EHA) at Vienna, Austria 2015.

Poster presentation accepted at 59th International society of thrombosis and hemostasis SSC Meeting, 23rd -26th June 2014, Milwaukee, USA.

Conference Abstract of American Society of Hematology (ASH) Published in Blood Journal titled Identification of Novel Mutations with Molecular Modelling of Missense Mutations of Congenital Afibrinogenemia Patients in Pakistan”

National

Poster presentation accepted in Pakistan society of Hematology Conference 2013 at Lahore, Pakistan.

xxv

Oral Presentation at 18th Annual Pakistan Society of Hematology conference, 5th -7th May 2016, Karachi, Pakistan.

Oral presentation accepted at Pakistan Association of Pathology (PAP) Conference 2014 at Peshawar, Pakistan

Oral Presentation accepted at Hematology Conference (OMICS) 2015 at Atlanta, USA.

xxvi

Prizes/Travel Awards

Received Travel Award Received Travel award from International Society from Japanese Society of of Thrombosis and Hematology (JSH) in 2018. Hemostasis (2016)

Travel award received from Travel award granted by International Society of European Society of Human Genomics (2016) Hematology(EHA) (2015)

xxvii

Abstract

Background

A hard beginning maketh a good ending

John Heywood

3 1. Introduction

1.1 Background

As rare as hen‘s teeth; congenital afibrinogenemia surfaced as a rare inherited bleeding disorder back in 1920 (1). It is an autosomal recessive Mandelian trait (Mandelian

Inheritance in Man, MIM #.202400), which has an incidence of one in one million population (2, 3). The ratio of occurrence of disease is equal in males and females (4). The affected individual is homozygous for the disease (5). The disease occurs due to complete absence of a protein known as Fibrinogen (6). The incidence of this diseases has gradually increased over the period of time so as the awareness regarding its clinical manifestations but still is considered as a rare disorder. This condition has strong affinity for consanguinity (7).

The entire phenomenon of this disease is based on disruption of physiological process of hemostasis in which blood loss is arrested from the site of injury in blood vessel wall in response to trauma through a series of steps which eventually result in formation of a clot on site and secure the flow of blood through the injured vessel lumen to maintain the normal circulation in vivo.

It has been hypothesized that congenital afibrinogenemia is no more a rare disorder as its incidence is swiftly increasing in Pakistan.

1.2 Disease Biology of CAF

Congenital afibrinogenemia is described as an inborn defect in which fibrinogen, a clotting protein is unable to be synthesized by liver cells due to pathogenic DNA variants. The normal levels of plasma fibrinogen ranges between 2 to 4g/L (3, 8), but in congenital 4 afibrinogenemia (CAF) it is available in negligible amount for clotting process to occur. The absence of fibrinogen affects both plasma and platelet linking fibrinogen which is required for platelet aggregation and for the formation of fibrin network for platelet plug followed by trauma. Studies have shown the fact that platelet loose plug is formed even in the absence of fibrinogen but in weak strength (9) and leads to bleeding as a result of failure to form a gel of blood to complete the coagulation process.

This inborn severe deficiency of fibrinogen leads to the impaired hemostasis and disrupts a delicate balance between coagulation and anticoagulation. The gene mutation occurs as a result of recessive inheritance pattern and can affect all or either three of fibrinogen genes which ultimately disrupts the entire mechanism clotting of blood leading to hemorrhagic diathesis.

1.3 Literature Review

International literature contains limited data on mutation analysis of congenital afibrinogenemia although the case toll is increasing gradually across the globe, specifically in high consanguineous regions, however the information on prevalence and phenotype of the disease is more readily available and getting more extensive with the passage of time. The story starts in 1920 with the reporting of a very first case officially in research literature by Rabe and Solomon (1). They reported the clinical findings and observations regarding this disease. Since then, 21 cases had been identified and reported in research literature and the count was raised to 22 in 1954 (10), as a young male was referred to a hospital in North Carolina, USA for the management of CAF. One case study from Srilanka mentioned the case of CAF presented at the age of 22years with

5 spontaneous hemoperitonium (11). In 1957, another study from Chicago, USA documented a new case of CAF in an infant and by that time the number of cases were reached to 31 in global literature (12). A study from Japan that was conducted in 1968, mentioned 14 cases identified in that region since the first case of congenital afibrinogenemia came into spot light in 1954 in Japan (13). In 1970, a research study from India reported a case of congenital afibrinogenemia. According to that study approximately 60 cases including three case report from India (14-16) were published in the global literature by that time (17). An inclination pattern can be observed in number of cases added from 1954 to 1970. In 1972, one study reported updated statistics of CAF cases increased to 80 which were identified and documented in the literature with major emphasis on clinical findings and frequent symptoms of CAF (18).

It has mentioned before that congenital afibrinogenemia is said to have a higher prevalence in countries where consanguineous wed locks are frequent and these affected regions predominantly includes the countries having muslim majorities like the Middle

East, Pakistan, Iran and south India. According to the research literature, in Iran the disease is more dominant as compared to other affected countries in the region. The western countries also have this disease due to the influx of migrants from the affected regions (19). In the Iranian registry, the incidence of fibrinogen disorders is seven times greater, comparing with other registries of Italy and England (20, 21). The North

American registry of inherited disorders published the data for all rare bleeding disorders

(RBDs) and reported a 24% frequency for inherited afibrinogenemia (22). A study from

North American Brazil published a comprehensive data of inherited coagulation disorders in 2007 and reported 10,982 patients with inherited coagulopathies in Brazil, of

6 which the most frequently occurring disorder was Hemophilia A (62.7%) followed by

Hemophilia B (11.9%) and VWD (21.2%). The study documented the frequency of other coagulopathies as 258 cases (2.4%). Out of these 258 registered cases of other coagulopathies, the prevalence of congenital afibrinogenemia was 11 (4.3%). The World

Hemophilia Organization reported Brazil as third largest of the affected countries of inherited coagulopathies (23).

The very first gene defect in CAF was identified in 1999 in a non-consanguineous family of Swiss origin; a homozygous deletion of ∼11 kb with removal of large sequence of the

FGA gene in four members of the same family (24). Succeeding research studies documented preponderance of FGA mutations. One article published the data of 16 unrelated patients in 2001 and out of that 13 mutations were identified in FGA (25). This provides a weight of significance of a mutation causing mechanism at the molecular level in the FGA gene in comparison to FGB and FGG. According to the published literature the frequently occurring mutations in subjects of European origin are both the 11-kb deletion and the IVS4 + 1 G > T donor splice mutation in FGA intron (26).

The European group on rare bleeding disorders (EN-RBD) reported 8% frequency for congenital afibrinogenemia (27). In 2006, Asselta et.al published a combined data containing a large bulk of reported variants in both afibrinogenemia and hypofibrinogenemia along with phenotypic expressions in CAF. That comprehensive data provided a detailed information specifically for CAF regarding mechanism of mutation effects on fibrinogen protein synthesis and frequent clinical presentations (28). Another review published in 2006, documented the details of total 62 mutations identified till that period and indicated that those mutations were responsible for afibrinogenemia. The 7 same published review also reported the identification of ten new additional genetic defects including eight mutations in FGA, one in FGG, and one in FGB reaching the total of 72 (29). The FGA gene is the most commonly affected gene in congenital afibrinogenemia and the majority of variants found were null mutations including large deletions, early truncating non sense, frameshift or splice site mutations. The FGB and

FGG mutations were also identified later and contributed to a significantly increasing mutation toll for congenital afibrinogenemia.

Although FGB was considered a less commonly affected gene in comparison to the other two genes but is of great importance as it was predicted to play role as a rate limiting polypeptide in fibrinogen production from liver. The majority of FGB mutations reported in the literature were either missense or late truncating nonsense mutations. Mostly, these mutations were located in a highly conserved region of C-terminal of the outer D domain of FGB gene (30). The progression of time has revealed the fact that Asian countries with relatively larger population of Muslims were proven to be the major hub for recessively inherited bleeding disorders due to the massive following for interfamily marriages tradition in Muslim communities for centuries but this practice is also not very uncommon in Hindu communities of southern parts of India particularly between uncles and nieces. As a result of this, recessively inherited coagulation disorders

(afibrinogenemia, prothrombin deficiency, factor V, VII, X and XIII deficiencies and combined factors deficiencies) are more frequent than in non-Muslim eastern or western countries (31). The registry of congenital coagulation disorders in Iran has been kept since 1970. Many studies in Iran revealed the fact that this country has a high rate of rare bleeding disorders (RBDs). Molecular analyses for factor I deficiency were undertaken in

8 a few patients and majority of those were evaluated from southeastern, southern and north-eastern Iran (32). In 2009, a study contributed data of 545 patients from southern Iran which stated that 79 patients out of 545 were affected with rare inherited bleeding disorder with a prevalence of 1.77 in a 100,000 population and the total population estimated was approximately 4.5 million. The data was analyzed from the southern Iranian registry from

1992 to 2007. In total 9 cases of inherited factor I deficiency were reported from this cross sectional data (33). A similar study from north eastern Iran was conducted with a sample size of 552 amongst which 85 (15.6%) of patients had rare inherited bleeding disorders.

Two patients (0.6%) with inherited afibrinogenemia were diagnosed in this cohort(34).

The study contributed from Iraq, Baghdad in 2011 had a frequency of rare inherited bleeding disorders around 9.9% with factor I deficiency detected in six patients (2.5%) in a total 243 patients (35). Other than Arab, Asian and Middle Eastern countries, some

African countries had also revealed the data initially based on phenotypic expression of congenital afibrinogenemia. In 2010, six patients from Cairo, Egypt were diagnosed with congenital afibrinogenemia and genetic analysis was performed. All six patients

(including four from same family) had homozygous novel truncating mutations in FGA

(36). In 2012, two different studies from Egypt reported the data of congenital afibrinogenemia. The first research study that was conducted over a period of 16 years

(1994-2009) in Egypt documented the data of 687 diagnosed cases of bleeding disorders; containing 187 cases of inherited bleeding defects. A total of four patients (2.3%) with congenital afibrinogenemia were reported (37). Another study from Egypt reported data of

70 patients amongst whom afibrinogenemia was the most frequently occurring inherited disorder with 23 cases identified (28.6%), followed by FVII and FX (38).

9 Another study from Jordan contributed a 14 years data from King Hussein Medical center comprised of 168 patients with one patient of afibrinogenemia (0.2%) (39). A case report based on a Syrian consanguineous family comprised of 13 family members was reported in 2011. All the members showed propensity towards bleeding but five out of thirteen family members had completely absent levels of plasma fibrinogen and were homozygous for the genetic mutations. The rest of the seven members had reduced levels

(1g/L) and were heterozygous for the identified mutation. The genetic analysis showed that all family members had same genetic variant which was identified in exon 5 of FGA gene and it was the significant C-terminal causative FGA mutation identified and reported in literature (40).

A comprehensive data was published in 2013 that documented a total of 39 identified mutations including 21missense, 9 nonsense, 3 frame shift and 6 splice site mutations in the FGB gene causing quantitative deficiency of fibrinogen (afibrinogenemia and hypofibrinogenemia). Of these 39, there were 8 missense, 4 nonsense, 3 splice site and one frameshift were identified purely in afibrinogenemia patient across the globe (30).

Scattered cases of congenital afibrinogenemia are also documented in research data from

Turkey (41), Tunisia and other countries in small numbers. Recently, a study published in

2016 reported two novel variants identified in two Tunisian families. The mutations were found in FGA gene was a nonsense truncating variant and FGB gene had a missense mutation (42).

Another study from Slovakia, which is the 28th largest country of Europe by area has reported a novel mutation in a Slovak family affected with CAF. The variant was

10 identified in FGB gene. This country has a very low incidence of one affected subject of

CAF in 20 million (43).

In Sub Continent region, India constitutes approximately one sixth of total world population. In India, the over representation of rare clotting factor deﬁciencies was reported frequently from south and west Indian parts and it is due to two reasons that includes a high prevalence of consanguineous marriages in southern India and certain communities in western India. A second factor is the availability of diagnostic facilities in these parts of the country. A large study from India in 2013 quoted 966 patients with bleeding disorders that enrolled in a study with inherited defects of coagulation were seen in 586 (60.6%).

Afibrinogenemia patients were detected in 5 (0.5%) out of 586 patients (44). Another study performed in India in 2013 with 27 patients from southern India suffering from inherited fibrinogen deficiency were enrolled for phenotypic as well as molecular analysis. Of 27 patients, thirteen had novel mutations which were identified in all three genes of fibrinogen. A further 14 mutations were also reported. The study also claimed to be the first in presenting largest data set with genotypic expression analysis in Indian registry to date (45).

In 2014, a large cohort was reported from different states of India expressing the data of

54 hemophilia centers (approx.16000 registered patients with hemophilia and rare inherited bleeding disorders. A total of 321 rare clotting factor deﬁciencies were analyzed in this study. The southern and western parts of India constituted the major bulk of patients (82%). Out of 39 identified cases of fibrinogen deficiencies, 24 had congenital afibrinogenemia (46).

11 In Pakistan, research studies contributed the data on CAF since 2001 and onwards but the major focus was remained to be either on frequency assessment or phenotypic expression for the disease. The genetic analysis was only performed in collaboration with international research studies and published as a multinational data. In a research data published by Neerman et al, the patients from different nationalities were evaluated for mutation analysis of congenital afibrinogenemia including Pakistan, but no mutation was detected in Pakistani patients at that time (25). In Pakistan, first case of CAF was reported in two affected families from Punjab province in 2001(47). Similarly another study reported three new cases from Punjab, Pakistan in 2005 by Anwar et.al (48). Both research studies reported the phenotypic data but no genotype was identified until that period.

In 2008, a tri-center study of was published; conducted in three medical institutes of

Karachi, Pakistan. The study had a focus on frequency of rare inherited bleeding disorder and their clinical spectrum and reported two afibrinogenemia cases out of 1100 patients

(3%). The age of presentation was early age with umbilical stump bleeding and hematomas due to minor trauma. A similar study reported a comparison of the relative frequency of congenital afibrinogenemia in Iran (70%), Italy (10%), UK (0.2%) and Pakistan (3%) (49).

In 2011, a study from Pakistan described the frequency of coagulopathy in screening of

376 patients and the bleeding disorders were confirmed in 318. Out of 318 (85%) only 13

(3.4%) patients were affected by rare inherited bleeding disorder and congenital afibrinogenemia was observed in 2 (0.53%) patients(50). Another study from Pakistan was undertaken with 1836 patients of bleeding disorders. Out of 1836, 435 patients 273

(62.8%) had coagulation factor deficiencies. Factor I deficiency was found in 8 (2.9%)

12 (51). A cross sectional descriptive study from Karachi, Pakistan presented the data, collected from a tertiary care hospital and evaluated 600 patients who visited this institute during a three years period (from 2010 to 2013). Total 64 patients were diagnosed with inherited clotting factor deficiencies with 15 patients having congenital afibrinogenemia along with grading of bleeding severities in these patients (52).

In 2014, the first genetic analysis of nine patients with congenital afibrinogenemia from

Pakistan was published as a part of a multinational data including patients of hypofibrinogenemia and afibrinogenemia from other countries as well including Iran,

Italy, Serbia, Russia, Greece and Finland. In Pakistani data set, five out of nine mutations were previously reported whereas four mutations were novel in that study and all identified mutations were homozygous nonsense. FGA exon 4 was observed to be the most frequently affected gene and had four mutations with one novel and three previously reported nonsense mutations, followed by FGA exon 5 and exon 1 had one novel nonsense mutation each. A total of three mutations were identified in FGB; two in exon 2 (nonsense reported) and one novel nonsense in exon 7. No mutations in FGG were reported (53).

In a most recent study from Pakistan (2017), a collective data of 429 cases was presented and 211 individuals were diagnosed cases of autosomal recessive bleeding disorders was published. The severe fibrinogen deficiency was diagnosed in a total 32 patients from

Pakistan that were mostly reported from Punjab province (20 cases) followed by Sindh province (11cases) and least explored in Khyber Pakhtunkhwa (3 cases) (54). A multicenter study published in 2018 from eight different branches of a non -profit organization of a

Hemophilia center, serving in Pakistan was conducted from 2015 to 2016. 13

40.00%

35.00%

30.00%

25.00%

20.00%

15.00%

10.00%

5.00%

0.00% Fibrinog FVII FV+VIII FXI FX FV FII FXIII en Percentile 37.50% 3% 26.50% 8% 9% 1.50% 6.50% 8%

Figure 1.1: A comprehensive survey documented by World Hemophilia Federation and European Network of Rare Bleeding Disorders showing the frequency of diseases in Europe. Fibrinogen was shown to have an 8% contribution to the rare bleeding disorder group (55).

14 In this one year study, the entire data of 1497 registered cases of hemophilia were included from all four provinces of Pakistan. The rare factor clotting deficiencies were diagnosed in 1256 (84%) cases and 6 (11%) patients were found to have congenital afibrinogenemia. The major cases were diagnosed from Sindh province (56).

The two major surveys conducted by world federation of Hemophilia (WFH) and the

European Network of Bleeding Disorders (ENRBDs). The WFH initiated the survey in

2004 and ENRBDs in 2007 and generated the data of clotting factor deficiencies in

Europe (55).

Since 1999, more than 200 mutations have been identified, accounting for afibrinogenemia or hypofibrinogenemia. Several new additions of entries on the Fibrinogen Mutation

Database (www.geht.org & http://www.hgmd.cf.ac.uk/ac/index.php) also can be accessed for mutation confirmation and quantity. Neerman et.al in their study summarizes the identified mutation numbers and phenotypes from collective data of different publications which makes approximately 208 mutations of afibrinogenemia so far (57).

15 Table 1.1: Presented the available reported variants in congenital afibrinogenemia databases (http://www.hgmd.cf.ac.uk/ac/index.php & www.geht.org), Seattle SNPs (http://pga.gs.washington.edu/) and published research literature

FGA S.No. Exon/ Intron Variant Protein Change Zygosity Type of Mutation Ref. 1 5 c.946G>T p.Gly316X Compound Heterozygous Nonsense (29) 2 5 c.1002G>A p.Trp334X (W334X) Compound Heterozygous Nonsense (29) 3 5 c.885G>A Trp295X Homozygous Nonsense (29) 4 4 c.448C>T Gln150X Homozygous Nonsense (29) 5 4 c.718C>T Q240X Homozygous Nonsense (36) 6 4 c.502C>T Arg168X (CGA→TGA) Homozygous Nonsense (29) 7 NA c.381C>A Tyr127X Homozygous Nonsense (45) 8 3 c.285 T>A Tyr95X Homozygous Nonsense (26, 29) 9 3 c.356C>G Ser119X Homozygous Nonsense (29) 10 1 c.54C>A p.Cys8X Homozygous Nonsense (53) 11 5 c.609_610insT Leu204X Homozygous Nonsense (29) 12 5 c.711_712insT Lys238X Homozygous Nonsense (29) 13 5 c.541C>T p.Arg181X Homozygous Nonsense (29) 14 2 c.94G>T Gly32X Homozygous Nonsense (58) 15 5 c.609_610insT Leu204X Homozygous Nonsense (29)

FGA S.No. Exon/ Intron Variant Protein Change Zygosity Type of Mutation Ref. 16 NA g.3174C>T p.Gln162X Homozygous Nonsense (53) 17 g.3873 p.Gln200X Homozygous Nonsense (53) 18 5 c.743 G>A p.Trp248X Homozygous Nonsense (29, 59) 19 4 c.502C>T Arg168X (CGA→TGA) Homozygous Nonsense (29) 20 4 c.385C>T p.Arg129X Homozygous Nonsense (58) 21 5 c.635T>G p.Leu212X Homozygous Nonsense (29) 22 Intron 2 c.180 +2 T>C NA Homozygous Splice site (29) 23 Intron4 c.510+1T >G IVS4 NA Homozygous Splice site (29, 45) +1G>T (11 Kb deletion) 24 Intron 3 c.364+1G>A NA Homozygous Splice site (29, 45) 25 NA c.510+2 T>G NA Homozygous Splice site (45) 26 Intron 3 g.1923_1926del GTAA NA NA Splice site (26) (IVS3 +1_+4delGTAA) 27 Exon1/ NA p. Met (-19)Val/ IVS4+1G>T Compound Heterozygous Missense/ Splice site (60) Intron 4 28 2 c.117delT p.Val40TrpfsX31 Homozygous Frameshift (29) 29 5 c.1055delC c.510+1G>T p.Pro352 LeufsX69 Compound Heterozygous Frameshift (29) 30 3 196_197inslT Ser66Phe fsX10 Homozygous Frameshift (29) 31 4 c.431_432delAA Lys144Ser fsx16 (3121delAA) Compound Heterozygous Frameshift (29) 32 5 c.1025delG p.Gly342GlufsX79 Homozygous Frameshift (29)

FGA S.No. Exon/ Intron Variant Protein Change Zygosity Type of Mutation Ref. 33 5 c.1037delA p.Asn346ThrfsX75 Homozygous Frameshift (29) 34 5 c.563_564insT Leu188PhefsX4 Homozygous Frameshift (29) 35 1 c.3_4insC Phe2Leu fsX21 Compound Heterozygous Frameshift (25, 29) 36 5 c.934delA p.Ser312AlafsX109 Compound Heterozygous Frameshift (29) 37 NA c.1398delT Thr466fsX17 Homozygous Frameshift (45) 38 NA c.887_894dup7 Asp296fsX59 Homozygous Frameshift (45) 39 5 c.943delA p.Ser312Alafs X109 Compound Heterozygous Frameshift (61) 40 5 c.786-789delGAGA p.Glu262AspfsX158 Homozygous Frameshift (61) 41 5 c.945delT p.Gly316GlufsX104 Homozygous Frameshift (61) 42 NA c.1725del A Lys575 fsX74 Homozygous Frameshift (45) 43 2 c.116delT (g.1215delT) Val40TrpfsX31 Compound Heterozygous Frameshift (26) 44 5 c.1846delA Thr616HisfxX32 Homozygous Frameshift (40) 45 5 NA p.Leul88PhefsX4 Homozygous Frameshift (60) 46 5 c.1084delC p.Pro362LeufsX59 Homozygous Frameshift (28)

Table 1.1: Continue…..

FGB Exon/ S.No. Variant Protein Change Zygosity Type of Mutation Reference Intron

1 1 c.139C>T Arg47X Homozygous Nonsense (28, 62)

2 8 c.1400G>A W467X Homozygous Nonsense (63)

3 3 c.352C>T p.Glu118X Homozygous Nonsense (30)

4 6 c. 895 T>C p.Tyr299His Homozygous Missense (30)

5 7 c.1129G>C or A Gly377Arg Compound Heterozygous Missense (64)

6 7 c.1148T>G p.Leu383Arg Homozygous Missense (65)

7 8 c.1289G>A Gly430Asp Homozygous Missense (65)

8 8 c.1399T>G Trp467Gly Homozygous Missense (66)

9 7 g.7228C>A Thr407Lys Homozygous Missense (53)

10 NA c.862G>A Gly288Ser Homozygous Missense (45)

11 NA 1334G>C Arg445Thr Homozygous Missense (45)

12 8 c.1330G>C Gly444Ser Compound Heterozygous Missense (62)

IVS4-1G>C 13 Intron 4 NA Heterozygous Splice site (30) (c.719-1G>C).

FGB Exon/ S.No. Variant Protein Change Zygosity Type of Mutation Reference Intron

14 Intron 7 IVS7 + 1G > T NA Homozygous Splice site (67)

15 Intron1 g.2659A>G IVS1+2076A>G Homozygous Splice site (68, 69)

g.6771C>T 16 Intron6 Glu321AsnfsX24 Homozygous Frameshift (67) IVS6+13C>T

17 4 g.5193delC Ser214LeufsX59 Homozygous Frameshift (53)

18 NA c.1241delG Gly384fsX2 Homozygous Frameshift (45)

19 2 c.605T>A Asp164Gly fsX13 Homozygous Frameshift (28, 70)

c.1346Del G 20 8 p.Gly449ValfsX17 Homozygous Frameshift (71) (g.7972DelG)

21 Intron 8 g.7253G>T Asn381X Homozygous Frameshift (67)

22 4 c.538C>T p.Gln180 Homozygous Frameshift (72)

Table 1.1: Continue…..

FGG S.No. Exon/ Intron Variant Protein Change Zygosity Mutation Type References

1 Intron 1 1876 + 5G>A NA Homozygous Splice site (73)

4 Intron 1 g.1881G>A Try27Val fsX17 Homozygous Frameshift (26)

5 2 c.98delA (g.1982delA) Asn33ThrfsX4 Homozygous Frameshift (26)

6 Intron 3 2395G>A Gly42IlefsX1 Homozygous Frameshift (74)

7 Intron 1 g.2018G>A (IVS2+1G>A) NA Homozygous Splice site (53)

8 Intron 2 IVS2–3C→G NA Homozygous Splice site (26)

9 NA c.851+1G>A NA Homozygous Splice site (45)

10 NA c.554delA Lys185fsX13 Homozygous Frameshift (45)

11 NA C.834_835delCT Asp278-279fsX17 Homozygous Frameshift (45)

12 NA c.243delA p.Ser81fsX5 Homozygous Frameshift (45)

13 NA c.1241delG p.414fsX2 Homozygous Frameshift (45)

14 7 g.5860G>T (c.769G>T) Glu231X Homozygous Nonsense (75)

15 4 c.400C>T (g.2602C>T) Arg134X Homozygous Nonsense (76)

16 7 c.667A>T (g.5758A>T) Arg223X Homozygous Nonsense (26)

These tables shows the data of reported variants documented in literature till date in all three fibrinogen genes (FGA, FGB and FGG). NA= not available.

1.4 Aim of the Study

The aim of this study comprises the assessment and identification of functional, genetic, structural and phenotypic findings and comprehensive analysis and evaluation of congenital afibrinogenemia in the Pakistani population for obtaining the specified and in depth knowledge and information regarding this particular disorder. In order to achieve the targeted aim, following objectives were set to acquire the desired results.

1.5 Objectives

. Collection of diagnosed cases of CAF from collaborative

centers in Pakistan to assess the prevalence of disease.

. To look for frequently occurring bleeding manifestations in congenital

afibrinogenemia (CAF) patients

. To identify gene defects in CAF in local population

. To look for the frequently affected gene and commonly occurring mutations.

. To contribute the identified set of mutations to the international literature for comparison and future research.

1.6 Rationale of the Study

Pakistan is highlighted among those countries on the globe where the trend of marriage with a close biological relative (cousin marriage) is common. As a consequence, this region is now considered as a hub for different inherited diseases.

Due to the escalating incidence of inborn Factor I deficiency in this country, a comprehensive study regarding genotype and phenotype was needed for the cases of congenital afibrinogenemia to look for frequently occurring clinical manifestations in order to differentiate it from other clotting factor defects (Hemophilia A and B). The available research data is still not too extensive on this particular variant of inherited fibrinogen defects (Afibrinogenemia).

This study will bridge the gap between locally identified DNA defects and the clinical signs and symptoms with international data to compare the diversities in findings exists amongst different countries within distinct cultures. It will provide a sound knowledge on genotype and phenotype of congenital afibrinogenemia thereby making a significant contribution towards establishing a genetic library of identified mutations in CAF exclusively found in Pakistani Population and will assist in setting up a local inherited factor I deficiency registry for the affected patients to maintain their records for follow up and treatment in future. This study data will also be helpful in finding the most commonly occurring gene defect or a mutation hotspot in our local population.

1.7 Hemostasis

Hemostasis is a series of events, strictly modulated by different components which includes blood vessel wall, platelets, fibrinogen and other clotting factors (clotting pathways) to harmonize the process. Normal hemostasis occurs in response to a collection of synchronized steps for executing two main tasks ;the first one is to maintain blood in a clot-free state and second task is to construct a confined plug of platelets rapidly at the site of blood vessel damage (77).

1.7.1 Hemostasis Development in Neonates

The hemostatic system is different in the fetus as compared to adults and is unique in many ways but it is absolutely physiologic and practically unmarked with no bleeding or abnormal clotting. In infants, from day 1 of birth until 6 months of age, the hemostatic system although continues to get mature postnatally but it is still significantly different between adults and children up to the age of 6 months (78, 79).

Lower levels of blood coagulation factors are expressed in fetal life than in adult life (80).

The coagulation factors are synthesized in the fetus, separately from the mother and they cannot cross the placental barrier to enter into the feto-maternal circulation (81, 82).

Numerous clotting proteins are produced in the initial phases of embryonic development and contribute to cell replication and distinction. The mRNA of clotting factors and transcribed into protein products and are detectable during the first month of gestational period in hepatic and endothelial cells and in the third month in the plasma of fetus (83).

A comprehensive study of RNA at 5 to 10 weeks of gestation revealed a similar design for three fibrinogen chains along with other coagulation factors (84).

1.7.2 Components of Hemostasis

The components of hemostasis includes an intact endothelium of blood vessels, platelets, clotting factors and anti-clotting factors which maintains this intricate balance and response to any damage by activating a chain of reactions.

1.7.3 Endothelium

Endothelium is a uniform single row of simple squamous cells lining the entire blood vessel lumen of the circulatory system. Its structure may vary according to the type of vessel (85).

Under physiologic conditions, a trauma free and healthy endothelium not only defines a lining between blood and tissue components to create and maintain a thrombo-resistance via different mechanisms including the anticoagulant pathway, antiplatelet factors or fibrinolysis but also it helps in maintaining the integrity of the entire vascular network irrespective of size or anatomical location in the human body. The involvement of endothelial cells is present in all hemostatic processes which become activated by any trauma to blood vessel and restrict the platelet clump formation at a site where hemostasis is required to maintain vessel integrity.

(a)

(b)

Figure 1.2: tructural representation of endothelium of blood vessel in two planes: longitudinal (a) showing the covering of vessel tube along with lumen and cross sectional (b) presenting the endothelial cell shape and size (https://healthdocbox.com/Heart_Disease /80978969-The- cardiovascular-system.html).

Figure 1.3: Interaction of endothelium with cellular elements and clotting factors (https://courses. washington.edu/conj/bloodcells/clotting.html)

1.7.4 Functions of Endothelium

 It maintains the vessel smooth muscles tone (86).

 Activation or inactivation of various vasoactive hormones (86).

 Endothelium releases nitric oxide (NO), also known as endothelial derived relaxing

factor (EDRF) along with prostaglandin (PGI2). Both are potent vasodilators and

also inhibits the activation, secretion and attachment of platelets with endothelium

(87).

 The endothelium also regulates the naturally existing anticoagulation process which

contains protein C and S, tissue factor pathway inhibitors (TFPI) and anti-thrombin

(88).

 Endothelial cells are the important reservoirs of von Willebrand factor (VWF) and

stores extreme large multimers in vacuoles called Weibel-Palade bodies. These

multimers are released upon stimulation so as to aggregate the platelets (89, 90).

 Endothelial cells also synthesizes and store Plasminogen Activator (tPA) and its

antagonist known as Plasminogen Activator Inhibitor-1 (PAI-1) (91).

 It allows transportation of nutrients and other substances through perforating the

endothelial membrane (92).

The endothelial cell surface contains an enzymatic activity that converts the ADP in to

AMP. The ADP secreted by activated platelets plays an important role in aggregation of

28 platelets and amplifying it. An ecto-ADPase functions as an antagonist and may restrict the process of platelet gathering on trauma location (93).

1.7.5 Platelets

1.7.5.1 Structure of Platelets

Platelets are the non-nucleated particles of megakaryocytes which are produced in the bone marrow (94). They are normally round to discoid in shape, covered by a smooth plasma membrane (95). with cytoplasm containing different granules (96). The life span of platelets in humans is approximately 8-10 days (97). The glycocalyx of platelets contains several receptors which provide interaction and adhesions for different molecules (98).

1.7.5.2 Platelet Receptors

The platelet‘s surface receptors are the major contributors in initiation and propagation of coagulation and hemostasis (99). These receptors either cause activation of platelets or serves as a link for adhesion with more platelets, binding with the affected endothelium or connecting platelets with leukocytes (100).

Figure 1.4: A normal platelet cell morphology describing the shape and intracellular contents REF. (Figure reproduced, with permission, from Jaun Carlos Zapata, PLOS 2014) (101) .

Platelet Receptors Family

Integrin Transmembrane Tetraspanin Selectins Lipid Tyrosine Immunoglobin Prostaglandin Leucine rich Receptor Receptor repeat receptors Receptor Kinase super family Agonist Receptor receptor Receptors β -1 α2β1 α5β1 α6β1 β -2 β-3

a. Integrin

Integrin are non-covalently bonded transmembrane protein receptors family of β1 (α2β1,

α5β1 and α6β1), β2 and β3 located on platelet surface (100).

(i) β-1 Receptor Functions

I. α2β1 Receptor (GPIa-IIa)

 Binds to collagen.

 Encourages platelet adhesion to collagen resulting in stabilization of platelet

cluster growth and further enhancement in coagulation activity.

 Initiates activation of platelet proteins after binding with collagen.

II. α5β1 Receptor

 Provides bridging of fibronectin to platelets.

III. α6β1 Receptor

 Provides an attachment of platelets with laminin which is present in

basement membranes and extracellular matrix. The interaction is facilitated

by cofactors including magnesium, manganese and cobalt.

 α6β1 takes part in signal transduction via phosphoinositide 3 kinase and

persuade the shape changes in platelets.

(ii) β2 Receptor Function

 αLβ2 or intercellular adhesion molecule 2 (ICAM-2)

 Located on platelet membrane and open canalicular system.

 Serves in platelet attachment to neutrophils and other leukocytes.

(iii) β3 Receptor Functions:

I. αIIbβ3 (CD41/CD61) (GPIIb-IIIa complex)

 Adhesion molecule involved in platelet aggregation.

 Only expressed on platelet membranes in a greater concentration and on α-

granules, dense granules and the canalicular system as well.

 Present in an inactive form on resting platelets.

 Upon activation of platelets by agonists, they move to the plasma membrane

after granules release reaction and allowing the binding of the fibrinogen

molecule.

 Other than fibrinogen they also binds with the fibrin network, vWF,

extracellular matrix glycoproteins including fibronectin, vitronectin and

thrombospondin (THBS1) and increase the formation of aggregates of platelet

cells (102).

II. GPIb-IX-V complex

 It is considered as one of the frequently occurring receptor on platelets after

integrin αIIbβ3.

 GPIb-IX-V complex is a vital contributory in commencing and dispensing of

both hemostasis as well as thrombosis.

b. Transmembrane Receptors Agonists

This vital agonist receptor family (ADP and thrombin receptors) constitute a major

portion on platelet surface and are well evident on platelet surface. These are membrane

spanning receptors that play a significant task in the activation and clumping of platelets.

i. Adenosine Diphosphate (ADP) receptors

 The ADP interacts with platelets through three purinergic receptors which

includes P2Y1, P2Y12 and P2X1.

 The ADP releases from platelet dense granules and damaged red cells.

 It is a significant physiologic agonist and promotes platelet aggregation via

P2Y1, which is also induced by other agonists and alterations in platelet shape.

 ADP through P2X1 involves in Ca2+ influx and shape changes.

 Release reaction and production of thromboxane A2 (TXA2).

Activating the immobilized fibrinogen and potentiates binding of platelets with αIIbβ3

(103). ii. Prostaglandin Receptors

Prostaglandin, a fat derivative synchronizes multiple normal functions in different

organ systems. Receptors for prostaglandins on platelets play important modulatory

functions. Prostaglandin receptors include thromboxane receptors, prostaglandins

(PGI2), Prostaglandin D2 and E2 (104).

a. Agonists

I. Thromboxane/ PG H2 Receptors

These receptors are involved in activating the phospholipase A2 and

phospholipase C via signal transduction with G coupled proteins. Their main

function is in the amplification of platelet activation and aggregation.

II. PGE2 Receptors

At lower concentrations of ADP and collagen, they begin platelet stimulatory

effect. On the contrary, higher concentrations causes inhibition of PGE2

mediated platelet activation.

III. Lipid Receptors

Platelet activating factor (PAF) receptors are phospholipids in nature. The role

of these receptors is to regulate the processes of inflammation, anaphylaxis,

gathering of platelets and degranulation.

IV. Lysophosphatidic Acid Receptors (LPA-R)

LPA receptors are transmembrane receptors on platelets. They are autocrine

agonist and participate in shape change, releasing reactions of granular

contents and platelet cluster formation (105). b. Antagonists

I. Prostacyclin Receptor (PGI2)

Functions as an inhibitory prostaglandin receptors for prostacyclin and

maintains the platelets‘ resting state by interacting with the G protein receptor

system to stimulate adenylate cyclase (106).

Immunoglobulin receptors family

It constitutes a collection of cell membrane proteins which perform actions like recognition of a molecule and binding within receptor socket of cells. GPVI belongs to this group of receptors and possesses an affinity for collagen in the sub-endothelial matrix of bare endothelium (107). It is a transmembrane glycoprotein and structurally consist of two molecules to form a complex on the surface of the platelet.

1.7.5.3 Platelet Adhesion and Activation

I. Gluing to collagen brings significant changes in cellular shape and function of

platelets. GPVI in the exposed endothelium fulfills a significant role in

procoagulant activity of collagen bonded platelets (108). The platelet

procoagulant reaction combines the steps of adhesion, activation and coagulation

for the localization of the hemostatic activity. This reaction is comprised of two

processes which involves the disruption of the phospholipid membrane

asymmetry which in turn activates phosphatidyl serine (PS), present on the outer

platelet membrane (109).

II. This causes the platelet membrane to produce blebs which may later shed off as

micro vesicles. Activation of phosphatidyl serine provides a negative surface

which helps in the conversion of inactivated clotting proteins into activated forms

and serve as a catalytic binding site for prothrombinase and tenase complexes

(110, 111).

A change in platelet shape from discoid to round or slightly irregular with

pseudopods occurs upon interaction with exposed sub endothelial matrix of vessel

as a result of injury (112).

Figure 1.5: Receptors located on the platelet surface for molecular interactions, (Marlis Zeiler, Molecular & Cellular Proteomics December 1, 2014) (113) .

A pivotal inceptive step in platelet adhesion involves the linking of the GP Ib-IX-V

complex receptor on the platelet surface with the A1 domain of vWF present in open

sub endothelium at the injury site vWF is a multimeric protein which has multiple

production sites including endothelium, alpha vesicles of platelets, present in plasma

(soluble state) and in the sub endothelial matrix (114).

The platelet receptors interact with collagen via GPVI and α2β1 integrin (115) which

initiates collagen to induce platelet activation alongside with GPIb-vWF interaction

(116). Other factors or agonists in sub endothelium also contribute to platelet

activation including thrombospondin, fibronectin, vitronectin and laminin via integrin

receptors on platelets (117), described earlier in this section.

` Figure 1.6: Changes in shape of platelets manifests: the OCS, pseudopods, spreading of platelet and blebs formation in the platelet membrane. (Magnification 40000) (118).

Figure 1.7: Shows interactions of exposed collagen from injured vessel sub endothelium with platelet surface receptors for collagen and Von Willebrand factor (119).

(Figure reproduced, with permission, from Gabor Szalai, Cellular and Molecular Life Sciences 2006)

Platelet placement along with degranulation of contents of alpha and dense granules to further propagates the aggregation thereby aiding in the cessation of bleeding (120). The pro-coagulation factors activate platelets by interacting with their specific receptors on the platelet membrane which ultimately raises the intracytoplasmic levels of calcium ions

(from intracytoplasmic stores and influx via the plasma membrane). An increased calcium concentration exerts changes in structure and function of platelet. The central alignment of granules which then pour their contents into the open canalicular system thereby releasing them outside platelets (121). Higher concentrations of platelet calcium enhances the membrane phospholipase A2 activity which in turn leads to the formation of thromboxane (TAX2). TAX2 is a potential activator of platelet cells. Membrane projections of platelets as a result of a shape change reaction allows the formation of aggregates by interacting with each other through those projections.

1.8 Clotting Factors

There are 13 clotting factors. Majority of clotting factors are produced by hepatocytes (122).

The rest of clotting proteins including FIV obtained from different sources including calcium from the diet, bone and gut,stored in the endoplasmic reticulum, tissue factor (FIII) is driven from damaged tissue (123, 124) and FVIII which is produced by platelets and endothelium

(125). They are given numbers in order of their discoveries (126). The majority of clotting factors are protein in nature and are synthesized in an inactivated form (127) and activation occurs when there is any trauma to blood vessel so as to secure the blood loss (128).

Clotting proteins are encoded by a specific gene (129). Out of 13 clotting proteins four are known as vitamin K dependent factors that include FII, FVII, FIX and FX (130).

Figure 1.8: Mechanism of platelet adherence following vessel injury leading to triggering and secreting phase. The receptor complex GPIb/V/IX on the outer surface of the platelet binds with von Willebrand factor and exposed collagen via αIIβ3 causes platelet activation and (1) a rise in intracellular calcium concentration resulting in (2) phosphatidylserine exposure and creating a negatively charged surface, (3) contents of platelet intracellular granules releases , (4) conversion of the GPIIb/IIIa receptor from quiescent to a high-affinity state , (5) thromboxane A2 generation and (6)cytoskeletal rearrangement. ADP, adenosine diphosphate (131).

These are the glutamic acid residues and are produced as inactive proteins. Vitamin K plays role as a co-factor to activate the proteins clotting process.

Anti- clotting squad comprises Anti S and Anti C proteins and plasminogen. Their major role is to prevent the overgrowth of clot formation in reaction to trauma. The excessive growth of fibrin clots may lead to the occlusion of the lumen of the vessel which in turn causes ischemia or complete blood flow obstruction through the repaired channels.

Calcium plays an integral role in the coagulation cascade at different levels.

Hemostasis is a broad term and encompasses multi steps involving different components significantly to prevent bleeding. Whenever there is a breach in the architecture of vessel wall followed by trauma, it leads to blood loss from the affected site. This is the initiating point for the coagulation mechanism to ensue and cease the bleeding from the damaged vessel wall.

1.9 Phases of Hemostasis

As a compact process, hemostasis is carried out in four steps (132), discussed in the following sections.

1.9.1 Vascular Smooth Muscle Contraction

Vascular endothelium is a single layered cellular lining of the entire circulatory network of vessels (85). Endothelium provides a non-thrombogenic covering for the circulating coagulation factors and (133) serves as a partition between sub endothelial matrix comprising collagen, vWF and other proteins including laminin, thrombospondin and vitronectins which are involved in platelet aggregation and attachment (134).

Figure 1.9: A compact view of components involved in primary and secondary Hemostasis. (https://quizlet.com/178508574/0103-hemostasis-wondisford-flash-cards/)

It is the first and foremost step, manifested in response to injury as contraction of smooth muscles of vessel walls after injury so as to minimize blood loss from the injured site

(135).

1.9.2 Loose Platelet Plug Formation

Soon after vascular muscle contraction, platelets become activated due to exposure with subendothelial collagen matrix by modifying their morphology and releasing their granular contents in to the periphery to attract more platelets and linking them together via fibrinogen. This will lead to a loose aggregation of platelets at the site of trauma, thereby constructing a temporary clot (136).

1.9.3 Secondary Platelet Plug Formation (Fibrin Deposition)

A compact, solid and long lasting plug of platelets and fibrin meshwork is formed which replaces the temporary clot (137).

1.9.4 Clot Resolution (Fibrinolysis)

This provides a check for an overgrown clot which may block the vessel lumen.

1.10 Types of Hemostasis

Hemostasis comprises three types. The first two types will explain the process of clotting and the third type will explain the fibrinolysis.

Figure 1.10: Elaborates the main steps of hemostasis after vessel injury (http://slideplayer.com/slide/15889126/)

1.10.1 Primary Hemostasis

Is defined as the formation of primary loose and porous platelet aggregates. It incorporates platelet activation and aggregation followed by vessel wall trauma (138).

The circulating platelets are attracted by bare collagen network and stored vacuoles of vWF in the damaged endothelial lining of the blood vessel. They bind themselves through surface receptors GPVI and ligand 1βα of the GPIX complex respectively to establish a connection between platelets and injured vascular endothelium (139).

Modification in morphology appears after platelets binding and degranulation of their membrane bound granular contents into plasma that favors the aggregation of circulating platelets thereby attracting more platelets interconnecting via fibrinogen crosslinking fibers connected to the surface receptors Gp IIb/IIIa on platelets (140).

1.10.2 Secondary Hemostasis

In continuity with primary hemostasis, activation of the coagulation cascade and fibrin monomers production is the hall mark of this phase (141). The fragile platelet bunch becomes more firm as the inactive fibrinogen in plasma gets activated by thrombin cleavage into insoluble fibrin filaments. These intricate lattice of fibrin threads provide stability and strength to the platelet plug and permanently seals off the breached area of vessel wall.

1.10.3 Tertiary Hemostasis

It is a final step in further stabilization of clot at the injured site. It involves the process of clot retraction and fibrinolysis (142, 143). The stable platelet plug formed will be

46 resolved so as to clear the lumen of repaired vessel for a smooth blood flow .This is carried out by another protein synthesized by liver called Plasminogen. This protein gets activated by tissue plasminogen activator. It clears the debris and all cellular material from the site of the repaired wound to maintain a smooth blood flow in previously partly occluded vessel. The platelets also retract back their granules and break links of fibrinogen via GP IIb/IIIa (136).

1.11 Mechanism of Hemostasis

The entire process of hemostasis encompasses two major process:

Exrinsic Pathway (Tissue Factor Pathway) Coagulation Pathway Intrinsic Pathway Hemostasis (Contact Activation Pathway) Fibrinolysis

Figure 1.11: Mechanism of Hemostasis

1.11.1 Coagulation Pathway

The detailed elucidation of the coagulation mechanism was presented by MacFarlane2 in

1964 (the "MacFarlane cascade") describing the basic concept of two pathways namely, tissue factor (TF) or extrinsic pathway and the intrinsic pathway (144). These two merge at the activation step of FX and convert prothrombin into thrombin which cleaves fibrinogen to form fibrin. Platelets were assumed to be independent in the coagulation cascade in basic theory.

Over a period of years, a new approach has been developed and implemented as follows:

 TF and FVII as a complex takes part in activation of FIX (145, 146)ref added and

highlight the connection between the two pathways from the very beginning of the

coagulation process.

 The process is completed in 3 consecutive phases including an initiation, amplification

and a propagation phase. The last two phases actively involves platelets and thrombin.

(i) Initial Phase

In an initial step, FX gets activated by tissue factor and FVII complex. FIX converts FII to thrombin (FIIa) in a limited quantity, not enough to complete the process of synthesis of fibrin.

(ii) Multiplication Phase

Thrombin generated in small quantity actively participates in activating FXI, FIX, FVIII,

FV and platelets. Simultaneously, these factors also attracted via chemotactic mechanisms on the platelets surface where extensive activation and amplification occurs.

(iii) Propagation Phase

The amplification of thrombin and platelets and the activation of all other factors allow large quantities of FX to be activated and form the prothrombinase complex to convert more prothrombin into thrombin and, through the action of thrombin, fibrinogen into fibrin. The final process, always occurs on the surface of the platelets, accelerates and leads to the massive production of large quantities of thrombin and fibrin.

It is subdivided into two parallel torrent series of activation of clotting factors which merges at a common pathway. Two complexes are formed in these pathways including the tenase complex in the extrinsic pathway and the prothrombinase complex in the intrinsic pathway (147).

1.11.2 Extrinsic Pathway

This pathway is the first to initiate the clotting mechanism via activation of tissue factor

(factor III) (148, 149). Tissue factor is expressed by the tunica adventitia of the vascular wall

(150). Some cells in the tunica media are also reported in the synthesis of tissue factor. Some cells in the tunica media are also reported in the synthesis of tissue factor (151). It is a transmembrane protein (152) which acts as a receptor for activated FVIIa (FVII/VIIa) (153).

They both form a complex in the presence of calcium ions and initiate the extrinsic clotting pathway. An intact endothelium serves as a separator for tissue factor which is a potent activator from its ligand FVII/FVIIa, circulating in the plasma. In this way endothelium avoids unnecessary activation of the clotting cascade. Any breach in endothelium leads to the release of tissue factor in the blood in turn activating the clot forming process.

Several studies have proposed the locations of tissue factor release in the human body which includes the surface of monocyte /macrophages, heart, brain, apoptotic cells and micro vesicles. These membranes bound micro vesicles increase in quantity in certain diseased conditions like sepsis or malignancy. One study has shown that the tissue factor is identified on membrane and matrix of α-granules of platelets. For this reason, it is called blood borne or plasma derived tissue factor (154). Tissue factor and the FVIIa complex along with calcium convert FX in to its activated form thereby leading to thrombin generation and ultimately formation of fibrin polymers by cleaving fibrinogen (155).

TF: VIIa: Xa TF:VIIa FX

FVII TF

Figure 1.12: Shows the extrinsic pathway with exposed tissue factor that binds FVII thereby activating FX to set in Common pathway

1.11.3 Intrinsic Pathway

Intrinsic pathway involves a series of activation of clotting factors as soon as the trauma causes the damaged site open. This embarks an extracellular torrent of activation of clotting zymogens in a sequence. The first factor to become activated in response to exposed collagen in the intrinsic pathway is FXII which is converted into activated FXII by exposed collagen of sub-endothelial tissue (156). FXIIa now activates FXI/FXIa (157) which along with cofactor VIII activates FIX/FIXa (158). The TF and FVII combination is activated through auto cleavage and activates FIX. This activated FIXa participates in the activation of FX (TF/FVIIa also activates FXa). Now FXa along with FVa (cofactor) converts prothrombin to thrombin which cleaves fibrinogen and allows it to polymerize as fibrin meshwork. Activation of FXIIIa stabilizes the fibrin clot by cross-linking. The

Intrinsic pathway initiates with activation of FXII via the prekallikerin system and thereby activates FXI and ultimately converging at the common pathway where factor X cleaves into FXa and the rest is same as mentioned above.

1.12 Fibrinolysis

Fibrinolysis is a two steps process:

(i) The conversion of plasminogen into plasmin by tissue plasminogen activators

(ii) The degradation and digestion of fibrin by activated plasmin (159). Mechanism of fibrinolysis comprises proteolytic enzymatic process in blood (160). The main function of this process includes the restriction of the growth of platelet plug formed as a result of injury to the vessel wall and to reopen vessels blocked by thrombosis to keep its lumen patent for a smooth circulation of blood. The fibrinolytic system consists of a proenzyme,

51 plasminogen (inactivated) converted to an activated form via plasminogen activators

(161) and inhibitors (162). Plasminogen is present in plasma and extracellular fluids converted into plasmin by tissue plasminogen activating factor. Plasmin breaks down the fibrin meshwork. An inhibitor, α2-antiplasmin (α2-AP) inhibits free plasmin concentrations in the plasma and prevents excessive fibrin degradation. High thrombin concentrations leads to the formation of fine clots with thin, compact bundles of fibers while low quantity results in rough clots with thick and porous strands of fibers. It has been proposed by some studies that thick fibers of fibrin lyse at a slower pace than thin fibers (163).

Figure 1.13: A comprehensive pictorial presentation of extrinsic and intrinsic pathway leading to fibrin formation (https://en.wikipedia.org/wiki/Coagulation)

Figure 1.14: Emphasizes the process of fibrinolysis and clot resolution (164)

1.13 Discovery of Fibrinogen

Fibrinogen was discovered as Fibrin threads back in 17th century (1666) by Marcello

Malpighi.

Malpighi (1628-1694) described the structure of a clot as a collection of red blood cells and a meshwork of fibrous texture after examining thrombi of cardiac and vessel origin under light microscopy.

In 1801, Fourcroy used the term fibrin for the fibrous texture in clot and explained the fact that soluble substances including the precursor of fibrin are present in plasma but not serum (165).

In 1847, Rudolf Virchow suggested the expression ‗fibrinogen‖ for a gradually clottable substance (166).

In 1872, Alexander Schmidt studied the conversion mechanism of fibrinogen into fibrin and explained it as enzymatic process (167).

In 1879, Olof Hammarsten made the first purified fibrinogen preparation through repeated precipitation with half-saturated NaCl. (https://www.fibrinogen.org)

1.14 What is Fibrinogen?

Fibrinogen is an inactivated protein produced in the liver (168). It is a soluble, 340 kDa protein molecule which is 45nm long (169) and contains different domains which are connected to each other through a twisted cord like structure (170). Its half‐ life is approximately four days (100 hours) (171). Although hepatocytes are the manufacturers

54 of fibrinogen but extrahepatic production has also been demonstrated by intestinal epithelial cells, cervical mucosa and lungs (28, 172). The synthesis of fibrinogen by megakaryocytes has remained in discussion in past years but now several studies have documented that fibrinogen stored in platelet alpha granules primarily come from plasma

(173).

1.14.1 Structure of Fibrinogen Molecule

The structure of fibrinogen was suggested by Hall and Slayter in 1959 (174). The molecular structure of fibrinogen was explained in the late 70s as three pairs of polypeptide chains namely Aα (610 amino acids), Bβ (461AA) and γ (411 AA) (174).

FGA Gene

Cytogenetic Location: 4q31.3 ( long (q) arm ,chromosome 4 ,position 31.3)

Figure 1.15: Fibrinogen Alpha gene (FGA) location on Chromosome No.4.(Ensembl: ENSG00000171560, OMIM: 134820 ,UniProtKB: P02671) (https://www.genecards.org/)

FGB Gene

Figure 1.16: Fibrinogen Beta gene (FGB) on chromosome 4.Cytogenetic Location is 4q31.3, Ensembl: ENSG00000171564, OMIM: 134830, UniProtKB: P02675 (https://www.genecards. org/)

FGG Gene

Figure 1.17: Fibrinogen gamma gene (FGG) on chromosome 4. Cytogenetic Location: 4q32.1, Ensembl: ENSG 00000171557, OMIM: 134850, UniProtKB: P02679 (https://www.genecards. org/)

Organization of Fibrinogen Gene Locus:

HNF-1

(Hepatic nuclear Factor) IL-6

IL-6 HNF-1

γ α Exons β

Direction of Transcription

Figure 1.18: Fibrinogen gene makeup for all three gene (FGA, FGB and FGG) encoding for polypeptides (175). (Figure reproduced, with permission, from Dr. Serap Tutgun Onrat, Genetics and Molecular Research 2009)

Fibrinogen protein structure comprises two twin halves (Dimer) which are a mirror image of each other. These two similar halves are connected to each other by 29 disulphide bonds (176). Each portion is comprised of three polypeptide chains which exist in pairs known as alpha, beta and gamma (173, 177). The polypeptides chains are encoded by genes present on long arm of chromosome 4(4q 23-32) (176, 177).

The three encoding genes for polypeptide α, β and γ are congregated in a 50Kb region

(178). The sequence of arrangement of polypeptide genes at the distal two-third of chromosome no.4 is γ, α and β. The γ and α genes are transcribed in the same direction while the β gene transcribed in the opposite direction (179). These medial ends (N-

Terminal) of polypeptide chains on either side are converged and connected at the center with E domain. The lateral edges of the chains are connected to a round structure located at the outer edge called the D domain in a twisted coil fashion(180). A detailed X-ray crystallographic focus revealed that the centrally located E domain is further comprised of four domains and each distal globular D region consist of seven domains. All polypeptide chains are distinct from each other (181). The linking of chains to their corresponding domains are in this pattern.

The remaining part of the polypeptide chains that extend beyond the D domain usually serve in polymerization linking (182).

D coiled –coil –E domain-coiled –D domain

Figure 1.19: Schematic composition of the fibrinogen molecule, showing all three polypeptide chains attached to the central E-domain and lateral D-domain (183).

1.14.2 Fibrinogen Gene Regulation

The fibrinogen alpha gene (gene symbol, FGA approved by HUGO Gene Nomenclature

Committee) is a 7.6 kb gene in size and is comprised of 6 exons, whereas the fibrinogen beta (β) gene (gene symbol, FGB) is comprised of an 8 kb region and has 8 exons and fibrinogen gamma (γ) chain (gene symbol, FGG) encompasses an 8.5 kb region and consist of 10 exons (184).

The main regulatory portions of the ﬁbrinogen gene are located in ﬁrst kilo base upstream from the transcription initiation site. All fibrinogen genes have promoter elements including TATA and CAAT boxes and they also expresses regulatory elements like hepatic nuclear factor 1 (HNF-1) present in the promoter region of FGA and FGB genes whereas interleukin 6 (IL-6) sensitive factors as a regulatory factor is present in all three genes of fibrinogen.

Fibrinogen biosynthesis as a result of injury and inflammation is significantly induced by

IL-6 at the transcriptional level. The production of the FGB polypeptide chain is considered as a rate limiting step in assembling and secretion of the fibrinogen protein.

The two contrasting factors including IL-6 receptive factors and bonding site of the

CCAAT box of enhancing binding protein are involved in transcription of FGB. Both of these factors are required for powerful induction by IL-6 (185). Fibrinogen gene transcription takes place in a closely coordinated manner. An upregulation signal, leads to an equivalent increase in transcription for all three genes. The elevation of expression of one gene may increase the transcription of the other two genes.

The circulating concentration of ﬁbrinogen is maintained by brief regulation of its production and degradation. It is still unclear that circulating levels of ﬁbrinogen itself affect its production.

The ﬁbrin degradation products may affect ﬁbrinogen gene transcription by stimulating

IL-6 production. HNF-1 is a transcription factor which mainly regulates the hepatic tissue specific expression of fibrinogen.

The CAAT enhancer binding protein (C/EBP) is also involved in basal expression of

ﬁbrinogen genes.

Lung ﬁbroblastic cell line (A549) has been found to manifest and release fibrinogen on stimulation of pro inﬂammatory cytokine IL-6.

These findings are suggestive of extra hepatic production of fibrinogen in lower amounts

(186).

1.14.3 Fibrinogen conversion to Fibrin

In clotting process the alpha and beta chains of the fibrinogen molecule cleaved at the N- terminal of the polypeptide chains in the E domain by thrombin and converted into a single fibrin thread (187, 188).

The location of breaking of FpA (residues 1–16) is at AαArg16-Gly17 peptide bond, while

FpB (residues 1– 14) cleaved at the BβArg14-Gly15 bond. The cleaved end of fibrinopeptides turns into a positively charged ends that fits into the negatively charged socket of the neighboring fibril for linking.

Figure 1.20: Elaborates the cleavage sites for thrombin as well as plasmin for the process of polymerization of fibrin and fibrinolysis (https://www.haemtech.com). Pm: Plasmin cleavage site, FIIa: thrombin cleavage sites.

FpA is cleaved off more rapidly than FpB, but as polymerization proceeds, the rate of release of FpB increases, suggesting that it is preferentially released from polymers. The N- terminal α-chain motif sequence of Gly-Pro-Arg gets exposed soon after the release of

FPAs (fibrinogen polypeptide alpha).The bare end of the polypeptide of N- terminal is now known as‖ Knob‖ of the fibrinogen Alpha chain . These knobs attaches to the holes known as ―a‖, located in the γ-nodules of another fibrin molecule. This establishes a connection between Knob A: Hole a. This bonding between two molecules of fibrinogen has proven to be very strong and stable (189). Similarly, when FpB got split from beta chain then beta chain attains a new arrangement of peptides at N-terminal as Gly-His-Arg-Pro (GHRP), forming knob called ‗B‘ which is capable of binding to hole ‗b‘ located in the β-nodule.

In this way, many fibrin threads assemble together to produce an insoluble network of fibrin filaments for platelet plug stabilization and formation of fibrin glue (190).

Cleavage of FpA without cleavage of FpB produces clots made up of thinner fibers than those initiated by cleavage of both fibrinopeptides. This suggests that B:b interactions are involved in the lateral aggregation of protofibrils, although lateral aggregation and fiber formation occurs without the cleavage of FpB (191). The cleaved fibrinopeptides A takes part in the linear assemblage of fibrin monomers and fibrinopeptides B contributes in an edgeways arrangement of the meshwork at a slower pace to make more thick fibrils

(192).

Figure 1.21: Fibrin polymerization in stepwise fashion. The figure shows stages from cleaving to release of fibrinopeptides, assemblage of monomers by knob: hole interactions to aggregation and of protofibrils. The oligomers also form lateral branching along with straight arrangements (189).

1.14.4 Functions of Fibrinogen a. Clotting

 Fibrinogen is a protein that acts significantly in the formation of a blood clot.

 Fibrinogen takes part in terminal steps of coagulation as fibrin threads.

 Fibrin filament monomers required for the formation of the hemostatic plug is the

substrate for fibrin clot formation (193). b. Acute Phase Protein

It also plays its role as a primary participant in the acute phase response to injury and stress (194). c. Wound Healing

Fibrinogen plays its role in wound healing (195). d. Fibrinolysis

Fibrin clots constructed as a result of vessel injury needs to be resolved to maintain the flow of blood. Fibrin present in the clot is degraded and digested by activated plasmin. A molecule of fibrin is split in to five parts by plasmin including two Alpha projections, two pieces containing lateral D domains, and the central E domain. A critical balance between clot formation and degradation is closely regulated. A fragile clot may result in a hemorrhage, while a strong one will lead to thrombosis and occlusion of the vessel lumen

(196).

1.14.5 Interactions of Fibrinogen

3.14.5.1Platelet Aggregation

Fibrinogen links platelets to each other via integrin receptors αIIb3 on activated platelets to form an aggregate. The mass of fibrin fibrils and platelets trap the blood cells, helping to increase the mass of the blood clot at the site of damage (197).

1.14.5.2 Other Interactions

Fibrinogen is involved in other intermolecular interactions through its binding to various proteins and cells.

It can bind to several different proteins, including:

 Fibronectin

 Albumin

 Thrombospondin

 von Willebrand Factor

 Fibrulin

 Fibroblast growth factor-2

 Vascular endothelial growth factor

 Interleukin-1

Fibrin binding to these proteins often results in a change to the structure and properties of the blood clot (198).

Clot Stiffness

Platelet Binding Fiber Density

Blood Viscosity Fibrinogen Fibrinolysis

RBC Binding Clot Pore Size

Inflammation

Figure 1.22: Explains the fibrinogen interaction and change of levels in plasma with respect to following mechanisms (199).

1.15 Plasma Fibrinogen Fluctuation

1.15.1 Low Fibrinogen Levels in Plasma

1.15.1.1 Acquired Causes

 Trauma: Excessive blood loss leads to hyperfibrinogenemia (200).

. Drugs

 Urokinase

 Anti- epileptic drugs

 Phenobarbitol.

 Pentoxifylline (201)

 Anabolic steroids (202)

. Illness

 Disseminated intravascular coagulation (203)

 Liver diseases (204, 205)

 Leukemia (206, 207)

1.15.1.2 Genetic Causes

 Congenital Afibrinogenemia (7)

 Congenital Hypofibrinogenemia (208).

 Fibrinogen Storage Disorder (209, 210).

1.15.1.3 High Levels of Fibrinogen

1.15.1.3.1 Acquired Causes

 Age

. Increasing age is shown to have an association with increased levels of

fibrinogen (211).

 Smoking

. Smokers and ex-smokers in a study were shown to have raised levels of

fibrinogen (212).

 Temperatures

. Cold temperatures has an impact on fibrinogen levels in plasma (213).

 Stress

. Stimulation by IL-6 causes more fibrinogen production at molecular levels

(214).

. Too much of stress has been shown to contribute in raising fibrinogen levels

(215).

. High cortisol levels as a result of stress is a known factor for high fibrinogen

levels (216).

 Oral Contraceptive Pills

. Increase fibrinogen levels due to the over expression of the FGG gene as a

result of oral contraceptives use (217).

 Pregnancy

. Fibrinogen levels rises up to threefold higher than normal so as to prevent

excessive bleeding during delivery (218).

. The levels return to normal within four to six weeks.

 Diet

. A high Sugary diet (glycemic) and iron consumption in terms of meat might

increase fibrinogen (219, 220).

 Obesity:

. People with excessive fat deposition have high levels of fibrinogen (221).

1.15.1.3.2 Genetic Causes of High Fibrinogen Plasma levels

. Mutations (222).

. Inherited Homocysteinuria (223).

1.16 Disorders of Fibrinogen

Disorders of fibrinogen are categorized into:

1. Congenital

2. Acquired

1.17 Types of Fibrinogen Deficiencies:

It is further classified in to:

i. Quantitative (type I) Deficiencies

ii. Qualitative defects (type II) Deficiencies (224).

If fibrinogen is deficient in either principle the clotting reaction is blocked prematurely and the blood clot does not form.

The three types of fibrinogen defects are different from each other as they all have different inheritance pattern, prevalence, circulating fibrinogen levels and clinical manifestations (225). Hypodysfibrinogenemia is a combination of manifestation of low levels along with malfunctioning fibrinogen protein in the plasma (226). The comparison of these types is described in table 1.2.

Hypofibrogenemia Afibrogenemia •Low levels of fibrinogen •Complete absence of between 0.2-0.8g/l. Fibrinogen levels, <0.2g/l. •Bleeding pattern mild to •Bleeding pattern is severe. moderate

Hypodysfibrogenemia Dysfibrogenemia Low levels of fibrinogen in Fibrinogen level is plasma along with usually normal (2-4g/l) in defective fibrinogen plasma. protein.

Figure 1.23: Shows the categorization of Fibrinogen disorder.

Table 1.2: Types of Quantitative and qualitative deficiencies of Fibrinogen disorder

Afibrinogenemia Hypo fibrinogenemia Dysfibrinogenemia

Transmission Autosomal recessive Autosomal dominant Autosomal pattern (parents are dominant carriers)

Impact One in one million Less than One in a million Afibrinogenemia

Fibrinogen <0.2 g/L in plasma 0.2 to 0.8 g/L in plasma 2 to 4 g/L in Level plasma

Symptoms -Umbilical bleeding - Umbilical stump No symptoms of bleeding hemorrhage. -Mucocutaneous - Mucocutaneous Thrombosis hemorrhages pattern of bleeding. - Gastrointestinal blood - Gastrointestinal loss hemorrhage - Intra-cranial - Intra-cranial hemorrhage bleeding(infrequent) - joint bleeding

(in <20% of subjects)

Treatment Fibrinogen Fibrinogen Fibrinogen Concentrates Concentrates Concentrates

Comparison between three types of afibrinogenemia (Factor I Deficiency. Canadian Hemophilia Society (CHS).2012)

1.18 Congenital Afibrinogenemia

Congenital afibrinogenemia is an autosomal recessive disorder (227). The prevalence is of this disease is one affected person in one million. The disorder occurs due to either complete absence or a negligible amount of fibrinogen protein in the plasma (228).

Fibrinogen is mainly produced by the liver and plays its major role in coagulation to prevent bleeding. The disorder of autosomal recessive trait presents with unusual clinical manifestations that in majority of cases are reported to be appeared at birth. The disease has shown strong associations with familial marriages in majority of the cases (229) and the disease mechanism affects both genders equally (230).

1.19 Etiology of Congenital Afibrinogenemia

As it is an inherited disorder; the defect lies in the fibrinogen gene (chromosome 4q) (57).

The affected subjects are homozygotes as heterozygotes do not manifest the absence of fibrinogen and usually remain asymptomatic. The different types of mutations present in the fibrinogen gene (alpha, beta and gamma) causes either cessation or production of a mutated protein which results in the absence of fibrinogen protein.

The aftermath of unavailability of fibrinogen in plasma are the bleeding manifestations which usually start their presentation immediately at or after birth and remains lifelong.

The hemorrhagic signs and symptoms shows variability in bleeding patterns. The very first and frequent symptom reported to appear in newborns is umbilical stump bleeding

(231) followed by circumcision bleeding in males, cutaneous hemorrhages, epistaxis and

74 muscle hematoma (232), menorrhagia, postpartum hemorrhages and spontaneous abortion in females (233) and post-surgical hemorrhages.

Several other bleeding presentations also exist is less frequent fashion including gum bleeds, hem arthrosis, gastrointestinal bleeds ,splenic hemorrhages, CNS hemorrhage, bone cyst formations, hemopericardium etc (7, 234, 235).

1.20 Diagnostic Criteria

Establishing the diagnosis of congenital afibrinogenemia:

CAF patients have a history of prolonged and intermittent hemorrhages along with prolonged PT and APTT. The test results of PT and APTT can be normalize in vitro through mixing of patient plasma with normal plasma in a ratio of 1:1, which excludes the presence of an inhibitor. The negligible fibrinogen levels in plasma can be measured with clauss quantitative assay (5, 27).

1.21 Treatment

Processed and virally inactivated fibrinogen concentrates are the treatment of choice for patients with afibrinogenemia. Cryoprecipitate is also an effective alternative and a source rich in fibrinogen, when concentrates are not available. A fibrinogen level at 100 to 150 mg/dl is usually sufficient for normal hemostasis (236).

Indications for prophylactic therapy:

 Surgeries

 Trauma

Diagnostic Workup For Congenital Afibrinogenemia

Coagualtion Comprehensive Screening History Profile

First Line test Specilized test - Sign & Symptoms of Physical bleeding. examination to -Past medical & drug look for hematomas or -CBC history to rule out cutaneous -ProthrombinTime(PT) Fibrinogen acquired causes. manifestations - Activated partial quantitative -Surgical history. Assay(Clauss thromboplastin Method) time(APTT)

Figure 1.24: A flow chart describing the diagnostic work plan for congenital afibrinogenemia

Figure 1.25: Formation of fibrin strands after administration of fibrinogen concentrates. https://www.riastap.com

For the prevention of miscarriage, the levels of fibrinogen should be at or above 60 mg/dl and must be maintained for the entire duration of pregnancy (237).

A single bag of cryoprecipitate contains 250 to 300 mg of fibrinogen and increases plasma fibrinogen level by 10 mg/dl/per bag (http://www.clinlabnavigator.com/cryopreci pitate.html).

There are multiple brands of fibrinogen concentrates currently available on a commercial basis.

1.21.1 Mechanism of Action of Fibrinogen Concentrates

Commercially available concentrates are lyophilized fibrinogen (coagulation Factor I). It is synthesized from human plasma and the route of administration is intravenously.

1.12.2 Complications

Complications of replacement therapy with factor 1 concentrates includes:

 Allergic reactions.

 Transmission of viral diseases.

 Development of antibodies against fibrinogen protein.

 Thrombotic phenomena has also been reported after transfusion of fibrinogen

concentrates which cause ischemia as a result foot lesions and stroke may occur.

The dislodged thrombus as an embolus might get blocked in the pulmonary

circulation. Obstruction of vessels of renal and ovarian veins by thrombus may

cause acute renal failure and infarction, other consequences includes deep venous

thrombosis and oral contraceptives dependent thrombosis (238).

The risk for thrombotic complications is greater with use of cryoprecipitate as it contains other clotting factors. The risk of thrombosis in patients with congenital afibrinogenemia receiving cryoprecipitate transfusion as a supportive therapy might become reduced if they are switched to fibrinogen concentrates. The possible suggested management to deal with thrombotic crises is a concomitant administration of low-dose heparin and compression stocking. If the complication persists then Lepirudin (direct thrombin inhibitor) appears to be effective for such cases (236).

Materials and Methods

General Schematic Presentation of Work Plan

Figure 2.1: Shows multi-step plan of work for Congenital Afibrinogenemia.

The idea of this research project was conceived and created at National Institute of Blood

Diseases and Bone Marrow Transplantation (NIBD), Karachi. Pakistan. The workup plan was designed and lined up diligently in a series of successive steps.

2.1 Contemplation /Survey

Congenital Afibrinogenemia is a rare inherited bleeding disease and to identify the cases of this rare disease, a vigorously comprehensive survey was conducted in hematological setups and hemophilia centers providing facilities to diagnose CAF in all four provinces.

The comprehensive and structured questionnaire was specifically designed for a detailed information regarding patients‘ demographics, medical history including clinical manifestations and physical examination by clinician to gathering all necessarily required details about the disease. Punjab province turned out to be the major contributor in sample collection as it is the mostly densely populated province followed by Sindh. Two patients were included from KP but Balochistan didn‘t had any diagnosed patient of CAF majorly because of law and order situation during last couple of years in Balochistan especially in

Quetta which receives major bulk of patients from all over province, lack of diagnostic facilities in healthcare centers, gender bias social taboo to bring female child for diagnosis and treatment, financial problems.

All hemophilia and hematological centers done the primary hemostatic screening at their respective laboratories and afterwards samples were shared for reconfirmation and extended analysis in reference center NIBD, Karachi.

People with bleeding symptoms

(n=382)

People diagnosed Hemophilia People diagnosed with RIBDs Undiagnosed

(n=40) (n=260) (n=82)

Cases of CAF Other clotting factor deficiencies

CAF CAF CAF

(22 cases) (n=60)

Figure 2.2: Survey scheme for identification and selection of CAF cases.

2.2 Study Design

The study design chosen for the project was descriptive cross sectional as the research study is based on observational analysis along with traverse study for assessment of prevalence of disease condition and frequency of mutations in our local population.

2.2.1 Ethical Guidelines

This study was assented by Ethics and Research Committee of National Institute of

Blood Diseases and Bone Marrow Transplantation (NIBD), Karachi and Liaquat

University of Medical and Health Sciences, Jamshoro.

As stated by declaration of Helsinki 2000, all the recruited patients were briefed about the sample collection, purpose and process of this research study. An easily understandable written consent printed in both English and Urdu was obtained from patients/guardians

(in case of minor) after describing the procedure and outcomes of the study.

2.2.2 Study Coalitions

This study was conducted at National Institute of Blood Diseases and Bone Marrow

Transplantation (NIBD); a specialized treatment and research center for hematological disorders (Benign and Malignant) and Bone Marrow Transplantation, located in the heart of Pakistan i.e. Karachi. This center of par excellence receives the major bulk of patients suffering from inherited bleeding disorders from the interior Sindh Province, Karachi and its suburban areas but also a large toll of patients from all over Pakistan with all types of suspected and diagnosed cases related to hematology and all other inherited diseases like lysosomal storage disorders. The diagnostic laboratory of NIBD is certified by NEQAS

(National External Quality Assessment Scheme, United Kingdom; https://ukneqas. org.uk), RIQAS (Randox International Quality Assessment Scheme; https://www. randox.com). The laboratory professionals follows the guidelines of CLSI (Clinical Lab and Standard Institute; https://clsi.org/).

Due to low awareness regarding the identification and diagnostic criteria for congenital afibrinogenemia not many centers either receive or have diagnostic facilities for such rare disorders. We collaborated with two major working medical institutes with a large turnover of inherited bleeding disorders patients especially from rural areas of Punjab and few close cities in KP province. The children‘s Hospital and Institute of Child Health,

Lahore were operationalized in 1995 and since then this institute caters for a large number of pediatrics cases ,providing the latest medical facilities and excelling in research advancements in parallel. Chughtai‘s Lab (https://chughtailab.com), a certified lab accredited and recognized by The American College of Pathologists, Punjab Health

Commission and Pakistan National accreditation Council has been working since 1983 and provides multidisciplinary facilities other than diagnostic services. It has a largest network of laboratory in Pakistan; densely located in Punjab. In this study, the major data contribution is from interior of the Punjab Province.

Chughtai's Lab,Lahore Children’s Hospital, Lahore (Collaborative Center) (Collaborative Center)

NIBD (Reference Center)

Figure 2.3: Details of Collaborative and Reference center for sample collection.

All three institutes are recognized by the Pakistan Medical and Dental Council (PMDC), College of Physician and Surgeons of Pakistan (CPSP).

2.3 Patient’s Selection

A total of 22 patients were selected for this study. The recruitment criteria were specified for identified diagnosed cases of congenital afibrinogenemia. No age or gender restrictions were applied. The male to female ratio was 1:1.

2.3.1 Exclusion Criteria

The exclusion of cases for this study was done under strict scrutiny following predefined selection requirements.

 Cases of inherited or acquired Hypofibrinogenemia and dysfibrinogenemias were

excluded from the study.

 Acute and chronic liver diseases which leads to clotting factors defects.

 Acquired causes of markedly reduced levels of fibrinogen (including disseminated

intravascular coagulation, severe infections, drugs etc.)

 Subjects that received transfusion of factor concentrates (Factor 1) one week prior

to this study.

 Subjects that received fresh frozen plasma or cryoprecipitate within one week

before recruitment for this study.

 Subjects received whole blood transfusion one week before included in this project.

2.3.2 Sample Size

Due to the rarity of these diseases, all the available patients registered at reference centers and NIBD were included in this study.

The sample size calculation was done on online calculator and found to be 18 (https:// www.surveysystem.com/sscalc.html).

2.3.3 Sampling Technique

A non-probability convenient sampling method was used in the study participants. A maximum possible number of cases was included to enhance the sample size.

2.3.4 Data Inference

A concise form covering demographics, past history, family history, clinical history, physical and clinical examinations and other necessary information details were collected from each subject/guardian (in case of minor).

The focus of information was age of subject at the time of first bleed and bleeding sites along with any complications that developed over the period of time prior to enrolment in the study.

Bleeding scores were calculated on the basis of clinical manifestations of bleeding patterns and correlated with identified mutations .A bleeding assessment tool based on the International Society of Thrombosis and Hemostasis (ISTH) and Tosetto et al was used.

Figure: 2.4: A standard pattern of pedigree for congenital afibrinogenemia

2.3.5 Pedigree Mapping

Pedigree of the study cases were made manually during history taking session, as per provided information by the subject/guardian in case of minor.

The protocols and abbreviations were adapted by Pedigree Standardization Task Force of the National Society of Genetic Counselors (239).

2.3.6 Sample Collection and Storage

 Material Utilized includes:

 Syringes, 3 mL BD Luer-Lok™ Syringe with BD Eclipse™ Safety 25 G x 5/8 in.

Thin Wall Needle, sterile single use, CE marked {BD® Hemogard™ (Becton

Dickinson and Company, Franklin Lakes, New Jersey, United States) {Reference

no.305781}

 EDTA K2 Vacutainer {BD® Hemogard™ (becton dickinson and company,

Franklin Lakes, New Jersey, United States) {Reference #367841}

 Plasma Tubes with 0.109M (3.2%) Buffered Sodium Citrate 2.7mL, 13x75mm,

BD Hemogard™ {Reference #363083}

 Serum 4.0ml, 13x75mm, BD Hemogard™ {Reference # 367812}

 Firstaid band Saniplast®

 Alcohal Swabs (Wuhan Youngsun Medical Import&Export Co.,Ltd,China)

 Torniquet

 Sample collection was carried out by phlebotomist under aseptic conditions

.Samples were drawn in 5 tubes from each patient including two citrate tubes of

2.7ml with 3.2% buffered sodium citrate solution (blue top) were used for

coagulation studies, two spray-coated K2EDTA Tubes (purple top) of 2ml used

for whole blood analysis and one plastic serum tubes (RST) of 5ml having spray-

coated silica (Red Top) were used for serum determinations in biochemistry

profile.

 All the samples drawn with parallel infusion of approximately one unit of FFPs

(250ml) which can raise fibrinogen levels 5-10mg/dl to the patients to avoid any

hemorrhage.

2.4 Sample Preparations

Serum (RST) was separated at 1000gx10 minutes for biochemistry analysis including liver profile (direct and indirect bilirubin levels, alanine transaminase (ALT), alkaline phosphatase (AST) and viral profile (HBsAg, Anti HCV and HIV). EDTA (K2EDTA) was used for complete blood count (CBC) and DNA extraction for PCR amplification and sequencing. Platelet poor plasma(PPP) was collected by centrifugation of citrate tubes at 4000g for 10 minutes .The PPP was removed from citrated tube and preserved in aliquots at -4 in a blast chiller and coagulation profile was performed including PT,

APTT and fibrinogen functional assays using the Clauss method.

2.5 Blood Parameters

Samples were collected in K2EDTA vacutainers and were used to perform CBC.

The parameters of CBC included Hemoglobin (Hb), MCV (Mean Corpuscular Volume),

MCHC (Mean Corpuscular Hemoglobin Concentration), RDW (Red Cell Distribution

Width), platelets count and white blood cell count on an automated hematology analyzer

(Sysmex XE-2100, Japan). It is a multi-parameter analyzer which can assess 32 parameters including WBC 5 parts differential and NRBCs simultaneously for patient‘s anticoagulated blood sample and can run up to 150 samples in one assay. The data is obtained on PC monitor attached with to a Sysmex XE-2100 analyzer through a data processing unit which operates WINDOWS NT functional unit to access result tables and graphs for flow cytometry (Table 2.1).

2.6 Assessment of Coagulation Parameters

2.6.1 Prothrombin Time (PT)

PT is a one stage test which is based on the time for fibrin clot formation on adding tissue factor (thromboplastin). The principle of the test is to assess the activity of clotting factors in the tissue factor pathway and common pathway. PT of patients and normal controls was assessed by taking 200µl of commercially prepared and available calcified thromboplastin named as Neoplastine R 15 (Diagnostic Stago) in glass tubes. The tubes were incubated in water bath at 37ºC (body temperature in vivo) for 2 minutes to activate the interaction of tissue factor thromboplastin in the presence of exogenous phospholipids which replaces platelet surface phospholipids and calcium for re-calcification.

Table 2.1 Principle of Detection Methods for Different Cellular Components of CBC by Sysmex (XE2100)

Parameters Principle of Detection

Red Blood Cells (RBCs) Sheath Flow DC Detection Method

White Blood Cells(WBCs) Flowcytomerty Method Using Semi-Conductor Laser

Hemoglobin SLS Hemoglobin Detection Method

Hematocrit(HCT) RBCs Cumulative Pulse Height Detection Method

MCV Calculation with RBCs and Hematocrit

MCH Calculation with RBCs and Hematocrit

MCHC Calculation with Hemoglobin and Hematocrit

Platelets Sheath Flow DC Detection Method/ Flowcytomerty

Method Using Semi-Conductor Laser

RDW SD & CV Analysis from Size Distribution Of RBCs

Shows the method applied for detection of different cellular components in CBC in an automated analyzer (Sysmex XE 2100)

After two minutes of incubation, 100µl of platelet poor plasma was added to the pre incubated reagent and observed for the initiation of clot formation by the tilt tube method and time was recorded by stop watch. The result was documented in seconds.

2.6.2 Activated Partial Thromboplastin Time (APTT)

APTT is also known as kaolin cephalin clotting time (KCCT). It is used to assess the intrinsic pathway and common pathway clotting defect. 100µl of patient‘s platelet poor plasma along with 100µl of APTT reagent named as cephascreen (Diagnostic Stago) taken in glass tube was incubated at 37ºC for three minutes. After incubation, 100µl of

CaCl2 (Diagnostic Stago) was added. The tilt tube method was used to observe the clot formation in water bath and the time was noted using stop watch. Results were recorded in seconds.

2.6.3 Fibrinogen Assay by Clauss Method

Fibrinogen levels were undertaken as a part of investigation carried out for bleeding disorders and unexplained prolongation of PT and APTT.

A quantitative assessment of fibrinogen in patient plasma was carried out by the Clauss

Method (http://www.practical-haemostasis.com/Screening%20Tests/fibrinogen.html)

The principle of this test is based on the dilution of standard normal plasma with known fibrinogen content. Clotting time is measured after the addition of thrombin. Clotting time is proportional to the concentration of fibrinogen and the 1/10th dilution is taken to represent the value in the standard preparation. A calibration curve is constructed with clotting time against fibrinogen concentration.

APTT

Figure 2.5: A comprehensive procedure demonstrating the basic principle of both PT and APTT test (https://www.perfusion.com/circuit_surfer_posts/hemostasis-assays/ )

20µl sample was diluted in 380µl of Owren Koller buffer (Diagnostic Stago). 200µl of diluted plasma was incubated at 37ºC for two minutes. 100 µl of reagent named as

Fibriprest (Diagnostic Stago) was added in pre incubated sample. Time was noted at 50% clot formation. Results were expressed in g/L.

2.7 DNA Extraction

Genomic DNA was extracted using QIAamp Blood DNA Mini Kit® manufactured by

Qiagen (Qiagen Ltd.Hilden Germany). In the process, peripheral blood leukocytes are lysed to release the DNA which binds to the spin column. The residual contaminants are washed away and the purified DNA is eluted out. The following procedure, as per manufacturer‘s guidelines were adopted.

2.8 PCR and Gene Sequencing

Regions of FGA, FGB and FGG were amplified by exon-specific polymerase chain reaction (PCR) using 50-100 ng (nano gram) of genomic DNA. PCR was performed in a

25ul final volume.

Figure 2.6: DNA Extraction Procedure by Spin column Method

EDTA preserved whole blood was thawed.

20μl QIAGEN Proteinase K solution was pipetted into a 1.5ml micro centrifuge tube.

A volume of 200μl blood and 200μl AL buffer (A lysis buffer) were added to the tube.

The mixture was then incubated at 56°C for 10 minutes.

To remove drops from inner aspect of the lid, the tubes were briefly spun.

A volume of 200μl 98% ethanol was the added to the mixture; followed by vortexing.

The mixture in tube (620μl) was then pipetted into a QIAamp Mini Spin column.

Centrifugation at 6000g (8000 rpm) for 1 minute followed.

The Spin Column was then transferred into a 2ml collection tube; the filtrate containing used collection tube was discarded.

This was followed by addition of 500μl AW1 buffer (wash buffer 1) to the QIAamp Spin Column without wetting the rim Centrifugation at 6000g (8000 rpm) for one minute followed.

The Spin Column was once again transferred to another collection tube (2ml) and the old tube containing filtrate was discarded.

500μl AW2 buffer (wash buffer 2) was carefully pipetted to the column.

The tube was then centrifuged at 20,000g (14,000 rpm) for 3 minutes.

The spin column was now transferred to a 1.5ml micro centrifuge tube and the old collection tube was discarded.

The QIAamp spin column was opened and 100μl AE (any elution) buffer was carefully added to it.

The tube was then incubated at room temperature for one minute.

DNA was eluted by centrifuging the tube at 8,000 rpm for 1 minute.

Yield of extracted DNA was taken.

The DNA so extracted was processed immediately or it was stored at <20°C till further analysis.

Table 2.2 The components of PCR reaction mix

Master Mix Components Quantity (×1)

1× PCR Buffer 22.6µl

Primer Mix F+R 2.1µl

Dream Taq Polymerase (Fermantas, Pittsburgh, USA) 0.2µl

Total 24µl

100ng/µL DNA or H2O blank 1µl

Total Volume 25µl

Table 2.2: Enlisted components of PCR reaction mix in a volume of 25ul.

This batch is mixed in a vortex mixer the reaction collected at the bottom of the vessel by short centrifugation all the rest was discarded.

Table 2.3 Composition of master mix

No. of Primer Forward/Reverse (Premix) Taq-Polymerase 1× PCR Buffer Samples 20pM/µl 5U/µl

1 22µl 1.2µl 0.2µl

22 484µl 26.4µl 4.4µl

Calculations of composition of master mix are according to study subjects. For each sample to be analyzed the following reagents are mixed in a correspondingly large sample vessel, briefly mixed and collected by centrifugation.

Table 2.4 Quantitation of 1X PCR buffer

1× PCR Buffer Components Quantity

10× buffer 5ml

dATP 100µl

dGTP 90µl

7-Deaza 10µl

dCTP 100µl

dTTP 100µl

50mM MgCl2-Lsg. 1.5 ml

Table 2.4: Shows quantity of 1x PCR buffer. Deoxyguanosine triphosphate (dGTP),

Deoxyadenosine triphosphate (dATP), Deoxycytidine triphosphate (dCTP), Deoxythymidine triphosphate (dCTP), Magnesium chloride (MgCl2), mM =milli molar The 1× Buffer was aliquoted in quantity of 2ml in Eppendorf vessel and stored at -20°Celsius

100

2.9 Primers for Fibrinogen Alpha, Beta and Gamma Genes

FGα Gene

s.# Exon Primer Sequence Annealing Temperature

1 FGα 1F CAG CCC CAC CCT TAG AAA 57°Celcius

2 FGα1R AGA TCT TCC TCT TGG TGT CA

3 FGα2F ACA TTG CTG TTG CTC TCT TT 57°Celcius

4 FGα2R CAT CAG AGG GAA GGA ATC TC

5 FGα3F CAT GGA AAC ATG CAA AAT TA 57°Celcius

6 FGα3R TGG GAT GAA ATT GTC ATA GA

7 FGα4F TGC TGG CAA TTA CAG ACA AA 57°Celcius

8 FGα4R GGG ACC ACA GCC ACA TAC TT

9 FGα5F TGA AAC CAG TTT CCA GAA GGA 57°Celcius

10 FGα5R CTG CCC CAT GTC TGT TAA TG

11 FGα5.1F CCA GTT CCA GAC TTG GTT CC 57°Celcius

12 FGα5.1R CCG GTA CTA CCA GGT CTA GGG

13 FGα5.2F TGG AAG TAC TGG AAG CTG GAA 57°Celcius

14 FGα5.2R TGG TTG TGC TAC CAG AGG TG

15 FGα5.3F GGA GAT AAA GAG CTC AGG ACT GG 57°Celcius

16 FGα5.3R ATT TAC CAC GGG AAG GGA AT

17 FGα5.4F AAG GAA TCC AGT TCT CAT CA 57°Celcius

18 FGα5.4R GGG AAC ACT GTG CAG AAA TA

101

FGβ Gene

S.# Exons Primer Sequence Annealing Temperatures

1 FGβ 1F TTC CTA GCA GAG GAC TCA GA 57°Celcius

2 FGβ 1R GAG GTT AAC AAT TCC ATT TCA

3 FGβ 2F AAA TGA GGG TGT TGG AAT AG 57°Celcius

4 FGβ 2R CAC CAC TGC AAG AAA CAT C

5 FGβ 3F CCA AAT CCT TCA TCT AAT GC 57°Celcius

6 FGβ 3R GAA TGG AAA GCA CAA GGA TA

7 FGβ 4F GTT TTA TGA GGC AAA AAT GC 57°Celcius

8 FGβ 4R TTG GTG TGT GAG TTC TTC TG

9 FGβ 5F TGT CAT AAA CCC CTG AAC AT 57°Celcius

10 FGβ 5R AAT ACA GGT ATG AGC CAC CA

11 FGβ 6F TGA CAT TAT TTG CTG TTG GT 57°Celcius

12 FGβ 6R TAA AAC AGG CTT CCA ACA AT

13 FGβ 7F GCA GTT TTT AGT TTC CCA AA 57°Celcius

14 FGβ 7R TTT AAA GCA GGA GCA AAG AG

15 FGβ 8F GGG TAA TCT GCA AAA CGT AA 57°Celcius

16 FGβ 8R TTG TCA CAT ACA GAA GAG CAA

102

FGγ S.# Exon Primer Sequence Annealing Temperatures

1 FGγ1F TGG GAA CCT GAC AGT ATA GG 57°Celcius

2 FGγ1R GCA GCA GTT GTC TCT GGT A

3 FGγ2F AGC AGT AAG TGT GCT CTT CA 57°Celcius

4 FGγ2R AAA GTT ACA AGT GCC AGA TGA

5 FGγ3F GCA TCT GTC TAC ATT TTT AAT CA 57°Celcius

6 FGγ3R CAG CAA AAG AAC TTC ACA GA

7 FGγ4F TCT GTG AAG TTC TTT TGC TG 57°Celcius

8 FGγ4R TAA ATC AGT CTT GCA GAG CA

9 FGγ5F TGA AAG GAA TAC TTA TTT TTG TCT T 57°Celcius

10 FGγ5R TGG GTA GCC ACT TTC TAA AC

11 FGγ6F TGC ATT TCA AAC CAC AGT AA 57°Celcius

12 FGγ6R GGT TCA CAA GGT GCT TAG AA

13 FGγ7F ATT TTC TCC TTT TGC TCT TG 57°Celcius

14 FGγ7R TTG GAA GTC ATT TCA AAA CA

15 FGγ8F TCA GCA TGT GAT GGT TGT AT 57°Celcius

16 FGγ8R CTT GGA ATC TAA GAA AGG AAA A

17 FGγ9F TGC AGA GGT AAA AAG ATT CC 57°Celcius

18 FGγ9R CAA ATC ATC CTC AGG GTA AA

19 FGγ10F TGT CTT TTT AAT ATG GTT TTT GTT TTG 57°Celcius

20 FGγ10R TGG CAA AAA GTG GTG GTT TT

103

2.10 Primers Reconstitution

Dissolved primers stock (100pM/µl) were stored at 20°Celsius .Dilutions are prepared from the stock solution (20pM) in 1.5ml tubes. This process was carried out collectively for all exons to be processed in each gene. 1X buffer, 1 U Dream Taq-Polymerase

(Fermantas, Pittsburgh, USA in Dream Taq buffer (Fermantas), primer concentrations of

200 mmol/l for forward and reverse and 200μM dNTPs (deoxyribonucleotide triphosphate). Annealing temperatures (Ta) were separately optimized for each pair of primers.

The qualitative detection of PCR products is carried out by 1% agarose gel and detection of DNA bands visualized by ethidium bromide fluorescence under gel documentation system on Alpha Imager® Mini (Protein Simple, San Jose, California, 95134 US).Direct gene sequencing was performed on both strands using the Big Dye Terminator v3.1

Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) according to manufacturer‘s protocol. Amplified fragments were purified by ethanol/sodium acetate precipitation and run on ABI PRISM® 3100 Genetic Analyzer (Applied Biosystems). It works on the principle of multi-color fluorescence-based DNA analysis system with 16 capillaries operating in parallel. Any sequence variants were sought on HGMD (Human

Gene Mutation Database http://www.hgmd.cf.ac.uk/ac/index.php & www.geht.org ) and

Seattle SNPs (http://pga.gs.washington.edu/).

104

Figure 2.7: Procedure of PCR product and reconstituted primer mix. (mixhttps://www.mediala binc.net/pcr_fundamentals.aspx)

105

The samples were transferred in to thermal cycler and the PCR is carried out according to the following scheme:

30sec

Figure 2.8: Steps of PCR in thermal cycler

106

DNA was denatured at 95 °C for 5 min

28 cycles of Holding at denaturation 10 °C (95°C for 30s

PCR

Primer 5 min final annealing extension at temperature 72°C (Ta) mostly 58°C for 30s Extension (72 °C for 30s

Figure 2.9: Programmed PCR cycles ran on 96-Well Fast Thermal Cycler of ABI Veriti™ (The Applied Biosystems® Veriti®, Catalog number: 4375305)

107

Figure 2.10: Diagrammatic presentation of entire process of PCR. (https://www.bosterbio.com/protocol-and-troubleshooting/molecular-biology-principle-pcr )

108

2.11 Biocomputing Analysis (Bioinformatics)

The results of sequencing were matched with The Human Gene Mutation Database

(HGMD®, www.hgmd.cf.ac.uk ) for reported and novel mutations.

The chromatogram was read through Color Fasta Format which represents a text-based format for nucleotide sequences/peptide sequences, in which nucleotides or amino acids are represented using single alphabet letter codes

2.12 Pathogenicity Scoring

Stability is an important characteristic which influences the function, activity and modulation of a protein molecule. For the assessment for protein structure stability and disease causing property, five pathogenicity scoring soft wares were applied.

2.12.1 MUpro (predictions of protein stability changes upon mutations)

MUpro is a software program (accessed on 20th April 2015) to predict the status of protein stability formed as a result of single nucleotide mutation (240).

2.12.2 Provean (Protein Variation Effect Analyzer) version 1.1, (accessed on 20th April

2015) (241) works via computing delta alignment for each supporting sequence (a cluster of closely related 30 cluster sequences). A predefined threshold is (-2.5). Provean score below this level for the protein variant is predicted to have a "deleterious" effect and above the threshold, the variant is predicted to have a "neutral" effect (241).new ref added

109

2.12.3 PolyPhen-2(polymorphism phenotyping v2)

The first computational tool called Polyphen-2 (accessed on 20th April 2015), works on naïve Bayes classifier rule, calculate and predict the functional impact of deleterious mutation.

This tool classifies the description of impact due to single nucleotide substitution in two formats:

i. Neutral

ii. Deleterious

Scores between 0 - 1 on numerical scale, labelled as neutral or have no effect on protein functional properties.

The term deleterious is used for those SNPs change which rate more than 1 on scoring scale of polyphen-2.

PolyPhen-2 (http://genetics.bwh.havard.edu) is an online upgraded tool of Polyphen software. It identifies the impact of single nucleotide change on protein structure stability and label result by comparing them with existed quick access data of previously calculated predictions through a database called WHESS.db (whole human exome sequence space data base; http://genetics.bwh.harvard.edu/pph2/dbsearch.shtml). This database contains all the annotations of single nucleotide change (SNPs) which are non- synonymous/missense in nature (242).

110

2.12.4 SIFT (Sorting Intolerant from Tolerant) (accessed 20th April 2015). It is a computational algorithms which predicts whether an amino acid substitution will affect protein function based on sequence homology and the physical properties of amino acids

(243). A SIFT score of less than 0.05 is predicted to be deleterious. A substitution with a score greater than or equal to 0.05 is predicted to be tolerated (http://www. exeterlaboratory. com/ molecular-genetics/).

2.12.5 SNP&GO (Single nucleotide polymorphism and GO) (accessed on 20th April

2015) (244).

Protein accession numbers were provided by Uniprot (Universal Protein Resource, http://www.uniprot.org/) and wild type color fasta sequence was first accessed

(http://pga.gs.washington.edu/data/fga/ on 27th January 2015 and later on 20th April

2015.

2.13 Molecular Modeling

Mutations in protein coding genetic sequence lead to partial or complete damage in either structure or function or both.

For the assessment of protein structural deformity and functional abnormality, two software‘s were utilized.

2.13.1 I-TASSER (Iterative Threading ASSEmbly Refinement)

It is a stratified approach to protein structure and function prediction. It first identifies structural templates from the PDB by multiple threading approach LOMETS, with full-

111 length atomic models constructed by iterative template-based fragment assembly simulations. Function insights of the target are then derived by threading the 3D models through protein function database BioLiP (Biologically relevant Ligand Protein).

The six novel missense mutations from this study were first evaluated for structural and functional assessment. The affected regions were modeled on I-TASSER (Iterative

Threading Assembly Refinement) threading modeling server (http:// zhanglab.ccmb. med.umich.edu/I-TASSER/; accessed on 12th November 2014). The protein structure of encoded protein previously available in databases were accessed and downloaded through

Protein Data Base file (PDB) (http://rcsb.org/pdb/ home/home. do;) accessed 20th

November 2014). All structural analysis and image rendering were performed with

YASARA (Yet another Scientific Artificial Reality Application) version 12.8.6

(www.yasara.org/).

112

113

All selected patients were previously diagnosed cases of congenital afibrinogenemia.

Figure 3.1 Schematic representation of accessibility and collection of data from subjects included in study

114

3.1 Demographic Details

The data set of this study included patients from different origins in Pakistan and the assessed frequency of majorly affected ethnic group has also been mentioned here on the basis of number of cases identified from each province (Figure 3.2).

Kashmir KP 2 patients

Kashmir: No cases were found due to difficult access and lack of diagnostic facilities.

Balochistan: Sindh No sample found due to 6 patients Law and Order situation and limited diagnostic facilities only available in few big cities of province.

Figure 3.2 Pictorial representation of frequency of cases contributed from each province of Pakistan.

Majority of cases were detected and included in this study from Punjab province.

Although it is the second largest province geographically but considered as the largest province in terms of thick population and have maximum numbers of developed cities.

Balochistan is a largest province with respect to geographical area but with very less population density and comes at fourth position on the basis of population figures. This

115 province had no identified cases of CAF reported in literature.

Sindh is the 3rd largest in size and second in population strength after Punjab. This province contributed six new cases from two ethnic groups those were from metropolitan city Karachi and interior Sindh.

Khyber Pakhtunkhwa (KP) province ;the fourth largest province in size and 3rd largest in population density had given two cases reported from different ethnic groups including one patient from Pathan and another from Hazarawal community that lives in northeastern part of KP province.

3.2 Patient’s Description

Total 22 diagnosed patients of CAF were selected with equal male to female ratio (1:1).

Mean age was ±11.3 years and SD 9.84 (https://www.calculator.net/standard-deviation- calculator.html). Out of 11 female patients, only two females were in reproductive age group and the rest were under age for menarche.

All subjects were unrelated proband with exceptions as two pairs of siblings were present in data subjects including two brothers and two sisters. One patient had more than one mutations in two different genes.

3.3 Baseline Screening Results and Phenotypic Manifestations

The initial i.e. CBC was performed at the respective collection centers and samples were sent to main center NIBD, Karachi along for sample tubes for further analysis. All the CBC results were with in normal ranges including platelet counts.

116

The baseline screening to reconfirm the diagnosis of congenital afibrinogenemia was performed at main center (NIBD), Karachi. These investigations included Prothrombin time (PT) and APTT (activated partial thromboplastin time). The results for PT and

APTT were found to be markedly prolonged in all samples along with circulating fibrinogen availability in plasma ranging from complete absence of negligible levels.

The status of patients in terms of consanguinity was also mentioned along with relationship. All patients were proband and unrelated to each other except two pairs of siblings in this data set.

The detailed history aided extensively in categorization and grading of bleeding manifestations with the help of bleeding score (245) thereby calculating the phenotype expression in study patients. The bleeding scores were calculated for the evaluation of bleeding pattern and extent of severity which was later correlated with the identified mutations.

The frequency of symptoms was assessed to set an impression for most common symptoms to be expressed in Pakistani congenital afibrinogenemia patients. The results have shown well that skin bleeding in form of petechial hemorrhages and bruises are found to be the most happening occurrence followed by bleeding or hematoma formation as a result of minor trauma .Umbilical bleeding was proved to be the first symptom appeared in majority at birth. Post circumcision bleeding after umbilical bleed at birth was remained to be the most evident finding in male data subset. Menorrhagia was noted in two females of grown age. The exact frequency of this particular symptom is still in question as majority of females in our study subject were below the age of menarche.

117

Table 3.1 Summarization the findings of Baseline screening tests in all selected study patients

Activated Fibrinogen Thrombin Prothrombin partial Bleeding Interfamilial Ethnic IP# Level Time Time (PT) thromboplastin Consanguinity Score Relation Origin (g/l) (Sec) (Sec) Time (APTT) (Sec) Urdu C1 0.01 23 >120 >180 20 positive Unrelated Speaking C2 0.02 24 >120 >180 21 positive Unrelated Punjabi C3 0 33 >120 >180 22 positive Unrelated Punjabi Urdu C4 0.1 24 >120 >180 17 positive Unrelated Speaking C5 0.02 31 >120 >180 20 positive Unrelated Sindhi Urdu C6 0.01 25 >120 >180 20 positive Unrelated speaking C7 0.02 29 >120 >180 22 positive Unrelated Sindhi C8 0.0 30 >120 >180 20 positive Unrelated Sindhi C9 0.0 32 >120 >180 22 positive Unrelated Punjabi C10 0.01 25 >120 >180 16 positive Unrelated Punjabi C11A 0.02 28 >120 >180 18 positive Punjabi ** C11B 0.01 24 >120 >180 16 positive Punjabi C12 0.0 30 >120 >180 21 positive Unrelated Punjabi C13 0.01 24 >120 >180 20 positive Unrelated Punjabi C14 0.0 26 >120 >180 21 positive Unrelated Punjabi C15A 0.02 24 >120 >180 20 positive Punjabi ** C15B 0.01 25 >120 >180 21 positive Punjabi C16 0.1 26 >120 >180 16 Positive Unrelated Punjabi C17 0.2 23 >120 >180 13 Positive Unrelated Punjabi C18 0.01 21 >120 >180 18 Positive Unrelated Pathan C19 0.1 22 >120 >180 16 Positive Unrelated Hazara C20 0.2 >120 >180 20 Positive Unrelated Saraiki

Describe comprehensive results of baseline coagulation profile including TT with prolonged duration followed by PT and APTT giving infinite range in seconds followed by quantitative assay results of circulating levels of fibrinogen in patients‘ plasma. The bleeding scores were calculated with the help of ISTH BAT and Tosetto et al (245). The ratio of consanguinity was noted cent percent in our set of data along with interfamilial relationship description for each patient along with ethnicity profile seconds (sec), **siblings.

118

Intracranial Bleed 1

Hemarthrosis 1

Menorrhagia in Females 2

Muscle Hematoma 10

Gum Bleed 8

Minor Trauma 20

Post Circumcisional Bleed in male 9

Cutaneous Bleed 20

Epistaxis 16

Clinical expression of bleeding in CF patients in CF bleedingof expression Clinical Umbilical Bleeding 22

0 5 10 15 20 25

Total Number of patients of congenital afibrinogenemia

Figure 3.3 Frequency of bleeding symptoms in congenital afibrinogenemia patients

119

Inline of regular manifestation of bleeding symptoms excluding umbilical bleed that occurs only at birth ;epistaxis was reported to be the third commonest symptom and also more frequent presentation at hospital for management of bleeding.

Small aged subjects under 5years were reported to develop hematomas due to crawling

(toddlers) or during play which was usually managed at home unless the size of hematoma isn‘t increasing or anatomical location is not dangerous. More commonly involved sites observed in our set of patients were appendicular body parts like upper and lower limbs. Gum bleeds were seen in some cases but mainly secondary to minor trauma during chewing or getting hurt by tooth brush. Hem arthrosis; a common finding factor deficiencies was a rare finding in this study. The most hazardous and rare symptom found was intracranial hemorrhage. No other rare manifestations like bone cyst or spleen rupture was observed. Gastrointestinal bleeding was also a rare finding in our set of data.

Phenotype determination for congenital afibrinogenemia is a real challenge .The clinical manifestations in CAF mimics with other clotting factor deficiencies and overlap in such a close manner that causes difficulty in differentiation with other rare inherited bleeding disorders.

The scoring of hemorrhagic symptoms was done for the assessment of severities in terms of numeric data which aided in sorting of patients on the basis of bleeding diathesis. The bleeding severity then correlated with identified mutations to establish a link between existing DNA defect and its presentation for future reference.

120

3.4 Genetic Analysis Results

In 22 study patients, 18 had mutations in different genes. Total 18 point mutations were identified in all three genes of fibrinogen (FGA, FGB and FGG), out of that 11 novel variants were found in all three genes (Table 3.2)

In FGA gene, total 13 mutations were identified. Amongst 13 variants, seven novel variants were present and two variants out of these seven were same but in two different unrelated proband. The rest of the six mutations in FGA were reported. The four reported mutations were similar in four unrelated proband.

In FGB, two novel and two reported variants were identified. As mentioned earlier in

FGA, FGB gene also had two reported similar genetic variant that were found in two sisters in contrast to mutations in FGA found in unrelated proband. The FGG gene had two similar novel mutations in two sisters. and the rest four patients didn‘t show any mutation .The occurrence of mutations were manifested in an interesting manner as there were two pairs of siblings having similar defects in same genes and there were two unrelated proband who had compound mutations with one novel and one reported and one subject had mutation in two different genes.

121

Figure 3.4 Pattern of identification of genetic variants in fibrinogen gene

122

Table 3.2 Genotypic expression of mutations in fibrinogen gene (FGA, FGB & FGG)

Amino Acid IP # Gene Exon Mutation Zygosity Mutation type change

C1 1 c.24C>A p.Cys8* Homozygous Nonsense (53)

p.Lys(AAA) Compound Novel C2 2 c.143_144 del AA Frame shift 48Arg fs9* Heterozygous

p.Thr 283Arg Compound Novel C3 5 c.846delG Frame shift fs138* Heterozygous

4 c.385C>T p.Arg129* Homozygous Nonsense (58)

C4 4 c.385 C>T p.Arg129* Homozygous Nonsense (58)

C5 4 c.385C>T p.Arg129* Homozygous Nonsense (28) FGA C6 4 c.385C>T p.Arg129* Homozygous Nonsense (28, 29)

C7 5 c.598C>T p.Gln200* Homozygous Nonsense (53)

C8 5 c.904C>G p.Pro302Ala Homozygous Missense Novel

C9 5 c.913A>G p.Thr 305 Ala Homozygous Missense Novel

C10 5 c.992A>G p.Thr331Ala Homozygous Missense Novel

C11i 5 c.992A>G p.Thr331Ala Homozygous Missense Novel

C12 5 c.973A>G p.Ser325Gly Homozygous Missense Novel

C13A 2 c.141C >T p.Arg47* Homozygous Nonsense (246)

C13B 2 c.141C>T p.Arg47* Homozygous Nonsense (246)

C14i FGB 8 c.1294T>A p.Trp 432Arg Homozygous Missense Novel

c.118_124 Dup C15 2 TTCTTCA Homozygous Frame shift TTCTTCA Novel

C16A 4 c.361A>T p.Lys121* Homozygous Nonsense Novel FGG C16B 4 c.361A>T p.Lys121* Homozygous Nonsense Novel

123

3.4.1 FGA Genetic Variants

The maximum number of variants were found in FGA (fibrinogen alpha gene). The most frequently involved exon in FGA gene was exon 5 as seven mutations were located in this particular exon but the most frequent type of existing point mutation was missense variant.

In FGA, three types of point mutations were observed in this study data. Though nonsense mutation constituted the larger portion with six reported defects identified in this gene but missense mutation stand out with five novel mutations. Out of five missense variants, one variant c.992A>G; p.Thr331Ala was similar in two unrelated proband. This particular variant was located in the coiled coil region of FGA gene. Four nonsense mutations were found in exon 4 followed by one in exon 1 and the other in exon 5.

Two frameshift mutations in FGA were found and both were novel and compound heterozygous. There was a deletion of a single nucleotide Guanine leading to the synthesis of error in DNA reading frame as the triplet codon are very important in transcription and translation of protein but this deletion caused the formation of defective protein manifesting malfunction. There was deletion of pair of adenine nucleotide forming lysine amino acid but due to deletion of these nucleotides an incomplete reading frame leads to formation of incorrect protein.

In FGA two mutations are found in one patient but in different exon. A Novel frameshift mutation in exon 5 along with nonsense mutation in exon 4. The identification of these two in same gene was later correlated with phenotypic manifestation for their functional impact, which was severe as intracranial bleed was noted in that patients.

124

3.4.2 FGB genetic Variants

The fibrinogen beta gene (FGB) was the second commonly affected gene in this set of patients with the sum of four mutations were seen in different exons of FGB gene but the frequently affected exon was exon no.2 as it showed two reported nonsense mutation in sibling and one novel frameshift mutation in a proband. One novel missense was also identified in exon 8 of FGB.

The reported mutation c.141C >T; p.Arg47X is an early truncating nonsense mutation numbered from the initiator ATG. This variant was predicted to cause the production of a severely truncated β chain or it might cause a defect in the FGB mRNA, by aberrant splicing (nonsense-associated alternative splicing) or by affecting the stability of the mRNA through nonsense-mediated mRNA decay. Whatever the mechanism involved, there is no reasonable doubt that this is a null mutation.

The frameshift mutation identified was a duplication or addition of seven nucleotides in one reading frame from 5‘ to 3‘. This duplication might result in synthesis of functionally deficient protein with some structural instability.

3.4.3 FGG genetic Variants

FGG gene appeared to be less affected amongst sequenced data. Two mutations identified which were novel homozygous nonsense mutation in a sibling. Both were located in exon 4.

125

Figure 3.5 Schematic representation of Fibrinogen gene with identified genetic variants. Exons represented by boxes and line portion demonstrate intron region of FGA, FGB and FGG gene of fibrinogen. (Figure reproduced, with permission, from Marguerite Neerman-Arbez, Seminars in Thrombosis and Hemostasis 2013;39:585–595.) (247).

126

3.5 Pathogenecity Scoring Results

For the assessment of novel genetic variants identified in proband, we utilized five different In Silico tools. There were six novel missense mutations with single substitution present and were evaluated for protein structure stability and functional impacts of non- synonymous single nucleotide change in protein sequence.

The online computational prediction tools used included:

 Polyphen-2

 MUpro

 SIFT

 Provean

 SNP & GO

 All six bioinformatics tools for prediction calculation analyzed the missense

mutations detected in six proband.

 Starting with Polyphen-2 (http://genetics.bwh.havard.edu ; accessed on 20th April

2015), five out of six missense single substitutions were neutral with

approximated normal functional protein according to polyphen-2 prediction

software.

127

The calculated score of six novel missense mutations are documented in Table 3.3.

Table 3.3 Pathogenicity Score of Missense Mutations

Softwares Polyphen- 2 Provean MUpro SNP&GO Sift

Missense Score prediction Score prediction SVM Protein structure Score Prediction Score Prediction Mutations score stability p.Pro302Ala 0.028 Benign -4.257 Deleterious -0.797 Decrease stability (0.4) Neutral 0.00 Benign p.Thr 305Ala 0.00 Benign -0.387 Neutral 0.134 Increase stability (0.05) Neutral 0.00 Benign p.Thr331Ala 0.025 Benign -1.100 Neutral 0.122 Increase stability (0.03) Neutral 0.00 Benign p.Thr331Ala 0.025 Benign -1.100 Neutral 0.122 Increase stability (0.03) Neutral 0.00 Benign p.Ser325Gly 0.014 Benign -2.331 Neutral -0.063 Decrease stability (0.1) Neutral 0.00 Benign p.Trp432Arg 1.00 Damaging -12.18 Deleterious -0.411 Decrease stability (0.8) Disease Na Na

Pathogenicity of missense mutations was calculated by five different software to check for the protein structure stability and deleterious effects. Na =not available

128

All the results documented on HumDiv Beysian probability model of polyphen-2.

Only one missense defect in FGB gene was found to have damaging function in polyphen

-2 software.

Provean prediction tool showed two mutations as deleterious. One located in FGA and formerly calculated by Polyphen-2 as neutral and the other was identified in FGB and labelled as damaging by polyphen-2

Sift and SNP&GO declared all neutral in nature when calculated for score. The prediction for missense mutation Tro432Arg wasn‘t calculated due to some error in software after repeated attempts so labelled as not available (Na).

MUpro manifested slight variation as it demonstrated similar results as Polyphen-2 and

Provean did but it score one new mutation as deleterious which p.ser325gly.

3.6 Molecular Modeling Analysis

Another significant bioinformatics tool was applied for the analysis of possible alteration in molecular structure of affected protein.

Among the six reported novel missense mutations from this study, four mutations were located in an area of the alpha chain that has no resolved crystal/NMR-based structure

(Nuclear magnetic Resonance). Thus, to assess the putative structural effect of these mutations, we modeled this region on the I-TASSER (Iterative Threading ASSEmbly

Refinement) threading modeling server (http://zhanglab.ccmb.med.umich.edu/I-

TASSER/; accessed on 12th November 2014). The model for this region was then joined

129 to the remaining beta chain for which the structure has already been determined and submitted in the protein structure database (PDB file ID: 3GHG; 2.9 Å resolution).

Model joining was performed by replacing the last two amino acid residues common to the model and the crystal structure (PDB file ID: 3GHG; chain A) (PDB: Protein Data

Base, ID: Identity, 3ghg is a 4-character unique identifier of every entry in the Protein

Data Bank) downloaded from the protein structure database (http://rcsb.org/pdb/ home/home.do;) accessed 20th November 2014) to maintain the dihedral angles for the full model at the point of joining the same. The complete model was refined by a short solvated simulation lasting 500 ps as described in Krieger et al, 2004 (Force field:

Yamber3, periodic boundary conditions, temperature: 298K, water density: 0.997 g/l, pH:

7.4). The local neighborhood of the wild type residue corresponding to the reported mutation was investigated to establish a logical hypothesis for the effect of the mutation.

An additional one missense mutations (p.Trp432Arg) in the beta chain lies on the structurally resolved region of the PDB file 3GHG; chain B). Similarly the local molecular environment for this wild-type residue was also inspected. All structural analysis and image rendering were performed with YASARA (Yet Another Scientific

Artificial Reality Application) version 12.8.6

Structural analysis of novel missense mutations using molecular modeling

3.6.1 Alpha chain missense mutations

All four novel missense mutations from the α-chain reported in this study were present in a region (residues 220-860) of the α-chain, which had no resolved/known crystal

130 structure. The region surrounding the reported mutations (residues 300-400) was relatively poorly conserved with most of it missing from some fibrinogen homologues.

Among the mutated residues, p.Pro302 was present in all homologues, which contained this part. The p.Ser325 residue was also conserved in all homologues with the exception of Musmusculus, where it was substituted by an Asn. The two Thr residues, p.Thr302 and p.Thr331, were relatively variable and substituted by Ser or Asp in a few homologues.

Only in the homologue from Canis lupus familiaris was one of the Thr residues

(p.Thr302) observed to be substituted by an Ala residue, which has been reported as a mutated residue for both Thr residues in our study. Modeling of this region showed that this region could be split into two central cores, each of which is organized as a beta sheet surrounded by flexible coils (Figure 3.6).

The two cores are connected by a central long helix. The first core, apart from being surrounded by flexible coils, also contains a few short helices. Three of the four reported mutated residues were located on these short helices with the exception that p.Ser325 is located on a short loop connecting one of the short helices to the central core. The residues p.Pro302, p.Thr305 and p.Thr331 are partially buried, with the p.Pro302 and p.Thr305 side chains oriented toward the central core beta sheets. The residues p.Pro302 and p.Thr305 participate in intra-helical hydrogen bonds with each other and with p.Ser399 and p.Arg308, respectively. The residue p.Thr331 lies at the edge of a short helix and also participates in intra-helical hydrogen bonding (p.Gly327). Interestingly, within the fold on which all four of these mutations reside, lysine residue p.Lys322 is known to be cross-linked to α-2 antiplasmin proteins and a glutamine residue, p.Gln347, which participates in inter-chain cross-links during clot formation.

131

Figure 3.6 Molecular modelling of novel missense variants identified in FGA gene of fibrinogen

132

3.6.2 Beta chain missense mutations

The one novel missense mutations (p.Trp432Arg) reported in the chain occurs in a highly conserved region (Figure 3.7).

The residues are completely conserved in homologues that have been used for the present alignment. The p.Trp432 residue lies completely in the densely packed hydrophobic core of the C-terminal region of chain. This densely packed hydrophobic core consists of a number of other aromatic acids, which are in close proximity to p.Trp432 (p.His400 and p.His438, p.Trp433 and p.Tyr434). The p.Trp432 residue hydrogen bond contacts with p.Tyr434 and p.Ser406.

133

Figure 3.7 Molecular modelling of novel missense variant in FGB gene of fibrinogen

134

An Investment in Knowledge Pays the Best Interest

Benjamin Franklin

135

Rare Inherited Coagulation Disorders (RICDs) by far have succeeded to grab the attention of researchers. The expanding spectrum of bleeding disorders has really come in lime light in the past few years. One of these disorders is the fibrinogen deficiency (factor

I). The congenital afibrinogenemia (CAF) is one of the moieties of fibrinogen defects in which there is a complete absence of fibrinogen in the plasma due to the presence of genetic variants in either of the three fibrinogen genes (FGA, FGB, and FGG).

Congenital fibrinogenemia is a rare inherited bleeding disorder with a vast variability in the phenotypic manifestations. Recently an effort was made to design a comprehensive and standardized reference table of clinical manifestations that were gathered from the extensive data of four international rare inherited bleeding disorder registries. All four registries developed a consensus on the symptoms that were found to be more frequent in patients of CAF.

According to this standard table of reference for CAF symptoms, umbilical stump bleeding at birth has now been proven and accepted to be the first and foremost bleeding manifestation and a hallmark for congenital afibrinogenemia. Majority of other small studies and individual cases reported in the literature apart from these large registries had reported the similar fact that the bleeding usually starts at birth or in the neonatal period with 85% of cases manifesting umbilical stump bleeding as a first clue. This study too has endorsed the above mentioned finding as the 21 patients (95.5%) had suffered prolonged umbilical stump bleeding and that was mentioned in their history.

The early bleeding events other than umbilical bleeding at birth included the development of hematomas due to traumatic delivery, spontaneous intracranial hemorrhage in neonate

136 or antenatal intracranial bleeding are still not very common to find there place in the international reference table but they are reported as solo events in research studies.

A case of antenatal intracranial bleeding at 29th week of gestation was reported in literature and the baby was diagnosed with congenital afibrinogenemia after complete laboratory test evaluation and correlation with symptomatic presentation. There was no visible external bleeding at birth (5). These rare early bleeding events except umbilical bleeding were absent in the patients of this study. The second frequently occurring symptom in this study were the spontaneous skin bruises (including point bruises and petechiae) that were either resolved spontaneously or in case of petechial hemorrhage or ecchymosis, FFPs transfusion aided in the resolution of this symptom. A very recent case on congenital afibrinogenemia, reported in literature from turkey stated the details of a female who was diagnosed with congenital afibrinogenemia at the age of 25 years and she was also 24 weeks pregnant. She had an unmarked and uneventful time throughout her life till she developed severe ecchymosis all over her body and went for a checkup where she got admitted for evaluation and treatment. Her PT and APTT was prolonged along with complete absence of fibrinogen in her plasma (Turkish Journal of Family

Medicine & Primary Care 2015;9 (4):188-190 doi: 10.5455/jfmpc.193401). This case not only contributed as an exception to the conventional presentation for CAF in terms of age and bleeding symptom but somehow supports the narrative of unusual and variable deflection of manifestations in congenital afibrinogenemia. Bleeding from minor trauma was also noted as a significant presentation and it was the third frequent bleeding symptom. A local study also documented the history of hematomas in two patients of

CAF after minor falls (6) followed by epistaxis, hematoma formation, gum bleed and

137 occasionally bleeding in lower limb joints (knee joints) in this study. Though epistaxis was not included as common event but it is a frequent presentation in CAF (248, 249) and also reported in a current study from Iran that epistaxis is the next frequent bleeding diathesis after umbilical stump bleeding that can be severely uncontrolled (250). A contradiction in few symptoms regarding frequency and severity may present in comparison to this study and international research data as it has been observed that the regions with high incidence of familial wedlock trends possess the most common existing bleeding patterns with more severity and probably similar mutation and the other parts of the world where the scenario is slight different. The rarest manifestations like intracranial bleeding was observed in one subject of this cohort, who had a novel compound heterozygous frameshift mutation in exon 5 of FGA gene and the other nonsense homozygous reported variant in exon 4 of same gene. It has been proposed that combination of these two mutation existing in same gene might led to the aggravated severity in the disease manifestation as previously reported variant in exon 4 i.e.

Arg129X was suggested to escape the nonsense mediated decay (NMD) pathway (28).

The intracranial bleeding was treated with cryoprecipitate and no complications in neurologic functions had been observed lately.

The congenital afibrinogenemia may also presented with gastrointestinal bleeding like hematemesis or occasional melena. These symptoms were absent in this cohort. Although an international study reported melena in one Pakistani patient and nonspecific gastrointestinal symptom in another out of nine patients of similar origin. The same study elaborated the phenotype of six female patients out of nine study subjects and only one female had menorrhagia and the remaining female subjects had bruises, nosebleeds and

138 bleeding after trauma were more common and the umbilical bleeding history was found in all patients (7).

In our set of female patients only two women index patients are in reproductive age group and were shown to have menorrhagia and there was a positive history of abortion in one female. The remaining nine female subjects were not reached the menarche yet.

Therefore, a close follow up and more female patients will be needed to study in large cohorts for the collection of a detailed information regarding frequency of the gynaecological symptoms in Pakistani adult women suffering from CAF.

Other than menorrhagia; menometrorrhagia, first-trimester abortions, ante-partum and postpartum hemorrhage, hemoperitoneum after corpus luteum rupture have also been reported in relevant research studies (20). Affected females of CAF generally conceive in a normal manner with successful implantation of embryo in uterus but they might suffer from spontaneous vaginal bleeding or miscarriage during the first trimester (usually 6 to

8weeks). The increasing incidence of spontaneous abortion has been markedly reduced with the transfusion of fibrinogen concentrates or cryoprecipitate to replenish the low to negligible levels of fibrinogen for maintaining an uneventful pregnancy and a successful delivery.

It has been recommended in literature to maintain higher levels of fibrinogen during gestational period due to its rapid clearance from plasma. Two independent events of thrombosis in gonadal and renal blood vessel within 24hours of receiving high dose of cryoprecipitate along with placental abruption and a retrochorionic hematoma formation

139 in a pregnant female during her first trimester with prophylaxis fibrinogen infusion has been reported (237).

Although rare but patients of congenital afibrinogenemia also seems to be peculiarly susceptible to develop spontaneous rupture of the spleen and a bone cyst formation (234,

251).

The most typical symptom, common to all rare inherited bleeding disorders, is the occurrence of excessive bleeding at the time of invasive procedures such as circumcision and dental extractions. Bleeding from mucosal tracts (particularly epistaxis and menorrhagia) is also a frequent feature; afibrinogenemia is sometimes associated with thrombotic episodes, thought to be triggered by thrombin-induced platelet aggregation in vivo due to the absence of the thrombin-neutralizing properties of fibrin.

Other bleeding manifestations included hemarthrosis that was found in only one patient of CAF in this research data. This finding is not very common in CAF then in

Hemophilia patients.

One of the local studies conducted in Pakistan reported three cases of congenital afibrinogenemia including two males and one female. Major presenting symptoms reported were severe bleeding from umbilical stump and hematoma formation at injection site. All three cases show history of consanguinity in their parents‘ marriage with no previous event of bleeding in the families. Two more families with diagnosed cases of congenital afibrinogenemia were also reported in province of Punjab with 5 out of 6 children in one family and one male child in other was affected. Both manifested approximately same phenotypic expression with history of spontaneous mucosal bleeding

140 in the form of repeated epistaxis and gum bleeding, prolonged bleeding after mild trauma and ecchymosis all over the body. There was no history of joint or muscle bleeding.

Parents in both families had consanguineous marriages and were asymptomatic. Another case of Pakistani afibrinogenemia patient was studied, a 3-year-old Pakistani child born from a consanguineous marriage with no bleeding complication occurred at birth, but after 3 weeks, the child presented with intracranial bleeding (subdural hematoma and ventricular hemorrhage) Plasma fibrinogen levels were immeasurable by clotting and immunologic assays in the proband at the time of the first diagnosis . The parents had reduced fibrinogen levels in plasma.

By far all the data reported by either local studies or contributed in international literature indicates the vast deflection in clinical symptoms, Due to the rarity of the disorder, the available data on the incidence of bleeding episodes, prevalent clinical manifestations and treatment modalities is scarce. Collectively the symptomatology is not so very different in our local setup with higher frequency of umbilical stump bleeding along with cutaneous bleeding (bruises and Petechae). Due to the rarity of disease and lack of diagnostic facilities and awareness about the disease except for large cities; a large chunk of undiagnosed patients still exists.

The major dataset contributed in this study of congenital afibrinogenemia was novel and not existed in literature previously. We also found some pre-existing defects in global literature like c.385C>T;p.Arg129*(59) in two unrelated proband. This homozygous nonsense mutation was located in exon 4 of FGA gene. One patient was an adult female and she had umbilical bleed at birth as a first symptom and now she reported menorrhagia and cutaneous manifestation followed by on & off episodes of epistaxis as

141 the most happening events to takes place. The other was male who had other mutation in same gene but different exon and manifested more severe bleeding complications.

This mutation resulted in the early truncation of protein synthesis as the stop codon appeared early during transcription by mRNA leading to the formation of a nonfunctional protein.

This mutation was previously reported by a study of Iranian patients and later same homozygous nonsense mutation was identified in three Pakistan patients in a collaborative study representing multinational data of congenital afibrinogenemia .All three patients were females with more or less similar bleeding manifestations like umbilical cord bleed, skin bruises and epistaxis but only one female was shown to have menorrhagia while the remaining two didn‘t as they hadn‘t reached the menarche yet.

Temporarily, this also creates laps in collecting evidential data for menorrhagia to establish as the frequently occurring symptom in our local population of affected females as most of them are in pediatric age group.

Frameshift mutations were found to be the less frequent defect as compared to nonsense or missense mutations. The deletion of single guanine nucleotide in c.846del G caused the truncation of protein synthesis which led to the formation of nonfunctional protein product. Same patients also had mutation in FGA exon 4 which was a nonsense mutant again resulted in early termination of protein coding. Now these patients had severe phenotypic manifestation of bleeding symptoms including intracranial hemorrhage.as he had more than one defect in FGA gene and loss of protein.

142

The second frameshift mutation was also a deletion of two adenine nucleotides but the phenotypic manifestations wasn‘t that catastrophic as compared to the first frameshift results.

Missense mutations were found to be the major part of novel mutations in FGA and also a novel finding in FGB. Three missense mutations (Pro302Ala, Thr305Ala and Thr331Ala) in the alpha chain reside on short helices surrounding a central beta sheet core. All these mutations are non-conservative in nature, i.e., the Pro302Ala substitution results in the replacement of a rigid imino group with a smaller, more flexible residue, and the

Thr305Ala and Thr302Ala substitutions result in the replacement of polar side chains by smaller but hydrophobic side chains. In addition, the introduction of alanine in these regions will most likely disrupt some of the intra-helical hydrogen bonds, thereby breaking the helical structure surrounding the central core. Because these short helices provide order and stability around an otherwise disordered coiled-coil region, their disruption might result in a loss of stability for this region and the alpha chain. The third mutation in this chain, Ser325Gly, is also non-conservative, i.e., it results in the substitution of a polar residue to a very small and flexible Gly residue. Because the wild- type residue already lies on the flexible loop, the introduction of a small residue will make this region more disordered and therefore unstable. Moreover, because all four missense mutations belong to a fold of the alpha chain that might be interacting with

Factor XIII (this fold also contains the Lys and Gln residues that participate in interchain cross linking and cross linking to alpha 2- antiplasmin), conformational changes induced by these mutations on this fold might interfere with the interaction of fibrinogen alpha chain with Factor XIII. The one beta chain missense mutation resides on a highly

143 conserved region of the beta chain, most likely because many of the residues of this region contribute to the stability of its densely packed hydrophobic core. The p.Trp432Arg substitution occurs in the middle of the hydrophobic core. The introduction of a large polar, positively charged residue instead of a hydrophobic aromatic one would destabilize the hydrophobic core of this region. Thus, the mutation affects the stability of the beta chain by disrupting its C-terminal hydrophobic core.

144

Conclusion

Great the art of beginning but greater is the art of ending…

Henry Wadsworth Longfellow

145

The study concluded that inherited afibrinogenemia is a prevalent disorder in Pakistan and now gradually losing its status of rarity. In past, most of the cases were misdiagnosed as hemophilia due to the overlapping clinical symptoms and lack of awareness regarding this disorder.

The collective work in this study presented the larger section of mutations identified as novel gene defect followed by reported mutations published earlier. The detailed analysis on diversity in phenotypic manifestations varying from patient to patients and its correlation with impact of mutation was also a highlight .The suspected hotspot of mutation was also identified in FGA gene. The identified mutation data will set a mark for a comparison of mutations that will be reported in future, specifically from our region.

The clinical manifestations of congenital afibrinogenemia in our local population are more or less same as reported in global literature. Few exceptions do exists as some of the rare complications like bone cyst, splenic rupture or joint deformity weren‘t found in this dataset. This data may contribute in establishing a standard table of symptoms for the cases of Pakistani origin in order of their frequencies to make the diagnosis of isolated factor I deficiency or congenital afibrinogenemia more convenient clinically.

146

Future Prospects

147

 Spreading awareness regarding this disorder through workshops, seminars, local

conferences of hematology, webinars and free availability of national and

international literature.

 Getting access to the far flung areas of all provinces in Pakistan to brief and train

the local practitioners for diagnosing and early management of this disorder.

 Exploration and Identification of new cases via extended coordination with

clinicians and hospitals across the country.

 Setting up a separate registry for inherited fibrinogen defects including congenital

afibrinogenemia, hypofibrinogenemia and fibrinogen storage disease to reduce the

chances of misdiagnosis for hemophilia and gathering a comparatively larger data

for research and study. It will help further in finding the facts regarding disease

phenotypes and genotypes.

 Establishing the carrier detection facility country wide to dig out maximum

possible no. of affected parents and siblings and will prevent the occurrence of

new cases.

 Genetic counsellors are needed to handle these cases and counsel them to

discourage the consanguineous marriages if there carrier status gets confirm. It

will surely bring down the rapid raising graph of this particular disease in the

country.

148

 Making the easy availability of fibrinogen concentrates and observing the effects

of fibrinogen concentrates in patients of congenital afibrinogenemia and to look

for any reaction of development of antibody against the concentrates.

 Gene therapy is the most advance and ultimate solution to this problem.

Designing a vector specifically according to our local genetic makeup and after

successful integration into patients DNA followed by observing the impact and

complications will be the next most interesting and crucial task to be achieved.

149

Limitations of the Study

There are no limitations to mind, except for those we acknowledge…

Napoleon Hill

150

Here are some temporary but significant situations which halted us to get more data and its processing.

Misdiagnosis of congenital afibrinogenemia for hemophilia restricted to get an

actual number of existing cases.

Lack of awareness in small cities, towns and villages regarding this disorder also

contributed as a major limiting factor in diagnosis.

Lack of diagnostic facilities at primary and secondary health centers in suburban

and rural areas of the country, even in some tertiary care centers the diagnostic

practice for congenital afibrinogenemia is not common.

Social taboo was also a hurdle especially if the affected person is a female child or

adult. They usually avoid to give consent for the study in trying to hide the issue.

Mostly people refuse to cooperate for study if counselling is given regarding

discoursing cousin marriages due to tribal, community or panchayat system.

Some religious issues also causes trouble as people don‘t agree for counselling of

no having children born with disease.

Accessibility to the distant areas was also restricted due to security risk and

deteriorating safety scenarios in last couple of years Pakistan faced.

Limited funds to reach out for advanced diagnostic modalities like sequencing

wasn‘t an easy task to fulfill.

151

152

Bibliography

1. Rabe F SE. Ueber-faserstoffmangel im Blute bei einem Falle von Ha mophilie. Arch Intern Med 1920;95:2-14.

2. Le Quellec S, Desjonqueres A, Rugeri L, Clavel HD, Farhat F, Mechtouff L, et al. Combined life-threatening thromboses and hemorrhages in a patient with afibrinogenemia and antithrombin deficiency. Thrombosis journal. 2018;16(1):6.

3. Atram S, Mundada SD. Management of a Case of Congenital Afibrinogenemia for Extradural Hematoma Evacuation with Fresh Frozen Plasma. Indian Journal of Clinical Anaesthesia. 2015;2(3):180-1.

4. Agarwal M, Sanzgiri P, Bhanotra P, Rao S, Mehta B, Shah M. Congenital disorders of fibrinogen. Journal of postgraduate medicine. 1981;27(3):178.

5. Hariharan G, Ramachandran S, Parapurath R. Congenital Afibrinogenemia presenting as antenatal intracranial bleed: a case report. Italian journal of pediatrics. 2010;36(1):1.

6. Malaquin S, Rebibo L, Chivot C, Badoux L, Mahjoub Y, Dupont H. Congenital afibrinogenemia: a case report of a spontaneous hepatic hematoma. Medicine. 2016;95(28).

7. Al‐Mondhiry H, Ehmann WC. Congenital afibrinogenemia. American journal of hematology. 1994;46(4):343-7.

8. Yatish S, Benakappa A, Mallesh K. AFIBRINOGENEMIA IN A YOUNG INFANT. JOURNAL OF THE IAP KARNATAKA STATE BRANCH.27.

9. Rahman M, Begum M. A case of hypofibrinogenemia presenting with submental hamatoma. Bangabandhu Sheikh Mujib Medical University Journal. 2014;7(1):65-7.

153

10. PRICHARD RW, VANN RL. Congenital afibrinogenemia: report on a child without fibrinogen and review of the literature. AMA American journal of diseases of children. 1954;88(6):703-10.

11. Fernando P, Dharmasena B. A case of congenital afibrinogenemia. Blood. 1957;5:474-9.

12. Grossman BJ, Carter RE. Congenital afibrinogenemia: Report on a newborn infant without fibrinogen. The Journal of pediatrics. 1957;50(6):708-13.

13. Yamagata S, Mori K, Kayaba T, Hiratsuka I, Kitamura T, Ishimori A, et al. A case of congenital afibrinogenemia and review of reported cases in Japan. The Tohoku Journal of Experimental Medicine. 1968;96(1):15-35.

14. Jain K, Parande A. Congenital afibrinogenemia (a case report). The Journal of the Association of Physicians of India. 1962;10:741-4.

15. Khatua S. CONGENITAL AFIBRINOGENAEMIA. Journal of the Indian Medical Association. 1964;42:183.

16. Basu A. Fibrinogen deficiency. Bull Cal Sch Trop Med. 1966;14:146.

17. Dube B, Agarwal S, Gupta M, Chawla S. Congenital deficiency of fibrinogen in two sisters. Acta haematologica. 1970;43(2):120-7.

18. Girolami A, Venturelli R, Bareggi G. A report of a case of congenital afibrinogenemia. Blut. 1972;24(1):23-31.

19. Peyvandi F, Mannucci P. Rare coagulation disorders. Thrombosis and haemostasis. 1999;82(10):1207-14.

20. Acharya S, Dimichele D. Rare inherited disorders of fibrinogen. Haemophilia. 2008;14(6):1151-8.

154

21. Chandan G, Annaji A, Bhatnagar S, Mohandas U, Dave P. Cellulitis on face in a patient with congenital afibrinogenemia. Journal of Indian Society of Pedodontics and Preventive Dentistry. 2011;29(1):46.

22. Acharya S, Coughlin A, Dimichele DM, Group NARBDS. Rare Bleeding Disorder Registry: deficiencies of factors II, V, VII, X, XIII, fibrinogen and dysfibrinogenemias. Journal of Thrombosis and Haemostasis. 2004;2(2):248-56.

23. Rezende S, Pinheiro K, Caram C, Genovez G, Barca D. Registry of inherited coagulopathies in Brazil: first report. Haemophilia. 2009;15(1):142-9.

24. Neerman-Arbez M, Honsberger A, Antonarakis SE, Morris MA. Deletion of the fibrogen alpha-chain gene (FGA) causes congenital afibrogenemia. The Journal of clinical investigation. 1999;103(2):215-8.

25. Neerman-Arbez M, De Moerloose P, Bridel C, Honsberger A, Scho nbo rner A, Rossier C, et al. Mutations in the fibrinogen Aα gene account for the majority of cases of congenital afibrinogenemia. Blood, The Journal of the American Society of Hematology. 2000;96(1):149-52.

26. Neerman-Arbez M, De Moerloose P, Honsberger A, Parlier G, Arnuti B, Biron C, et al. Molecular analysis of the fibrinogen gene cluster in 16 patients with congenital afibrinogenemia: novel truncating mutations in the FGA and FGG genes. Human genetics. 2001;108(3):237-40.

27. Peyvandi F. Epidemiology and treatment of congenital fibrinogen deficiency. Thrombosis research. 2012;130:S7-S11.

28. Asselta R, Duga S, Tenchini ML. The molecular basis of quantitative fibrinogen disorders. Journal of Thrombosis and Haemostasis. 2006;4(10):2115-29.

29. Neerman‐Arbez M, De Moerloose P. Mutations in the fibrinogen gene cluster accounting for congenital afibrinogenemia: an update and report of 10 novel mutations. Human mutation. 2007;28(6):540-53.

155

30. Casini A, Lukowski S, Quintard VL, Crutu A, Zak M, Regazzoni S, et al. FGB mutations leading to congenital quantitative fibrinogen deficiencies: an update and report of four novel mutations. Thrombosis research. 2014;133(5):868-74.

31. Mannucci PM. Thrombosis and bleeding disorders outside Western countries. Journal of Thrombosis and Haemostasis. 2007;5:68-72.

32. Dorgalaleh A, Alavi SER, Tabibian S, Soori S, Moradi Eh, Bamedi T, et al. Diagnosis, clinical manifestations and management of rare bleeding disorders in Iran. Hematology. 2017;22(4):224-30.

33. Karimi M, Haghpanah S, Amirhakimi A, Afrasiabi A, Dehbozorgian J, Nasirabady S. Spectrum of inherited bleeding disorders in southern Iran, before and after the establishment of comprehensive coagulation laboratory. Blood coagulation & fibrinolysis. 2009;20(8):642-5.

34. Mansouritorghabeh H, Manavifar L, Banihashem A, Modaresi A, Shirdel A, Shahroudian M, et al. An investigation of the spectrum of common and rare inherited coagulation disorders in North-Eastern Iran. Blood Transfusion. 2013;11(2):233.

35. Abdul-Karim ET, Mohammed SF. Study of clinical characteristics, presentation, and complications among patients with congenital coagulation disorders. Saudi Med J. 2010;31(3):299-303.

36. Wahab MA, De Moerloose P, Fish RJ, Neerman-Arbez M. Identification and functional characterization of a novel nonsense mutation in FGA accounting for congenital afibrinogenemia in six Egyptian patients. Blood coagulation & fibrinolysis. 2010;21(2):164-7.

37. Mokhtar GM, Tantawy AA, Adly AA, Telbany MA, El Arab SE, Ismail M. A longitudinal prospective study of bleeding diathesis in Egyptian pediatric patients: single-center experience. Blood coagulation & fibrinolysis. 2012;23(5):411-8.

156

38. Abdelwahab M, Khaddah N. Rare coagulation disorders: a study of 70 cases in the Egyptian population. Haemophilia. 2012;5(18):e386-e8.

39. Eid SS, Kamal NR, Shubeilat TS, Wael A-GM. Inherited bleeding disorders: a 14-year retrospective study. Clinical Laboratory Science. 2008;21(4):210.

40. Levrat E, Aboukhamis I, De Moerloose P, Farho J, Chamaa S, Reber G, et al. A novel frameshift mutation in FGA (c. 1846 del A) leading to congenital afibrinogenemia in a consanguineous Syrian family. Blood coagulation & fibrinolysis. 2011;22(2):148-50.

41. Balter M. India‘s Fragmented Society Was Once a Melting Pot. 2013.

42. Amri Y, Toumi NEH, Fredj SH, de Moerloose P. Congenital afibrinogenemia: Identification and characterization of two novel homozygous fibrinogen Aα and Bβ chain mutations in two Tunisian families. Thrombosis research. 2016;143:11- 6.

43. Simurda T, Zolkova J, Snahnicanova Z, Loderer D, Skornova I, Sokol J, et al. Identification of two novel fibrinogen Bβ chain mutations in two Slovak families with quantitative fibrinogen disorders. International journal of molecular sciences. 2018;19(1):100.

44. Gupta M, Bhattacharyya M, Choudhry V, Saxena R. Spectrum of inherited bleeding disorders in Indians. Clinical and applied thrombosis/hemostasis. 2005;11(3):325-30.

45. Sumitha E, Jayandharan G, Arora N, Abraham A, David S, Devi G, et al. Molecular basis of quantitative fibrinogen disorders in 27 patients from I ndia. Haemophilia. 2013;19(4):611-8.

46. Shetty S, Shelar T, Mirgal D, Nawadkar V, Pinto P, Shabhag S, et al. Rare coagulation factor deficiencies: a countrywide screening data from India. Haemophilia. 2014;20(4):575-81.

157

47. Ahmed P, Mahmood A, Azim W. Congenital afibrinogenaemia in two families. JOURNAL-PAKISTAN MEDICAL ASSOCIATION. 2001;51(4):160-2.

48. Anwar M, Iqbal H, Gul M, Saeed N, Ayyub M. Congenital afibrinogenemia: report of three cases. Journal of Thrombosis and Haemostasis. 2005;3(2):407-9.

49. Khalid S, Bilwani F, Adil SN, Khurshid M. Frequency and clinical spectrum of rare inherited coagulopathies--a tricenter study. Journal of the Pakistan Medical Association. 2008;58(8):441.

50. Borhany M, Shamsi T, Naz A, Khan A, Parveen K, Ansari S, et al. Congenital bleeding disorders in Karachi, Pakistan. Clinical and applied thrombosis/hemostasis. 2011;17(6):E131-E7.

51. Naziar K, Ahmed S, Kamran S, Anwar J. Frequency of bleeding disorders diagnosed at Armed Forces Institute of Pathology Rawalpindi. Pakistan Armed Forces Medical Journal. 2011;61(3).

52. Borhany M, Shamsi T, Fatima N, Fatima H, Naz A, Patel H. Rare bleeding disorders are not so rare in Pakistan. Journal of Hematology & Thromboembolic Diseases. 2013.

53. Asselta R, Platè M, Robusto M, Borhany M, Guella I, Soldà G, et al. Clinical and molecular characterisation of 21 patients affected by quantitative fibrinogen deficiency. Thrombosis and haemostasis. 2015;113(03):567-76.

54. Naz A, Jamal MY, Amanat S, Najmuddin A, Patel H, Raziq F, et al. Autosomal recessive inherited bleeding disorders in Pakistan: a cross-sectional study from selected regions. Orphanet journal of rare diseases. 2017;12(1):66.

55. Palla R, Peyvandi F, Shapiro AD. Rare bleeding disorders: diagnosis and treatment. Blood, The Journal of the American Society of Hematology. 2015;125(13):2052-61.

158

56. Hussain S, Baloch S, Parvin A, Najmuddin A, Musheer F, Junaid M, et al. Inherited Bleeding Disorders—Experience of a Not-for-Profit Organization in Pakistan. Clinical and applied thrombosis/hemostasis. 2018;24(8):1241-8.

57. Neerman-Arbez M, Casini A. Clinical consequences and molecular bases of low fibrinogen levels. International journal of molecular sciences. 2018;19(1):192.

58. Asselta R, Duga S, Spena S, Santagostino E, Peyvandi F, Piseddu G, et al. Congenital afibrinogenemia: mutations leading to premature termination codons in fibrinogen Aα-chain gene are not associated with the decay of the mutant mRNAs. Blood, The Journal of the American Society of Hematology. 2001;98(13):3685-92.

59. Monaldini L, Asselta R, Duga S, Peyvandi F, Karimi M, Malcovati M, et al. Mutational screening of six afibrinogenemic patients: Identification and characterization of four novel molecular defects. Thrombosis and haemostasis. 2007;97(04):546-51.

60. Smith N, Bornikova L, Noetzli L, Guglielmone H, Minoldo S, Backos DS, et al. Identification and characterization of novel mutations implicated in congenital fibrinogen disorders. Research and Practice in Thrombosis and Haemostasis. 2018;2(4):800-11.

61. Robert-Ebadi H, De Moerloose P, El Khorassani M, El Khattab M, Neerman- Arbez M. A novel frameshift mutation in FGA accounting for congenital afibrinogenemia predicted to encode an aberrant peptide terminating 158 amino acids downstream. Blood coagulation & fibrinolysis. 2009;20(5):385-7.

62. Vu D, Bolton-Maggs PH, Parr JR, Morris MA, De Moerloose P, Neerman-Arbez M. Congenital afibrinogenemia: identification and expression of a missense mutation in FGB impairing fibrinogen secretion. Blood. 2003;102(13):4413-5.

63. Neerman-Arbez M, Vu D, Abu-Libdeh B, Bouchardy I, Morris MA. Prenatal diagnosis for congenital afibrinogenemia caused by a novel nonsense mutation in

159

the FGB gene in a Palestinian family. Blood, The Journal of the American Society of Hematology. 2003;101(9):3492-4.

64. Fang Y, Wang H-L, Wang X-F, Fu Q-H, Wang W-B, Xie S, et al. Inherited afibrinogenemia caused by compound heterozygous mutations in the beta beta- chain of fibrinogen. Zhongguo shi yan xue ye xue za zhi. 2005;13(6):1086-9.

65. Duga S, Asselta R, Santagostino E, Zeinali S, Simonic T, Malcovati M, et al. Missense mutations in the human β fibrinogen gene cause congenital afibrinogenemia by impairing fibrinogen secretion. Blood, The Journal of the American Society of Hematology. 2000;95(4):1336-41.

66. Spena S, Asselta R, Duga S, Malcovati M, Peyvandi F, Mannucci PM, et al. Congenital afibrinogenemia: intracellular retention of fibrinogen due to a novel W437G mutation in the fibrinogen Bβ-chain gene. Biochimica et Biophysica Acta (BBA)-Molecular Basis of Disease. 2003;1639(2):87-94.

67. Spena S, Duga S, Asselta R, Malcovati M, Peyvandi F, Tenchini ML. Congenital afibrinogenemia: first identification of splicing mutations in the fibrinogen Bβ- chain gene causing activation of cryptic splice sites. Blood. 2002;100(13):4478- 84.

68. Attanasio C, De Moerloose P, Antonarakis SE, Morris MA, Neerman-Arbez M. Activation of multiple cryptic donor splice sites by the common congenital afibrinogenemia mutation, FGA IVS4+ 1 G→ T. Blood, The Journal of the American Society of Hematology. 2001;97(6):1879-81.

69. Dear A, Daly J, Brennan S, Tuckfield A, George P. An intronic mutation within FGB (IVS1+ 2076 a→ g) is associated with afibrinogenemia and recurrent transient ischemic attacks. Journal of Thrombosis and Haemostasis. 2006;4(2):471-2.

160

70. Asselta R, Duga S, Spena S, Peyvandi F, Castaman G, Malcovati M, et al. Missense or splicing mutation? The case of a fibrinogen Bβ-chain mutation causing severe hypofibrinogenemia. Blood. 2004;103(8):3051-4.

71. Xu X, Zhou R, Wu J, Fang Y, Wang X, Zhai Z, et al. Fibrinogen beta chain gene mutation contributes to one congenital afibrinogenemia. Zhonghua xue ye xue za zhi= Zhonghua xueyexue zazhi. 2005;26(3):137-9.

72. Moerloose M, editor. Fibrinogen Martin: a novel mutation in FGB (Gln180Stop) causing congenital afibrinogenemia. Seminars in Thrombosis & Hemostasis; 2016.

73. Asselta R, Duga S, Simonic T, Malcovati M, Santagostino E, Giangrande PL, et al. Afibrinogenemia: first identification of a splicing mutation in the fibrinogen gamma chain gene leading to a major gamma chain truncation. Blood, The Journal of the American Society of Hematology. 2000;96(7):2496-500.

74. Margaglione M, Santacroce R, Colaizzo D, Seripa D, Vecchione G, Lupone MR, et al. A G-to-A mutation in IVS-3 of the human gamma fibrinogen gene causing afibrinogenemia due to abnormal RNA splicing. Blood, The Journal of the American Society of Hematology. 2000;96(7):2501-5.

75. Iida H, Ishii E, Nakahara M, Urata M, Wakiyama M, Kurihara M, et al. A case of congenital afibrinogenemia: fibrinogen Hakata, a novel nonsense mutation of the fibrinogen γ-chain gene. Thrombosis and haemostasis. 2000;84(07):49-53.

76. Neerman-Arbez M, Germanos-Haddad M, Tzanidakis K, Vu D, Deutsch S, David A, et al. Expression and analysis of a split premature termination codon in FGG responsible for congenital afibrinogenemia: escape from RNA surveillance mechanisms in transfected cells. Blood. 2004;104(12):3618-23.

77. Gale AJ. Continuing education course# 2: current understanding of hemostasis. Toxicologic pathology. 2011;39(1):273-80.

161

78. Andrew M, Paes B, Milner R, Johnston M, Mitchell L, Tollefsen DM, et al. Development of the human coagulation system in the full-term infant. 1987.

79. Gibson B. Neonatal haemostasis. Archives of disease in childhood. 1989;64(4 Spec No):503.

80. Manco-Johnson M, Nuss R. Hemostasis in the Neonate. Neoreviews. 2000;1(10):e191-e5.

81. Pichler E, Pichler L. The neonatal coagulation system and the vitamin K deficiency bleeding–a mini review. Wiener Medizinische Wochenschrift. 2008;158(13-14):385-95.

82. Cade J, Hirsh J, Martin M. Placental barrier to coagulation factors: its relevance to the coagulation defect at birth and to haemorrhage in the newborn. Br Med J. 1969;2(5652):281-3.

83. Manco-Johnson MJ. Development of hemostasis in the fetus. Thrombosis research. 2005;115:55-63.

84. Hassan H, Leonardi A, Chelucci C, Mattia G, Macioce G, Guerriero R, et al. Blood coagulation factors in human embryonic-fetal development: preferential expression of the FVII/tissue factor pathway. 1990.

85. Alberts B, Johnson A, Lewis J, Raff M, Roberts K, Walter P. Blood vessels and endothelial cells. Molecular Biology of the Cell 4th edition: Garland Science; 2002.

86. Sandoo A, van Zanten JJV, Metsios GS, Carroll D, Kitas GD. The endothelium and its role in regulating vascular tone. The open cardiovascular medicine journal. 2010;4:302.

87. Mitchell JA, Ali F, Bailey L, Moreno L, Harrington LS. Role of nitric oxide and prostacyclin as vasoactive hormones released by the endothelium. Experimental physiology. 2008;93(1):141-7.

162

88. Yau JW, Teoh H, Verma S. Endothelial cell control of thrombosis. BMC cardiovascular disorders. 2015;15(1):1-11.

89. Michels A, Swystun LL, Mewburn J, Albánez S, Lillicrap D. Investigating von Willebrand factor pathophysiology using a flow chamber model of von Willebrand factor-platelet string formation. JoVE (Journal of Visualized Experiments). 2017(126):e55917.

90. Randi AM, Smith KE, Castaman G. von Willebrand factor regulation of blood vessel formation. Blood. 2018;132(2):132-40.

91. Oliver JJ, Webb DJ, Newby DE. Stimulated tissue plasminogen activator release as a marker of endothelial function in humans. Arteriosclerosis, thrombosis, and vascular biology. 2005;25(12):2470-9.

92. Félétou M, editor. The endothelium, Part I: Multiple functions of the endothelial cells--focus on endothelium-derived vasoactive mediators. Colloquium Series on Integrated Systems Physiology: From Molecule to Function; 2011: Morgan & Claypool Life Sciences.

93. Marcus AJ, Broekman MJ, Drosopoulos J, Islam N, Alyonycheva TN, Safier LB, et al. The endothelial cell ecto-ADPase responsible for inhibition of platelet function is CD39. The Journal of clinical investigation. 1997;99(6):1351-60.

94. Nurden AT. Platelets and tissue remodeling: extending the role of the blood clotting system. Endocrinology. 2007;148(7):3053-5.

95. White JG, Michelson A. Platelet structure. Platelets. 2007;2:45-71.

96. Thon JN, Italiano JE. Platelets: production, morphology and ultrastructure. Antiplatelet Agents: Springer; 2012. p. 3-22.

97. Bautista A, Buckler P, Towler H, Dawson A, Bennett B. Measurement of platelet life‐span in normal subjects and patients with myeloproliferative disease with

163

indium oxine labelled platelets. British journal of haematology. 1984;58(4):679- 87.

98. Fritsma GA. Platelet structure and function. Clinical Laboratory Science. 2015;28(2):125.

99. Yun S-H, Sim E-H, Goh R-Y, Park J-I, Han J-Y. Platelet activation: the mechanisms and potential biomarkers. BioMed research international. 2016;2016.

100. Saboor M, Ayub Q, Samina Ilyas M. Platelet receptors; an instrumental of platelet physiology. Pakistan journal of medical sciences. 2013;29(3):891.

101. Zapata JC, Cox D, Salvato MS. The role of platelets in the pathogenesis of viral hemorrhagic fevers. PLoS Negl Trop Dis. 2014;8(6):e2858.

102. Joo S-J. Mechanisms of platelet activation and integrin αIIβ3. Korean circulation journal. 2012;42(5):295-301.

103. Murugappa S, Kunapuli SP. The role of ADP receptors in platelet function. Front Biosci. 2006;11:1977-86.

104. Ushikubi F, Hirata M, Narumiya S. Platelet prostaglandin receptors. Platelets and Their Factors: Springer; 1997. p. 135-54.

105. Khandoga A, Fujiwara Y, Goyal P, Pandey D, Tsukahara R, Bolen A, et al. Lysophosphatidic acid-induced platelet shape change revealed through LPA1–5 receptor-selective probes and albumin. Platelets. 2008;19(6):415-27.

106. Woulfe D. REVIEW ARTICLES: Platelet G protein‐coupled receptors in hemostasis and thrombosis. Journal of Thrombosis and Haemostasis. 2005;3(10):2193-200.

107. Xu XR, Carrim N, Neves MAD, McKeown T, Stratton TW, Coelho RMP, et al. Platelets and platelet adhesion molecules: novel mechanisms of thrombosis and anti-thrombotic therapies. Thrombosis journal. 2016;14(1):29.

164

108. Siljander P, Farndale RW, Feijge MA, Comfurius P, Kos S, Bevers EM, et al. Platelet adhesion enhances the glycoprotein VI–dependent procoagulant response: Involvement of p38 MAP kinase and calpain. Arteriosclerosis, thrombosis, and vascular biology. 2001;21(4):618-27.

109. Lhermusier T, Chap H, Payrastre B. Platelet membrane phospholipid asymmetry: from the characterization of a scramblase activity to the identification of an essential protein mutated in Scott syndrome. Journal of Thrombosis and Haemostasis. 2011;9(10):1883-91.

110. Heemskerk J, Mattheij N, Cosemans J. Platelet‐based coagulation: different populations, different functions. Journal of Thrombosis and Haemostasis. 2013;11(1):2-16.

111. Reddy EC, Rand ML. Procoagulant Phosphatidylserine-Exposing Platelets in vitro and in vivo. Frontiers in Cardiovascular Medicine. 2020;7.

112. Kamath S, Blann A, Lip G. Platelet activation: assessment and quantification. European heart journal. 2001;22(17):1561-71.

113. Zeiler M, Moser M, Mann M. Copy number analysis of the murine platelet proteome spanning the complete abundance range. Molecular & Cellular Proteomics. 2014;13(12):3435-45.

114. Bryckaert M, Rosa J-P, Denis CV, Lenting PJ. Of von Willebrand factor and platelets. Cellular and Molecular Life Sciences. 2015;72(2):307-26.

115. Nieswandt B, Brakebusch C, Bergmeier W, Schulte V, Bouvard D, Mokhtari‐ Nejad R, et al. Glycoprotein VI but not α2β1 integrin is essential for platelet interaction with collagen. The EMBO journal. 2001;20(9):2120-30.

116. Clemetson KJ. Platelets and primary haemostasis. Thrombosis research. 2012;129(3):220-4.

165

117. Rumbaut RE, Thiagarajan P. Platelet-vessel wall interactions in hemostasis and thrombosis. Synthesis Lectures on Integrated Systems Physiology: From Molecule to Function. 2010;2(1):1-75.

118. Swanepoel AC, Pretorius E. Ultrastructural analysis of platelets during three phases of pregnancy: a qualitative and quantitative investigation. Hematology. 2015;20(1):39-47.

119. Szalai G, LaRue A, Watson D. Molecular mechanisms of megakaryopoiesis. Cellular and Molecular Life Sciences CMLS. 2006;63(21):2460-76.

120. Nieswandt B, Watson SP. Platelet-collagen interaction: is GPVI the central receptor? Blood. 2003;102(2):449-61.

121. Massini P, Käser-Glanzmann R, Lüscher E. Movement of calcium ions and their role in the activation of platelets. Thrombosis and haemostasis. 1978;40(01):212- 8.

122. Lechner K, Niessner H, Thaler E, editors. Coagulation abnormalities in liver disease. Seminars in thrombosis and hemostasis; 1977.

123. Dashty M, Akbarkhanzadeh V, Zeebregts CJ, Spek CA, Sijbrands EJ, Peppelenbosch MP, et al. Characterization of coagulation factor synthesis in nine human primary cell types. Scientific reports. 2012;2:787.

124. Morrissey JH. Tissue factor: a key molecule in hemostatic and nonhemostatic systems. International journal of hematology. 2004;79(2):103-8.

125. Lenting PJ, van Mourik JA, Mertens K. The life cycle of coagulation factor VIII in view of its structure and function. Blood, The Journal of the American Society of Hematology. 1998;92(11):3983-96.

126. Graham J. Stuart Factor: discovery and designation as factor X. Journal of Thrombosis and Haemostasis. 2003;1(5):871-7.

166

127. Berg JM, Tymoczko JL, Stryer L. Many Enzymes Are Activated by Specific Proteolytic Cleavage. Biochemistry: The Molecular Design of Life. 2002;5:10.5.

128. Palta S, Saroa R, Palta A. Overview of the coagulation system. Indian journal of anaesthesia. 2014;58(5):515.

129. Peyvandi F, Kunicki T, Lillicrap D. Genetic sequence analysis of inherited bleeding diseases. Blood, The Journal of the American Society of Hematology. 2013;122(20):3423-31.

130. Pauli R, Lian J, Mosher D, Suttie J. Association of congenital deficiency of multiple vitamin K-dependent coagulation factors and the phenotype of the warfarin embryopathy: clues to the mechanism of teratogenicity of coumarin derivatives. American journal of human genetics. 1987;41(4):566.

131. Haley KM, Recht M, McCarty OJ. Neonatal platelets: mediators of primary hemostasis in the developing hemostatic system. Pediatric research. 2014;76(3):230-7.

132. Becan-McBride K. Phlebotomy Handbook: Blood Collection Essentials: Prentice Hall; 1999.

133. McGuigan AP, Sefton MV. The influence of biomaterials on endothelial cell thrombogenicity. Biomaterials. 2007;28(16):2547-71.

134. Bergmeier W, Hynes RO. Extracellular matrix proteins in hemostasis and thrombosis. Cold Spring Harbor perspectives in biology. 2012;4(2):a005132.

135. Bacakova L, Travnickova M, Filova E, Matějka R, Stepanovska J, Musilkova J, et al. The role of vascular smooth muscle cells in the physiology and pathophysiology of blood vessels. Muscle Cell Tissue Curr Status Res Field. 2018;229.

167

136. Periayah MH, Halim AS, Saad AZM. Mechanism action of platelets and crucial blood coagulation pathways in hemostasis. International journal of hematology- oncology and stem cell research. 2017;11(4):319.

137. Hiller E. Basic principles of hemostasis. Modern Hematology: Springer; 2007. p. 327-45.

138. Broos K, Feys HB, De Meyer SF, Vanhoorelbeke K, Deckmyn H. Platelets at work in primary hemostasis. Blood reviews. 2011;25(4):155-67.

139. Andrews RK, Berndt MC. Platelet physiology and thrombosis. Thrombosis research. 2004;114(5-6):447-53.

140. Davì G, Patrono C. Platelet activation and atherothrombosis. New England Journal of Medicine. 2007;357(24):2482-94.

141. Everts P, Knape J, Weibrich G, Schnberger J, Hoffmann J, Overdevest E, et al. Platelet-rich plasma and platelet gel. A review.2006:38.

142. McEWAN A. Aspects of bleeding after cardiac surgery in children. Pediatric Anesthesia. 2007;17(12):1126-33.

143. Guzzetta NA, Miller BE. Principles of hemostasis in children: models and maturation. Pediatric Anesthesia. 2011;21(1):3-9.

144. Macfarlane R. An enzyme cascade in the blood clotting mechanism and its function as a biochemical amplifier. Nature. 1964;202(4931):498-9.

145. De Caterina R, Husted S, Wallentin L, Andreotti F, Arnesen H, Bachmann F, et al. General mechanisms of coagulation and targets of anticoagulants (Section I). Thrombosis and haemostasis. 2013;109(04):569-79.

146. Smith SB, Gailani D. Update on the physiology and pathology of factor IX activation by factor XIa. Expert review of hematology. 2008;1(1):87-98.

168

147. Romney G, Glick M. An updated concept of coagulation with clinical implications. The Journal of the American Dental Association. 2009;140(5):567- 74.

148. Nakagaki T, Foster DC, Berkner KL, Kisiel W. Initiation of the extrinsic pathway of blood coagulation: evidence for the tissue factor dependent autoactivation of human coagulation factor VII. Biochemistry. 1991;30(45):10819-24.

149. Nemerson Y. Tissue factor and hemostasis [published erratum appears in Blood 1988 Apr; 71 (4): 1178]. 1988.

151. Wilcox JN, Smith KM, Schwartz SM, Gordon D. Localization of tissue factor in the normal vessel wall and in the atherosclerotic plaque. Proceedings of the National Academy of Sciences. 1989;86(8):2839-43.

152. Edgington TS, Mackman N, Brand K, Ruf W. The structural biology of expression and function of tissue factor. Thrombosis and haemostasis. 1991;65(01):067-79.

153. Morrissey JH. Tissue factor: an enzyme cofactor and a true receptor. Thrombosis and haemostasis. 2001;86(07):66-74.

154. Müller I, Klocke A, Alex M, Kotzsch M, Luther T, Morgenstern E, et al. Intravascular tissue factor initiates coagulation via circulating microvesicles and platelets. The FASEB Journal. 2003;17(3):1-20.

155. Smith SA, Travers RJ, Morrissey JH. How it all starts: Initiation of the clotting cascade. Critical reviews in biochemistry and molecular biology. 2015;50(4):326- 36.

169

156. Chaudhry R, Babiker HM. Physiology, coagulation pathways. StatPearls [Internet]: StatPearls Publishing; 2019.

157. Kaplan AP, Silverberg M. BL0 OD. Blood. 1987;70(1):1-15.

158. Bouma BN, von dem Borne PAK, Meijers JC. Factor XI and protection of the fibrin clot against lysis–a role for the intrinsic pathway of coagulation in fibrinolysis. Thrombosis and haemostasis. 1998;80(07):24-7.

159. Kolev K, Longstaff C. Bleeding related to disturbed fibrinolysis. British journal of haematology. 2016;175(1):12-23.

160. Blinc A, Francis CW. Transport processes in fibrinolysis and fibrinolytic therapy. Thrombosis and haemostasis. 1996;75(04):481-91.

161. Lolck H, Goenner-Radig C, Ostermann H, Theissen J, der Meer J, Born V, et al. Recent Literature on Fibrinolysis. Variations. 1993;52(6):443-8.

162. Wiman B, Collen D. Molecular mechanism of physiological fibrinolysis. Nature. 1978;272(5653):549-50.

163. Bannish BE, Chernysh IN, Keener JP, Fogelson AL, Weisel JW. Molecular and physical mechanisms of fibrinolysis and thrombolysis from mathematical modeling and experiments. Scientific reports. 2017;7(1):1-11.

164. Bhattacharjee P, Bhattacharyya D, Kolev K. An insight into the abnormal fibrin clots—its pathophysiological roles. Fibrinolysis and Thrombolysis. 2014:1-29.

165. Costa-Filho R, Hochleitner G, Wendt M, Teruya A, Spahn DR. Over 50 years of fibrinogen concentrate. Clinical and applied thrombosis/hemostasis. 2016;22(2):109-14.

166. Copley AL. The Robin Fåhraeus Memorial Lecture Robin Fåhraeus—the Scientist and the Person. Upsala journal of medical sciences. 1989;94(3):223-53.

170

167. Doolittle RF. Some important milestones in the field of blood clotting. Journal of Innate Immunity. 2016;8(1):23-9.

168. Tennent GA, Brennan SO, Stangou AJ, O'Grady J, Hawkins PN, Pepys MB. Human plasma fibrinogen is synthesized in the liver. Blood. 2007;109(5):1971-4.

169. Hedrich HC, Gulle H. Fibrin Glues and Bandages. Production of Plasma Proteins for Therapeutic Use. 2013:137-45.

170. Mosesson MW, Siebenlist KR, Meh DA. The structure and biological features of fibrinogen and fibrin. Annals of the New York Academy of Sciences. 2001;936(1):11-30.

171. Negrier C, Rothschild C, Goudemand J, Borg J, Claeyssens S, Alessi M, et al. Pharmacokinetics and pharmacodynamics of a new highly secured fibrinogen concentrate. Journal of Thrombosis and Haemostasis. 2008;6(9):1494-9.

172. Wang N-Y, Patras KA, Seo HS, Cavaco CK, Rösler B, Neely MN, et al. Group B streptococcal serine-rich repeat proteins promote interaction with fibrinogen and vaginal colonization. The Journal of infectious diseases. 2014;210(6):982-91.

173. Rox JM, Müller J, Pötzsch B. Absence of fibrinogen α‐, β‐and γ‐chain mRNA in human platelets. British journal of haematology. 2005;130(4):647-8.

174. Fuss C, Palmaz JC, Sprague EA. Fibrinogen: structure, function, and surface interactions. Journal of Vascular and Interventional Radiology. 2001;12(6):677- 82.

175. Onrat S, Emmiler M, Sivaci Y, Söylemez Z, Ozgöz A, Imirzalioğlu N. A patient with ascending aortic dilatation, similar to phenotypes of connective tissue disorders. Genet Mol Res. 2009;8(2):426-34.

176. Hoeprich Jr PD, Doolittle RF. Dimeric half-molecules of human fibrinogen are joined through disulfide bonds in an antiparallel orientation. Biochemistry. 1983;22(9):2049-55.

171

177. Xu W-f, Chung DW, Davie EW. The Assembly of Human Fibrinogen The Role of the Amino-Terminal and Coiled-Coil Regions of the Three Chains in the Formation of the αγ and βγ Heterodimers and αβγ Half-Molecules. Journal of Biological Chemistry. 1996;271(44):27948-53.

178. Fort A, Fish RJ, Attanasio C, Dosch R, Visel A, Neerman-Arbez M. A liver enhancer in the fibrinogen gene cluster. Blood, The Journal of the American Society of Hematology. 2011;117(1):276-82.

179. Chung DW, Harris JE, Davie EW. Nucleotide sequences of the three genes coding for human fibrinogen. Fibrinogen, thrombosis, coagulation, and fibrinolysis: Springer; 1990. p. 39-48.

180. Mosesson MW. Structure and functions of fibrinogen and fibrin. Recent Advances in Thrombosis and Hemostasis 2008: Springer; 2008. p. 3-26.

181. Tiscia GL, Margaglione M. Human fibrinogen: molecular and genetic aspects of congenital disorders. International journal of molecular sciences. 2018;19(6): 1597.

182. Collet J-P, Moen JL, Veklich YI, Gorkun OV, Lord ST, Montalescot G, et al. The αC domains of fibrinogen affect the structure of the fibrin clot, its physical properties, and its susceptibility to fibrinolysis. Blood. 2005;106(12):3824-30.

183. Bratek-Skicki A, Żeliszewska P, Ruso JM. Fibrinogen: a journey into biotechnology. Soft Matter. 2016;12(42):8639-53.

184. Herrick S, Blanc-Brude O, Gray A, Laurent G. Fibrinogen. The international journal of biochemistry & cell biology. 1999;31(7):741-6.

185. Gervois P, Vu-Dac N, Kleemann R, Kockx M, Dubois G, Laine B, et al. Negative regulation of human fibrinogen gene expression by peroxisome proliferator- activated receptor α agonists via inhibition of CCAAT box/enhancer-binding protein β. Journal of Biological Chemistry. 2001;276(36):33471-7.

172

186. Fuller GM, Zhang Z. Transcriptional control mechanism of fibrinogen gene expression. Annals of the New York Academy of Sciences. 2001;936(1):469-79.

187. Greenberg CS, Miraglia CC, Rickles FR, Shuman MA. Cleavage of blood coagulation factor XIII and fibrinogen by thrombin during in vitro clotting. The Journal of clinical investigation. 1985;75(5):1463-70.

188. Tsurupa G, Yakovlev S, McKee P, Medved L. Noncovalent interaction of α2- antiplasmin with fibrin (ogen): localization of α2-antiplasmin-binding sites. Biochemistry. 2010;49(35):7643-51.

189. Weisel JW, Litvinov RI. Mechanisms of fibrin polymerization and clinical implications. Blood, The Journal of the American Society of Hematology. 2013; 121(10):1712-9.

190. Bhagavan NV. Medical biochemistry: Academic press; 2002.

191. Weisel JW, Litvinov RI. Fibrin formation, structure and properties. Fibrous proteins: structures and mechanisms: Springer; 2017. p. 405-56.

192. Riedel T, Suttnar J, Brynda E, Houska M, Medved L, Dyr JE. Fibrinopeptides A and B release in the process of surface fibrin formation. Blood, The Journal of the American Society of Hematology. 2011;117(5):1700-6.

193. Sørensen B, Tang M, Larsen OH, Laursen PN, Fenger-Eriksen C, Rea CJ. The role of fibrinogen: a new paradigm in the treatment of coagulopathic bleeding. Thrombosis research. 2011;128:S13-S6.

194. Davidson SJ. Inflammation and acute phase proteins in haemostasis. Acute Phase Proteins. 2013:30-54.

195. Laurens N, Koolwijk Pd, De Maat M. Fibrin structure and wound healing. Journal of Thrombosis and Haemostasis. 2006;4(5):932-9.

173

196. Chapin JC, Hajjar KA. Fibrinolysis and the control of blood coagulation. Blood reviews. 2015;29(1):17-24.

197. Bennett JS. Platelet‐fibrinogen interactions. Annals of the New York Academy of Sciences. 2001;936(1):340-54.

198. Weisel JW. Fibrinogen and fibrin. Advances in protein chemistry: Elsevier; 2005. p. 247-99.

199. Ariëns RA. Elevated fibrinogen causes thrombosis. Blood, The Journal of the American Society of Hematology. 2011;117(18):4687-8.

200. Besser MW, MacDonald SG. Acquired hypofibrinogenemia: current perspectives. Journal of blood medicine. 2016;7:217.

201. McCarty MF, O'Keefe JH, DiNicolantonio JJ. Pentoxifylline for vascular health: a brief review of the literature. Open Heart. 2016;3(1).

202. Kahn NN, Sinha AK, Spungen AM, Bauman WA. Effects of oxandrolone, an anabolic steroid, on hemostasis. American journal of hematology. 2006;81(2):95- 100.

203. Venugopal A. Disseminated intravascular coagulation. Indian journal of anaesthesia. 2014;58(5):603.

204. Görlinger K. Coagulation management during liver transplantation. Hämostaseologie. 2006;26(S 02):S64-S75.

205. Arif S, Khan AS, Khan AR. Changes in fibrinogen level in liver cirrhosis. Journal of Ayub Medical College Abbottabad. 2002;14(2).

206. Lou Y, Suo S, Tong H, Qian W, Mai W, Meng H, et al. Hypofibrinogenemia as a clue in the presumptive diagnosis of acute promyelocytic leukemia. Leukemia research. 2016;50:11-6.

174

207. Weltermann A, Pabinger I, Geissler K, Jäger U, Gisslinger H, Knöbl P, et al. Hypofibrinogenemia in non-M3 acute myeloid leukemia. Incidence, clinical and laboratory characteristics and prognosis. Leukemia. 1998;12(8):1182-6.

208. Platè M, Asselta R, Spena S, Spreafico M, Fagoonee S, Peyvandi F, et al. Congenital hypofibrinogenemia: characterization of two missense mutations affecting fibrinogen assembly and secretion. Blood Cells, Molecules, and Diseases. 2008;41(3):292-7.

209. Al-Hussaini A, Altalhi A, El Hag I, AlHussaini H, Francalanci P, Giovannoni I, et al. Hepatic fibrinogen storage disease due to the fibrinogen γ375 Arg→ Trp mutation ―fibrinogen Aguadilla‖ is present in Arabs. Saudi Journal of Gastroenterology: Official Journal of the Saudi Gastroenterology Association. 2014;20(4):255.

210. Brennan SO, Wyatt J, Medicina D, Callea F, George PM. Fibrinogen Brescia: Hepatic endoplasmic reticulum storage and hypofibrinogenemia because of a γ284 Gly→ Arg mutation. The American Journal of Pathology. 2000;157(1):189- 96.

211. Mari D, Ogliari G, Castaldi D, Vitale G, Bollini EM, Lio D. Hemostasis and ageing. Immunity & Ageing. 2008;5(1):12.

212. Sinha S, Luben RN, Welch A, Bingham S, Wareham NJ, Day NE, et al. Fibrinogen and cigarette smoking in men and women in the European Prospective Investigation into Cancer in Norfolk (EPIC-Norfolk) population. European Journal of Cardiovascular Prevention & Rehabilitation. 2005;12(2):144-50.

213. Neild PJ, Syndercombe-Court D, Keatinge W, Donaldson G, Mattock M, Caunce M. Cold-induced increases in erythrocyte count, plasma cholesterol and plasma fibrinogen of elderly people without a comparable rise in protein C or factor X. Clinical Science. 1994;86(1):43-8.

175

214. Margaglione M, Grandone E, Mancini FP, Di Minno G. Genetic modulation of plasma fibrinogen concentrations: possible importance of interleukin-6. Journal of Thrombosis and Thrombolysis. 1996;3(1):51-6.

215. Ellins EA, Rees DA, Deanfield JE, Steptoe A, Halcox JP. Increased fibrinogen responses to psychophysiological stress predict future endothelial dysfunction implications for cardiovascular disease? Brain, Behavior, and Immunity. 2017; 60:233-9.

216. Lippi G, Franchini M, Salvagno GL, Montagnana M, Guidi GC. Higher morning serum cortisol level predicts increased fibrinogen but not shortened APTT. Journal of Thrombosis and Thrombolysis. 2008;26(2):103.

217. Middeldorp S, Meijers JC, van den Ende AE, van Enk A, Bouma BN, Tans G, et al. Effects on coagulation of levonorgestrel-and desogestrel-containing low dose oral contraceptives: a cross-over study. Thrombosis and Haemostasis-Stuttgart-. 2000;84(1):4-8.

218. Adler G, Duchinski T, Jasinska A, Piotrowska U. Fibrinogen fractions in the third trimester of pregnancy and in puerperium. Thrombosis research. 2000;97(6):405- 10.

219. James S, Vorster HH, Venter CS, Kruger HS, Nell TA, Veldman FJ, et al. Nutritional status influences plasma fibrinogen concentration: evidence from the THUSA survey. Thrombosis research. 2000;98(5):383-94.

220. Miura K, Nakagawa H, Ueshima H, Okayama A, Saitoh S, Curb JD, et al. Dietary factors related to higher plasma fibrinogen levels of Japanese-Americans in Hawaii compared with Japanese in Japan. Arteriosclerosis, thrombosis, and vascular biology. 2006;26(7):1674-9.

221. Bo M, Raspo S, Morra F, Cassader M, Isaia G, Poli L. Body fat is the main predictor of fibrinogen levels in healthy non-obese men. Metabolism. 2004; 53(8):984-8.

176

222. Jacquemin B, Antoniades C, Nyberg F, Plana E, Müller M, Greven S, et al. Common genetic polymorphisms and haplotypes of fibrinogen alpha, beta, and gamma chains affect fibrinogen levels and the response to proinflammatory stimulation in myocardial infarction survivors: the AIRGENE study. Journal of the American College of Cardiology. 2008;52(11):941-52.

223. Tofler GH, D‘Agostino RB, Jacques PF, Bostom AG, Wilson PW, Lipinska I, et al. Association between increased homocysteine levels and impaired fibrinolytic potential: potential mechanism for cardiovascular risk. Thrombosis and haemostasis. 2002;88(11):799-804.

224. Korte W, Poon M-C, Iorio A, Makris M. Thrombosis in inherited fibrinogen disorders. Transfusion Medicine and Hemotherapy. 2017;44(2):70-6.

225. Yamanaka Y, Takeuchi K, Sugimoto M, Sato A, Nakago S, Maruo T. Dysfibrinogenemia during pregnancy treated successfully with fibrinogen. Acta obstetricia et gynecologica Scandinavica. 2003;82(10):972-3.

226. Song KS, Park NJ, Choi JR, Doh HJ, Chung KH. Fibrinogen Seoul (FGG Ala341Asp): a novel mutation associated with hypodysfibrinogenemia. Clinical and applied thrombosis/hemostasis. 2006;12(3):338-43.

227. Falsoleiman H, Daloee MH, Dehghani M, Rohani A, Bayani B. Percutaneous coronary intervention in a case of afibrinogenemia. Asian Cardiovascular and Thoracic Annals. 2013;21(3):358-9.

228. Rodriguez RC, Buchanan GR, Clanton MS. Prophylactic cryoprecipitate in congenital afibrinogenemia. Clinical pediatrics. 1988;27(11):543-5.

229. Ataoglu E, Duru NS, Celkan T, Civilibal M, Yavuz SC, Elevli M, et al. Spontaneous intracranial bleeding in a neonate with congenital afibrinogenemia. Blood coagulation & fibrinolysis. 2010;21(6):592-4.

231. Casini A, De Moerloose P, Neerman-Arbez M, editors. Clinical features and management of congenital fibrinogen deficiencies. Seminars in thrombosis and hemostasis; 2016: Thieme Medical Publishers.

232. Naz A, Biswas A, Khan TN, Goodeve A, Ahmed N, Saqlain N, et al. Identification of novel mutations in congenital afibrinogenemia patients and molecular modeling of missense mutations in Pakistani population. Thrombosis journal. 2017;15(1):1-8.

233. Cetinkaya SE, Pabuccu EG, Ozmen B, Dokmeci F. Recurrent massive hemoperitoneum due to ovulation as a clinical sign in congenital afibrinogenemia. Acta obstetricia et gynecologica Scandinavica. 2011;90(2):192-4.

234. Arcagök BC, Özdemir N, Tekin A, Özcan R, Eliçevik M, Şenyüz OF, et al. Spontaneous splenic rupture in a patient with congenital afibrinogenemia. Turkish Archives of Pediatrics/Türk Pediatri Arşivi. 2014;49(3):247.

235. Fettah A, Gökçebay DG, Çulha V, Yaralı N, Tunç B, Özbek N. A rare complication of congenital afibrinogenemia: bone cysts. Turkish Journal of Hematology. 2017;34(2):183.

236. Tziomalos K, Vakalopoulou S, Perifanis V, Garipidou V. Treatment of congenital fibrinogen deficiency: overview and recent findings. Vascular Health and Risk Management. 2009;5:843.

237. Aygören‐Pürsün E, Martinez Saguer I, Rusicke E, Louwen F, Geka F, Ivaskevicius V, et al. Retrochorionic hematoma in congenital afibrinogenemia: resolution with fibrinogen concentrate infusions. American journal of hematology. 2007;82(4):317-20.

238. Bornikova L, Peyvandi F, Allen G, Bernstein J, Manco‐Johnson M. Fibrinogen replacement therapy for congenital fibrinogen deficiency. Journal of Thrombosis and Haemostasis. 2011;9(9):1687-704.

178

239. Bennett RL, French KS, Resta RG, Doyle DL. Standardized human pedigree nomenclature: update and assessment of the recommendations of the National Society of Genetic Counselors. Journal of genetic counseling. 2008;17(5):424-33.

240. Cheng J, Randall A, Baldi P. Prediction of protein stability changes for single‐site mutations using support vector machines. Proteins: Structure, Function, and Bioinformatics. 2006;62(4):1125-32.

241. Choi Y, Chan AP. Provean web server: a tool to predict the functional effect of amino acid substitutions and indels. Bioinformatics. 2015;31(16):2745-7.

242. Adzhubei IA, Schmidt S, Peshkin L, Ramensky VE, Gerasimova A, Bork P, et al. A method and server for predicting damaging missense mutations. Nature methods. 2010;7(4):248.

243. Ng PC, Henikoff S. SIFT: Predicting amino acid changes that affect protein function. Nucleic acids research. 2003;31(13):3812-4.

244. Capriotti E, Calabrese R, Fariselli P, Martelli PL, Altman RB, Casadio R. WS- SNPs&GO: a web server for predicting the deleterious effect of human protein variants using functional annotation. BMC genomics. 2013;14(S3):S6.

245. Rodeghiero F, Tosetto A, Abshire T, Arnold D, Coller B, James P, et al. ISTH/SSC bleeding assessment tool: a standardized questionnaire and a proposal for a new bleeding score for inherited bleeding disorders. Journal of Thrombosis and Haemostasis. 2010;8(9):2063-5.

246. Asselta R, Spena S, Duga S, Peyvandi F, Malcovati M, Mannucci PM, et al. Analysis of Iranian patients allowed the identification of the first truncating mutation in the fibrinogen Bbeta-chain gene causing afibrinogenemia. haematologica. 2002;87(8):855-9.

179

247. De Moerloose P, Casini A, Neerman-Arbez M, editors. Congenital fibrinogen disorders: an update. Seminars in thrombosis and hemostasis; 2013: Thieme Medical Publishers.

248. Peyvandi F, Haertel S, Knaub S, Mannucci P. Incidence of bleeding symptoms in 100 patients with inherited afibrinogenemia or hypofibrinogenemia. Journal of Thrombosis and Haemostasis. 2006;4(7):1634-7.

249. Köker SA, Köker A, Neerman-Arbez M, Tunçer GÖ, Akbas Y, Kara TT, et al. A Novel Frameshift Mutation in the FGA Gene (c. 196 delT) Leading to Congenital Afibrinogenemia. Journal of Pediatric Hematology/Oncology. 2020;42(2):e98-e9.

250. Lak M, Toogheh G, Keyhani M. Bleeding and Thrombosis In 90 Iranian Patients with Inherited Afibrinogenemia. American Society of Hematology; 2010.

251. Ra'Anani P, Levi Y, Varon D, Gitel S, Martinowitz U. Congenital afibrinogenemia with bleeding, bone cysts and antibodies to fibrinogen. Harefuah. 1991;121(9):291-3.

180

Informed Consent: (Annexure A) Informed consent form must be properly explained to the patient and written in local language (Urdu) to make it understand for all those who cannot read it in English. Questionnaire: (Annexure B) Please enter maximum information .Do not leaves blanks if possible. If there is any information missing of not taken, please enter N/D (not done) or N/A (not available).

Consent Form

You are being asked to take part in a research study titled “Frequency, Phenotypic and Haemostatic Expressions In Fibrinogen Based Inherited Coagulopathies In Pakistani Population”. Please read this form carefully and ask any questions you may have before agreeing to take part in the study.

What the study is about: The purpose of this study is to screen the Pakistani families for phenotypic,haemostatic and genetic expression of afibrinogenemia.

What we will ask you to do: If you agree to be in this study, we will conduct an interview with you. The interview will include questions about the particular inherited fibrinogen deficiency which you or your family is suffering from and how is this affecting your or their life. The interview will take about 30 minutes to complete. With your permission, we would also like to tape-record an interview.

Risks and benefits

"I do not anticipate any risks to you participating in this study other than those encountered in day-to-day life."

There are certain benefits to you as this study aims to investigate and thus have better understanding of the inherited fibrinogen disorder in the population. It will also have some beneficial effects on the treatment and the outcome of the disease.

181

Your answers will be confidential. The records of this study will be kept private. In any sort of report we make public we will not include any information that will make it possible to identify you. Research records will be kept in a locked file; only the researchers will have access to the records. If we tape-record the interview, we will destroy the tape after it has been transcribed, which we anticipate will be within two months of its taping.

Taking part is voluntary: Taking part in this study is completely voluntary. You may skip any questions that you do not want to answer. If you decide not to take part or to skip some of the questions we will respect it. If you decide to take part, you are free to withdraw at any time.

If you have questions: This study has been conducted by Dr. Tehmina Nafees Sonia Khan, under the supervision of Dr. Arshi Naz Assistant Professor at NATIONAL INSTITUTE OF BLOOD DISEASES, Karachi. Please ask any questions you have now. If you have questions later, you may contact national institute of blood diseases organization that serves as a liaison between the University and the person bringing the complaint so that anonymity can be ensured.

You will be given a copy of this form to keep for your records.

Statement of Consent:

I have read the above information, and have received answers to any questions I asked. I consent to take part in the study.

182

Your Signature ______Date ______

Your Name (printed) ______

In addition to agreeing to participate, I also consent to that this information can be used in other research project/projects for the benefit of mankind in general.

Your Signature ______Date ______

Signature of person obtaining consent ______Date ______

Printed name of person obtaining consent ______Date ______

183

184

185

QUESTIONNAIRE Annexure B:

Study Code: ______

Date: ______

Name of Patient: ______Age: ______Date of birth: ______Father’s/Guardian: ______Address : ______

City, state, zip: ______Telephone: Home: ______Cell #: ______Work: ______Sex Female ( ) Male ( )

Background

1. Ethnic origin (check only one): � Urdu speaking. � Baluchi � Makrani. � Pathan � Punjabi � Sindhi � Other: ______

2. Are you currently (check only one): � Married � Single � Other

186

History of Consanguinity: (cousin marriage) ______

History of Bleeding: Oral Cavity

0 No 1 Reported atleast one. 2 Consultation only 3 Surgical hemostasis or antifibrinolytics 4 Blood transfusion or Replacement Therapy or desmopressin

Cutaneous Bleeding:

0 No or Trivial (<1cm) 1 >1 cm and no trauma 2 Consultation only

Epistaxis:

0 No or trivial bleeding 1 >5 or more than 10 2 Consultation 3 Packing or cauterization of Antifibrinolytics 4 Blood Transfusion o replacement Therapy or Desmopressin.

187

Gastrointestinal Bleeding:

0 No 1 Associated with Ulcer and Portal Hypertension,Hemorrhoids,angiodysplasia. 2 Spontaneous 3 Surgical hemostasis or Blood Transfusion or replacement Therapy or Desmopressin or Antifibrinolytics.

Postpartum Haemorrhage:

-1 No bleeding in atleast 2 deliveries 0 No deliveries or no bleeding in one delivery 1 Consultation only 2 Curettage or iron therapy or antifibrinolytics 3 Blood transfusion or replacement therapy or desmopressin 4 Hysterectomy

Bleeding from Minor Trauma:

0 No or trivial (less than 5) 1 5 or more than 5 2 Consultation only 3 Surgical haemostasis 4 Blood Transfusion or replacement therapy or desmopressin

188

Surgery:

-1 No bleeding in atleast 2 surgeries 0 Not done or no bleeding in 1 surgery 1 Reported in < 25% of all surgeries 2 Reported in >25% of all surgeries , no intervention (yes) 3 Surgical hemostasis or antifibrinolytics 4 Blood transfusion or replacement therapy or desmopressin (yes)

Menorrhagia:

0 No 1 Consultation only 2 Antifibrinolytics or pill use 3 Curettage or iron therapy 4 Blood transfusion or replacement therapy or Desmopressin

Muscle hematoma:

0 Never 1 Post trauma no therapy 2 Spontaneous no therapy 3 Spontaneous or traumatic requiring Desmopressin or replacement therapy 4 Spontaneous or traumatic requiring surgical intervention or blood transfusion

189

Haemarthrosis:

CNS bleeding:

0 Never 1 Mild 2 Severe 3 Sub dural , any intervention 4 Intracerebral , any intervention.

History of Trauma:

If any: ______

Past Medical History: ______

Past Surgical History: ______

190

Drug history:

. Use of Heparin ______NSAIDs. ______. Warfarin ______Injectable/oral .Vit K ______. Aspirin ______

Family history:

No. of family members.

Siblings Brothers

Sisters

Other members:

Affected: ______Died of this or any other disease: ______History of any disease in family: ______

History of Blood Transfusion:

Time /year: ______

Product transfused: a) Packed Cells ______b) Platelets ______c) Cryoprecipitate ______

191

General Health:

1. In general, would you say your health is: (Circle one) Excellent ...... 1 Very good...... 2 Good...... 3 Fair ...... 4 Poor...... 5

Sign and Symptoms: ( please tick if any) Yes No 1 Fever 2 Hemoptysis 3 Hemetamesis. 4 Bloody Dirrhea 5 Signs of hypovolemia or hemorrhagic shock 6 Pregnancy or recent delivery 7 Signs of infection or sepsis

Medical Care

1. When you visit your doctor, how often do you do the following (please tick one number for each question):

Never Sometimes fairly often very often always y y y y

2 . In the past 6 months, how many times did you visit a physician?

Do not include visits while in the hospital or the hospital emergency department.

192

Visits

1. In the past 6 months, how many times did you go to a hospital emergency department? ______

2. In the past 6 months, how many times were you hospitalized for one night or longer?

______

Thank you for your help!

193

RODEGHIERO, F., TOSETTO, A., ABSHIRE, T., ARNOLD, D. M., COLLER, B., JAMES, P.,

NEUNERT, C., LILLICRAP, D. AND ON BEHALF OF THE ISTH/SSC JOINT VWF AND

PERINATAL/PEDIATRIC HEMOSTASIS SUBCOMMITTEES WORKING GROUP (2010), ISTH/SSC BLEEDING ASSESSMENT TOOL: A STANDARDIZED QUESTIONNAIRE AND A PROPOSAL FOR A

NEW BLEEDING SCORE FOR INHERITED BLEEDING DISORDERS. JOURNAL OF THROMBOSIS

AND HAEMOSTASIS, 8: 2063–2065.

SUPPLEMENTARY MATERIAL TO THE OFFICIAL COMMUNICATION

OF THE SSC (LAST REVISION: 19 JULY 2011)

Background

The clinical appreciation of the presence and severity of bleeding symptoms is a fundamental step in the evaluation of patients referred for a possible bleeding disorder. In an attempt to improve the collection and reproducibility of the bleeding history, several Bleeding

Assessment Tools (BAT) have been proposed and used. Currently available BAT have some limitations, particularly regarding the lack of pediatric-specific symptoms in some of them and the predominance of the severity of bleeding symptoms over other potentially clinically important features, such as the frequency of symptoms.

To overcome the above-mentioned limitations and to promote the standardization of the available BATs, a Working Group was established within the framework of the ISTH/SSC

Subcommittees on VWF and on Perinatal/Pediatric Hemostasis (ISTH/SSC-BAT) during the

53rd SSC Annual Meeting held in Geneva in 2007. Members of the group first met in Toronto on January 2008 and then regularly at each subsequent SSC meeting. This paper presents a structured questionnaire and its clinical use agreed on by the ISTH/SSC-BAT together with a proposal for a new BS system to undergo validity and reliability testing in future studies.This new BAT is intended for inherited bleeding disorders in children and adults. The questionnaire should be collected by a physician or another adequately trained health-

1 professional. Only symptoms and related treatments, if any, before and/or at diagnosis should be reported. Refer to the full text for additional instructions.

Minimal criteria defining a significant bleeding

For each specific bleeding symptom, the ISTH/SSC joint working group proposed minimal criteria in order to classify a symptom as significant and thus receive a score of 1 or more (see also Table 1):

1. Epistaxis: Any nosebleed, especially occuring after puberty, that causes patient concern

(e.g., interference or distress with daily or social activities) is considered significant. In general, epistaxis should not be considered significant when it lasts less than 10 minutes, has a frequency of < 5 episodes/year, has a seasonal occurence, or is associated with infections of the upper respiratory tract or other identifiable cause (e.g., dusty dry air).

2. Cutaneous bleeding: Bruises are considered significant when 5 or more (> 1cm) in exposed areas; petechiae when adequately described by the patient or relatives; or hematomas when occurring without trauma.

3. Minor cutaneous wound: Any bleeding episode caused by superficial cuts (e.g., by shaving razor, knife, or scissors) or that requires frequent bandage changes is considered significant.

Insignificant bleeding from wounds includes those of duration < 10 minutes and lesions that usually require stitches in normal subjects (e.g., under the chin). Symptoms should also be manifest on more than one occasion to be considered significant.

4. Oral cavity bleeding: Gum bleeding should be considered significant when it causes frankly bloody sputum and lasts for 10 minutes or longer on more than one occasion. Tooth eruption or spontaneous tooth loss bleeding should be considered significant when it requires assistance or supervision by a physician, or lasts at least 10 minutes (bleeding associated with tooth extraction is considered separately). Bleeding occurring after bites to lips, cheek, and tongue should be considered significant when it lasts at least 10 minutes or causes a swollen tongue or mouth.

5. Hematemesis, melena, and hematochezia: Any gastrointestinal bleeding that is not explained by the presence of a specific disease should be considered significant.

2 6. Hematuria: Only macroscopic hematuria (from red to pale-pink urine) that is not explained by the presence of a specific urologic disease should be considered significant.

7. Tooth extraction: Any bleeding occurring after leaving the dentist’s office and requiring a new, unscheduled visit or prolonged bleeding at the dentist’s office causing a delay in the procedure or discharge should be considered significant.

8. Surgical bleeding: Any bleeding judged by the surgeon to be abnormally prolonged, that causes a delay in discharge, or requires some supportive treatment is considered significant.

9. Menorrhagia: Any bleeding that interferes with daily activities such as work, housework, exercise or social activities during most menstrual periods should be considered significant.

Criteria for significant bleeding may include any of the following: changing pads more frequently than every 2 hours; menstrual bleeding lasting 7 or more days; and the presence of clots > 1 cm combined with a history of flooding. If a patient has previously made a record of her menstrual loss using a pictorial blood loss assessment chart (PBAC), a PBAC score higher than 100 also qualifies for a score of 1.

10. Post-partum bleeding. Vaginal bleeding or uterine discharge (lochia) that lasts for more than 6 weeks. Any bleeding of lesser duration that is judged by the obstetrician as abnormally heavy or prolonged, that causes a delay in discharge, requires some supportive treatment, requires changing pads or tampons more frequently than every 2 hours, or causes progressive anemia is also considered significant

11. Muscle hematomas or hemarthrosis. Any spontaneous joint / muscle bleeding (not related to traumatic injuries) is considered significant.

12. CNS bleeding. Any subdural or intracerebral hemorrhage requiring diagnostic or therapeutic intervention is scored 3 or 4, respectively.

13. Other bleeding symptoms. When these bleeding symptoms occur during infancy, they are scored 1 or more. Their presence when reported by either the patient or a family member should always prompt detailed laboratory investigation.

3 Only symptoms and treatment BEFORE and AT diagnosis should be considered

1. Epistaxis

1.1 Have you ever had spontaneous  Yes  No or trivial (skip to 2) epistaxis? 1.2 Have the symptom ever required  Yes  No (resolve medical attention ? spontaneously; skip to 1.6)

1.3 If answer to 1.2 is yes, please  Consultation only

specify  Cauterization

 Packing  Antifibrinolytics  Iron therapy

 Treatment with desmopressin

 Treatment with plasma

 Treatment with platelet concentrate  Treatment with factor concentrates

 Blood (RBC) transfusion

1.4 How many times in your life did  1 - 2 you receive any of the above  3 to 5 treatments (# 1.3)?  6 to 10  more than 10  Before 1 year 1.5 At what age did you first have symptoms?  Between 1-5 years of age  Between 6-12 years of age  Between 13-25 years of age  After 25 years of age

1.6 Approximate number of episodes  less than 1 per year NOT requiring medical attention  1 per year  2-5 every year  1-3 every month  1 every week

1.7 Duration of average single  1 minute or less episode (min.) NOT requiring  1 - 10 minutes medical attention  more than 10 minutes

2. Cutaneous bleeding (Bruising, ecchymoses, purpura, subcutanueos hematomas)

2.1 Have you ever had any of the above  Yes  No or trivial skip to 3 cutaneous bleeding? 2.2 Have the symptom ever required  Yes No  skip to 2.6 medical attention?

If answer to 2.2 is yes, please  Consultation only

2.3 specify

 Treatment with desmopressin

 Treatment with plasma

 Treatment with platelet concentrate  Treatment with factor concentrates

 Blood (RBC) transfusion

2 How many times in your life did  1 - 2 2.4 you receive any of the above  3 to 5 treatments (# 2.3)?  6 to 10  more than 10  Before 1 year 2.5 At what age did you first have symptoms?  Between 1-5 years of age  Between 6-12 years of age  Between 13-25 years of age  After 25 years of age

2.6 Approximate number of episodes  less than 1 per year NOT requiring medical attention  1 per year  1-5 every six month  1-3 every month  1 every week

2.7 Type of bleeding  Petechiae  Bruises  Hematomas 2.8 Location  Exposed sites  Unexposed sites  Both 2.9 Common size  ≤ 1 cm  >1 cm  Extensive (palm sized or larger)

2.10 How many bruises >1 cm in  ≤ 5 exposed areas in the most severe  > 5 manifestation? 2.11 Location of petechiae  Limited to lower limbs  Diffuse

3. Bleeding from minor wounds (not requiring stitches in the average patient)

3.1 Have you ever had prolonged  Yes  No or trivial skip to 4 bleeding from minor wounds? 3.2 Have the symptom ever required  Yes  No skip to 3.6 medical attention ?

3.3 If answer to 3.2 is yes, please  Consultation only specify

 Surgical hemostasis

 Treatment with desmopressin

 Treatment with plasma

 Treatment with platelet concentrate  Treatment with factor concentrates

 Blood (RBC) transfusion

3.4 How many times in your life did  1 - 2 you received any of the above  3 to 5 treatments (# 3.3)?  6 to 10  more than 10  Before 1 year 3.5 At what age did you first have symptoms?  Between 1-5 years of age  Between 6-12 years of age  Between 13-25 years of age  After 25 years of age

3.6 Approximate number of episodes  less than 1 per year NOT requiring medical attention  1 per year  2-5 every year  1-3 every month  1 every week

3.7 Duration of average single  1 to 10 minutes episode (min.)  more than 10 minutes

4. Hematuria

4.1 Have you ever had hematuria ?  Yes  No skip to 5 4.2 If answer to 4.1 is yes, please specify

Presence of associated Yes  (skip to 5) No  urologic disease Specify:  Stones  Infection  Kidney/ bladder disease

Please answer the following questions only for SPONTANEOUS symptoms (answer No to 4.1)

4.3 Have the symptom ever required Yes  No  skip to 4.7 medical attention ?

4.4 If answer to 4.3 is yes, please  Consultation only specify  Surgery  Iron therapy

 Treatment with desmopressin

 Treatment with plasma

 Treatment with platelet concentrate  Treatment with factor concentrates

 Blood (RBC) transfusion

4.5 How many times in your life did  1 - 2 you received any of the above  3 to 5 treatments (# 4.4)?  6 to 10  more than 10  Before 1 year 4.6 At what age did you first have symptoms?  Between 1-5 years of age  Between 6-12 years of age  Between 13-25 years of age  After 25 years of age

4.7 Approximate number of episodes  less than 1 per year NOT requiring medical attention  1 per year  1-5 every six month  1-3 every month  1 every week

5. Gastrointestinal bleeding (Hematemesis, Melena, Hematochezia)

5.1 Have you ever had gastrointestinal  Yes  No skip to 6 bleeding ?

5.2 If answer to 5.1 is yes, please specify

Type of bleeding  Hematemesis  Melena  Hematochezia

Presence of associated Yes  No  GI disease Specify:  Ulcer  Portal hypertension  Angiodysplasia Please answer to the following questions only for SPONTANEOUS symptoms

5.3 Have the symptom ever required Yes  No  skip to 5.7 medical attention ?

5.4 If answer to 5.3 is yes, please  Consultation only specify

 Surgical haemostasis  Antifibrinolytics

 Treatment with desmopressin

 Treatment with plasma

 Treatment with platelet concentrate  Treatment with factor concentrates

 Blood (RBC) transfusion

5.5 How many times in your life did  1 - 2 you received any of the above  3 to 5 treatments (# 5.4)?  6 to 10  more than 10  Before 1 year 5.6 At what age did you first have symptoms?  Between 1-5 years of age  Between 6-12 years of age  Between 13-25 years of age  After 25 years of age

5.7 Approximate number of episodes  less than 1 per year NOT requiring medical attention  1 per year  1-5 every six month  1-3 every month  1 every week

6. Oral cavity bleeding (Tooth eruption, spontaneous or after brushing/flossing, gum bleeding, bleeding after bites to lip & tongue)

6.1 Have you ever had oral cavity  Yes  No or trivial skip to 7 bleeding ? 6.2 Have the symptom ever required Yes  No  skip to 6.6 medical attention ?

6.3 If answer to 6.2 is yes, please  Consultation only specify  Surgical hemostaisis (dental packing, suture,

cauterization)  Antifibrinolytics

 Treatment with desmopressin

 Treatment with plasma

 Treatment with platelet concentrate  Treatment with factor concentrates

 Blood (RBC) transfusion

6.4 How many times in your life did  1 - 2 you received any of the above  3 to 5 treatments (# 6.3)?  6 to 10  more than 10  Before 1 year 6.5 At what age did you first have symptoms?  Between 1-5 years of age  Between 6-12 years of age  Between 13-25 years of age  After 25 years of age

6.6 Approximate number of episodes  less than 1 per year NOT requiring medical attention  1 per year  1-5 every six month  1-3 every month  1 every week

6.7 Duration of average single  1 to 10 minutes episode (min.)  more than 10 minutes

7. Bleeding after Tooth/ Teeth extraction

7.1 Have you ever had bleeding after  Yes  No tooth (teeth) extraction ? If no extractions, skip to 7.2 Please specify number of extractions  section 8

Please fill in one of the following forms for each tooth extraction

Age at extraction Type of extraction  Deciduous   Permanent  Molar Actions taken to prevent  None bleeding  Antifibrinolytics  Desmopressin  Plasma or clotting factor concentrates  Platelet infusion

Bleeding after extraction? Yes  No 

Actions taken to control  None bleeding  Resuturing  Packing  Antifibrinolytics  Desmopressin  Plasma or clotting factor concentrates  Platelet infusion  Blood (RBC) transfusion

8. Bleeding after Surgery or Major Trauma

8.1 Have you ever had bleeding after  Yes  No surgery or major trauma ?

Please specify number of surgeries/ If no Surgery or Trauma 8.2 major trauma  skip to section 9

Please fill in one of the following forms for each surgery or major trauma episode

Age at intervention/trauma Type of surgery   Major-abdominal  Major-thoracic  Tonsillectomy/Adenoids  Major-gynecology  Pharynx/Nose  Other Actions taken to prevent  None bleeding  Antifibrinolytics  Desmopressin  Plasma or clotting factor concentrates  Platelet infusion

Bleeding after intervention? Yes  No 

Actions taken to control  None bleeding  Surgical hemostasis  Antifibrinolytics  Desmopressin  Plasma or clotting factor concentrates  Platelet infusion  Blood (RBC) transfusion

9. Menorrhagia

9.1 Have you ever had very heavy  Yes  No or trivial skip to 10 menstrual bleeding (menorrhagia)? If answer to 9.1 is yes, please Changing pads/tampons more frequently than specify every 2 hours Bleeding more than 7 days Clot and flooding

Impairment of daily Never or rarely activities (work, Most menses housework, exercise, social activities): 9.2 Have the symptom ever required  Yes  No skip to 9.6 medical attention ?

9.3 If answer to 9.2 is yes, please a  Consultation only specify

b  Pictorial Bleeding Score ______Assessment c  Antifibrinolytic therapy

d  Iron therapy e  Hormonal therapy f  Combined antifibrinolytics & Hormonal therapy

g  Hysterectomy / endometrial ablation / D & C  Treatment with desmopressin h  Treatment with plasma  Treatment with platelet concentrate  Treatment with factor concentrates i  Blood (RBC) transfusion l  Hospital admission and emergency treatment

9.4 How many times in your life did  1 - 2 you received any of the above  3 to 5 treatments (# 9.3 a-l)?  6 to 10  more than 10  At menarche 9.5 At what age did you first have symptoms?  Between 14-25 years of age  After 25 years of age

9.6 Have you had time off  < twice a year work/school for menorrhagia?  > twice a year  Since menarche 9.7 Duration of menorrhagia  > 12 months  < 12 months

9.8 Have you had acute menorrhagia  Yes  No requiring emergency How many times: ____ treatment/hospital admission

10. Post-partum hemorrhage

10.1 Number of successful pregnancies (live births)  10.2 Have you ever had post-partum  Yes  No or trivial skip to 11 haemorrhage? 10.3 Did it occur  In the first 24 hours after delivery (Primary)  Between 24 hours and 6 weeks postpartum

(Secondary)  Both Primary and Secondary 10.4 How long did vaginal discharge  < 6 weeks (lochia) last?  > 6 weeks 10.5 Did it require changing  Yes  No pads/tampons more frequently than every 2 hours? 10.6 Did this bleeding cause delay of  Yes  No hospital discharge/ readmission to hospital? 10.7 Have the symptom ever required  Yes  No medical treatment?

10.8 If answer to 10.7 is yes, please  Consultation only /oxytocin i.v. infusion specify

 Additional uterotonic medications

 Iron therapy

 Antifibrinolytic therapy

 Treatment with desmopressin

 Treatment with plasma  Treatment with platelet concentrate  Treatment with factor concentrates  Blood (RBC) transfusion

 Any procedure requiring examination under anaesthesia  Uterine balloon/package to tamponade the uterus  Any procedure requiring critical care or surgical intervention (includes: hysterectomy, internal iliac artery legation, uterine artery embolization, uterine brace sutures)

10.9 Number of deliveries that required any of the above  treatments (# 10.8)?

11. Muscle hematomas (spontaneous)

11.1 Have you ever had muscle  Yes  No or trivial skip to 12 hematomas or hemarthrosis ? If yes, was it spontaneous or after  Yes, spontaneous  No, trauma-related trauma? 11.2 Have the symptom ever required  Yes  No skip to 11.6 medical attention ? 11.3 If answer to 11.2 is yes, please  Consultation only specify

 Surgical draining

 Treatment with desmopressin

 Treatment with plasma

 Treatment with platelet concentrate  Treatment with factor concentrates

 Blood transfusion

11.4 How many times in your life did  1 - 2 you receive any of the above  3 to 5 treatments (# 11.3)?  6 to 10  more than 10  Before 1 year 11.5 At what age did you first have symptoms?  Between 1-5 years of age  Between 6-12 years of age  Between 13-25 years of age  After 25 years of age

11.6 Approximate number of episodes  less than 1 per year NOT requiring medical attention  1 per year  1-5 every six month  1-3 every month  1 every week

12. Hemarthrosis

12.1 Have you ever had muscle  Yes  No or trivial skip to 13 hematomas or hemarthrosis ? If yes, was it spontaneous or after  Yes, spontaneous  No, trauma-related trauma? 12.2 Have the symptom ever required  Yes  No skip to 12.6 medical attention ? 12.3 If answer to 12.2 is yes, please  Consultation only specify

 Surgical draining

 Treatment with desmopressin

 Treatment with plasma

 Treatment with platelet concentrate  Treatment with factor concentrates

 Blood transfusion

12.4 How many times in your life did  1 - 2 you receive any of the above  3 to 5 treatments (# 11.3)?  6 to 10  more than 10  Before 1 year 12.5 At what age did you first have symptoms?  Between 1-5 years of age  Between 6-12 years of age  Between 13-25 years of age  After 25 years of age

12.6 Approximate number of episodes  less than 1 per year NOT requiring medical attention  1 per year  1-5 every six month  1-3 every month  1 every week

13. CNS bleeding (spontaneous)

13.1 Have you ever had cranial or spinal  Yes  No or trivial skip to 14 bleeding? If yes, was it spontaneous or after  Yes, spontaneous  No, trauma-related trauma?

13.2 If answer to 13.1 is yes, please specify

Type of bleeding  Subdural  Intracerebral  Subarachnoid

Was the diagnosis made by  CT scan  MNR  Angiography

13.3 Type of treatment

 Consultation

 Surgical draining

 Treatment with plasma, platelet or factor concentrates

 Before 1 year 13.4 At what age did you have CNS bleeding?  Between 1-5 years of age  Between 6-12 years of age  Between 13-25 years of age  After 25 years of age

14 Other bleedings

14.1 Have you ever had one of the following? Excessive umbilical stump bleeding  Yes  No Cephalohematoma  Yes  No Bleeding at circumcision  Yes  No Venipuncture bleeding  Yes  No Suction Bleeding  Yes  No Ovulation bleeding(in women)  Yes  No 14.2 Have one of these symptoms ever  Yes  No required medical attention?

14.3 If answer to 12.2 is yes, please  Consultation only specify

 Antifibrinolytics

 Surgery

 Treatment with desmopressin

 Treatment with plasma

 Treatment with platelet concentrate  Treatment with factor concentrates

 Blood (RBC) transfusion

14.4 How many times in your life did  1 - 2 you receive any of the above  3 to 5 treatments (# 12.3) for this  6 to 10 symptom?  more than 10

Table 1. Bleeding score

SYMPTOMS SCORE (up to the time of diagnosis) 0§ 1§ 2 3 4 1.1.1.1.

Epistaxis No/trivial - > 5/year Consultation only* Packing or cauterization or Blood transfusion or replacement or antifibrinolytic therapy (use of hemostatic blood - more than 10 components and rFVIIa) or minutes desmopressin

Cutaneous No/trivial For bruises 5 or Consultation only* Extensive Spontaneous hematoma requiring more (> 1cm) in blood transfusion exposed areas

Bleeding from minor No/trivial - > 5/year Consultation only* Surgical hemostasis Blood transfusion, replacement wounds or therapy, or desmopressin - more than 10 minutes

Oral cavity No/trivial Present Consultation only* Surgical hemostasis or Blood transfusion, replacement antifibrinolytic therapy or desmopressin

GI bleeding No/trivial Present (not Consultation only* Surgical hemostasis, Blood transfusion, replacement associated with antifibrinolytic therapy or desmopressin ulcer, portal hypertension, hemorrhoids, angiodysplasia)

18 Hematuria No/trivial Present Consultation only* Surgical hemostasis, Blood transfusion, replacement (macroscopic) iron therapy therapy or desmopressin

Tooth extraction No/trivial or Reported in <25% Reported in >25% of Resuturing or packing Blood transfusion, replacement none done of all procedures, no all procedures, no therapy or desmopressin intervention** intervention**

Surgery No/trivial or Reported in <25% Reported in >25% of Surgical hemostasis or Blood transfusion, replacement none done of all procedures, no all procedures, no antifibrinolytic therapy or desmopressin intervention** intervention**

Menorrhagia No/trivial Consultation only* - Time off - Requiring combined - Acute menorrhagia requiring or work/school > 2/year treatment with hospital admission and - Changing pads or antifibrinolytics and hormonal emergency treatment more frequently - Requiring therapy or than every 2 hours antifibrinolytics or or - Requiring blood transfusion, or hormonal or iron - Present since menarche Replacement therapy, - Clot and flooding therapy and > 12 months Desmopressin, or or - PBAC score>100# - Requiring dilatation & curretage or endometrial ablation or

hysterectomy)

Post-partum No/trivial or Consultation only* - Iron therapy - Requiring blood transfusion, - Any procedure requiring critical hemorrhage no deliveries or or replacement therapy, care or surgical intervention (e.g. - Use of syntocin - Antifibrinolytics desmopressin hysterectomy, internal iliac artery or or legation, uterine artery - Lochia > 6 weeks - Requiring examination embolization, uterine brace under anaesthesia and/or the sutures) use of uterin balloon/package to tamponade the uterus

Muscle hematomas Never Post trauma, no Spontaneous, no Spontaneous or traumatic, Spontaneous or traumatic, therapy therapy requiring desmopressin or requiring surgical intervention or replacement therapy blood transfusion

19 Hemarthrosis Never Post trauma, no Spontaneous, no Spontaneous or traumatic, Spontaneous or traumatic, therapy therapy requiring desmopressin or requiring surgical intervention or replacement therapy blood transfusion

CNS bleeding Never - - Subdural, any intervention Intracerebral, any intervention

Other bleedings^ No/trivial Present Consultation only* Surgical hemostasis, Blood transfusion or replacement antifibrinolytics therapy or desmopressin

In addition to the guidance offered by the table, it is mandatory to refer to the text for more detailed instructions.

§ Distinction between 0 and 1 is of critical importance. Score 1 means that the symptom is judged as present in the patient’s history by the interviewer but does not qualify for a score 2 or more * Consultation only: the patient sought medical evaluation and was either referred to a specialist or offered detailed laboratory investigation ** Example: 1 extraction/surgery resulting in bleeding (100%): the score to be assigned is 2; 2 extractions/surgeries, 1 resulting in bleeding (50%): the score to be assigned is 2; 3 extractions/surgeries, 1 resulting in bleeding (33%): the score to be assigned is 2; 4 extractions/surgeries, 1 resulting in bleeding (25%): the score to be assigned is 1 # If already available at the time of collection ^ Include: umbilical stump bleeding, cephalohematoma, cheek hematoma caused by sucking during breast/bottle feeding, conjunctival hemorrhage or excessive bleeding following circumcision or venipuncture. Their presence in infancy requires detailed investigation independently from the overall score

20 Acknowledgments

We wish to acknowledge the collaboration of the other members of the ISTH/SSC Joint

VWF and Perinatal/Pediatric Hemostasis Subcommittees Working Group: Christoph Bidlingmaier

(Germany), Victor Blanchette (Canada), George Buchanan (USA), Jorge DiPaola (USA), Gili

Kenet (Israel), Robert Montgomery (USA), James Riddel (USA), Margaret Rand (Canada), and

Nicole Schlegel (France).

We are also most grateful to the members of the Menorrhagia Working Group of the

Women's Issues SSC/ISTH Subcommittee: Rezan A. Kadir (UK), Peter Kouides (USA), Christine

Lee (UK), Flora Peyvandi (Italy), Claire Philipp (USA), and Rochelle Winikoff (Canada) for their valuable contributions in the formulation of the questionnaire and BS, for menorrhagia and postpartum bleeding.

In addition, we wish to thank the personal contribution of Andra H. James (USA) and

Sabine Eichinger (Austria).

Naz et al. Thrombosis Journal (2017) 15:24 DOI 10.1186/s12959-017-0143-3

RESEARCH Open Access Identification of novel mutations in congenital afibrinogenemia patients and molecular modeling of missense mutations in Pakistani population Arshi Naz1* , Arijit Biswas2,6, Tehmina Nafees Khan1,7, Anne Goodeve3,8, Nisar Ahmed4, Nazish Saqlain4, Shariq Ahmed1,7, Ikram Din Ujjan5, Tahir S Shamsi1,7 and Johannes Oldenburg2,9

Abstract Background: Congenital afibrinogenemia (OMIM #202400) is a rare coagulation disorder that was first described in 1920. It is transmitted as an autosomal recessive trait that is characterized by absent levels of fibrinogen (factor I) in plasma. Consanguinity in Pakistan and its neighboring countries has resulted in a higher number of cases of congenital fibrinogen deficiency in their respective populations. This study focused on the detection of mutations in fibrinogen genes using DNA sequencing and molecular modeling of missense mutations in all three genes [Fibrinogen gene alpha (FGA), beta (FGB) and gamma (FGG)] in Pakistani patients. Methods: This descriptive and cross sectional study was conducted in Karachi and Lahore and fully complied with the Declaration of Helsinki. Patients with fibrinogen deficiency were screened for mutations in the Fibrinogen alpha (FGA), beta (FGB) and gamma (FGG) genes by direct sequencing. Molecular modeling was performed to predict the putative structure functional impact of the missense mutations identified in this study. Results: Ten patients had mutations in FGA followed by three mutations in FGB and three mutations in FGG, respectively. Twelve of these mutations were novel. The missense mutations were predicted to result in a loss of stability because they break ordered regions and cause clashes in the hydrophobic core of the protein. Conclusions: Congenital afibrinogenemia is a rapidly growing problem in regions where consanguinity is frequently practiced. This study illustrates that mutations in FGA are relatively more common in Pakistani patients and molecular modeling of the missense mutations has shown damaging protein structures which has profounding effect on phenotypic bleeding manifestations in these patients. Keywords: Afibrinogenemia, Molecular modeling, FGA gene, Consanguinity, Inherited bleeding disorder

Background from congenital or acquired causes. Congenital afibrino- Hemostasis is the normal physiological response that pre- genemia (OMIM #202400) is a rare coagulation disorder vents blood loss following vascular injury. It is dependent that was first described in 1920 [1]. It is as a recessive on an intricate series of events involving platelets and spe- autosomal inherited trait characterized by the absence of cific coagulation factors. Inherited bleeding disorders can fibrinogen (factor I) in plasma [2]. The disease has a be grouped into abnormalities of primary and secondary worldwide prevalence of 1–2 per million in the general hemostasis. Fibrinogen (Factor I) deficiency can originate population [3]. Fibrinogen is a 340 KDa hexameric protein of hepatic origin with multiple functions including roles in platelet aggregation and platelet plug formation and is an * Correspondence: [email protected]; [email protected] 1National Institute of Blood Diseases and Bone Marrow Transplantation, acute phase reactant [4]. It is secreted as zymogen similar Karachi University of Bonn, ST 2/A, Block-17, Gulshan-e-Iqbal KDA scheme, 24, to all other clotting factors and needs to be activated prior Karachi, Pakistan to its participation in the coagulation cascade. It consist of Full list of author information is available at the end of the article

© The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Naz et al. Thrombosis Journal (2017) 15:24 Page 2 of 8

three pairs (Aα,Bβ and Gγ) of polypeptide chains [5] symptoms. A diagnosis was made on the basis of history encoded by three genes (FGA, FGB and FGG)clusteredin and quantitative analysis. All subjects were registered at a region of approximately 50 kb on chromosome 4q28- Hemophilia Society of Pakistan. Samples from all centers q31 [6, 7]. The normal plasma levels of fibrinogen are were collected and initially processed and saved at the 4 g/l [8, 9] and its half-life is approximately 100 h/4 days National Institute of Blood Diseases (NIBD) for coagula- [10]. The main role of fibrinogen in hemostasis is to tion profile, biochemistry tests including liver profile and strengthen the platelet plug by converting into its poly- viral markers. DNA sequencing was performed in NIBD meric insoluble form called fibrin by thrombin [11]. The genome department, Karachi. fibrin meshwork traps red blood cells and platelets to form a plug which stops bleeding from site of injury. The Sample collection and lab assays absence of fibrinogen may result in excessive blood loss Blood samples from patients were collected in 3.2% so- after a trauma. Moreover spontaneous bleeding events dium citrate for coagulation profile in serum (RST) for can occur. Fibrinogen defects are classified as quantitative biochemistry analysis, including liver profile and viral (Hypofibrinogenemia and Afibrinogenemia, depending profile, (HBsAg, Anti HCV and HIV) and in K2EDTA upon the partial or complete absence of fibrinogen) or for complete blood count and DNA extraction for amp- qualitative (Dysfibrinogenemia and Hypodysfibrinogen- lification and sequencing. All sampling was performed emia) [12]. The most common symptom associated with with supportive infusion of cryoprecipitate to avoid fibrinogen deficiency is umbilical stump bleeding with bleeding. Platelet-poor plasma was collected by centri- other secondary bleeding manifestations including epi- fugation of citrate tubes at 4000×g for 10 min and co- staxis, gum bleeding, cutaneous bleeding, muscle agulation profile was performed, including PT, APTT hematoma and haemarthrosis [13]. and fibrinogen assay, using the Clauss method. Liver Congenital fibrinogen deficiency is considered as function tests (direct and indirect bilirubin, ALT, AST rare coagulation disorder but its incidence is growing and alkaline phosphatase) and viral markers (HBsAg, higher in those regions where consanguineous partner- anti HCV and HIV) were performed to exclude any ac- ships are common [14, 15]. Pakistan is the country quired cause of afibrinogenemia. with high ratio of consanguinity resulting in increasing Genetic analysis was performed after isolation of geno- numbers of rare inherited bleeding disorders including mic DNA using standard protocols, exons and intron- congenital afibrinogenemia. Our focus was to identify exon junctions of the fibrinogen genes were amplified by the mutations, assess the possible structure functional polymerase chain reaction [16] and sequenced [17] as impact of affected protein by using molecular model- previously described. ing/silico analysis tools. In addition to this, the study also encompasses the insight for possible mutational Pathogenecity scoring spectrum in frequently involved fibrinogen gene which Pathogenecity scoring was done by five prediction tools may contribute for future prenatal diagnosis of carriers of to predict the possible structure functional impact of af- these defects in Pakistani population. fected protein in identified novel missense mutations. The prediction software tool Poly-phen2 (polymorphism Methods phenotyping v2), (http://genetics.bwh.havard.edu/pph2/ Patient inclusion and exclusion criteria accessed on 20th April 2015) was used to assess the pos- This study, involving human subjects, was performed ac- sible impact of substitution on structure and function in cording to the Declaration of Helsinki, 1975, revised in human SNPs (Single nucleotide polymorphism). MUPRO 2000, and was approved by the relevant institutional (predictions of protein stability changes upon mutations), Ethical Committee. Patients with congenital afibrinoge- (http://mupro.proteomics.ics.uci.edu/ accessed on 20th nemia i.e. absent or undetectable levels of fibrinogen April 2015) utilizes an SVM (support vector machines) antigen (0–0.1 g/dl) and it activity in plasma were se- model to predict the changes in stability as a result of lected for this study. These low levels excluded acquired single-site mutations, primarily from sequential informa- causes of fibrinogen deficiency, such as liver disease and tion, and optionally provided structural information. The consumptive coagulopathies, leukemia or other factor result only predicts whether the alteration in single amino deficiencies. Patients from across Pakistan were re- acid will lead to destabilization or not. MUPRO predic- cruited from centers including Karachi (Sindh) and tions are reported with the confidence score (C score). A Lahore (Punjab). A written informed consent was taken positive score indicates higher stability whereas a negative from patients and guardians incase of minor. Sampling score shows the mutation decreases the protein stability was performed independent of sex or age. A comprehen- (http://mupro.proteomics.ics.uci.edu/ accessed on 20th sive questionnaire was completed containing informa- April 2015). SNP&GO (Single nucleotide polymorphism tion about the patient’s demographics and disease and GO terms, http://snps.biofold.org/snps-and-go Naz et al. Thrombosis Journal (2017) 15:24 Page 3 of 8

accessed on 20th April 2015). SIFT (Sorting Intolerant image rendering were performed with YASARA (Yet from Tolerant, http://sift.jcvi.org accessed 20th April 2015) Another Scientific Artificial Reality Application) version are algorithms which predict whether an amino acid substi- 12.8.6 (www.yasara.org/). tution will affect protein function based on sequence homology and the physical properties of amino acids. A SIFT Results score of less than 0.05 is predicted to be deleterious. A sub- Mutations were identified in all 13 patients. The major stitution with a score greater than or equal to 0.05 is pre- bulk of identified mutations is present in FGA gene dicted to be tolerated (http://www.exeterlaboratory.com/ which tends to be the most frequently occurring mu- molecular-genetics/). Provean (http://provean.jcvi.org/ tation site in our study population. Ten patients who about.php) accessed on 27th January 2015) has the default have mutations in FGA gene are individual unrelated threshold of −2.5 that means if the score of a variant is probands. equal or below this threshold then the mutation is said to Mutations in FGB gene are less frequent as compared be deleterious and if the threshold is above −2.5, the score to FGA. of variant is said to have neutral effects. Protein accession In FGA gene, eight mutations were identified as novel numbers were provided by Uniprot (Universal Protein and the remaining two were reported mutations. Eight Resource, http://www.uniprot.org/) and wild type color novel mutations include five missense, one nonsense fasta sequence was first accessed (http://pga.gs.washingto- and two frameshift mutations including homozygous n.edu/data/fga/fga.Colorfasta.html) on 27th January 2015 and a compound heterozygous frameshift mutation. The and later on 20th April 2015. two nonsense mutations in FGA are reported in literature. There is one more mutation with reported status in Structural analysis of novel missense mutations using proband (C3). This patient had compound heterozygous molecular modeling mutation with frameshift as novel mutation and non- Among the six reported novel missense mutations from sense as reported. this study, four mutations were located in an area of the We identified three mutations in FGB including one alpha chain that has no resolved crystal/NMR-based novel missense mutation (C9) and two homozygous structure (Nuclear magnetic Resonance). Thus, to assess nonsense mutations reported in siblings. the putative structural effect of these mutations, we mo- The FGG gene mutations are the rarest of all three deled this region on the ITASSER (Iterative Threading fibrinogen genes. We detected three novel mutations ASSEmbly Refinement) threading modeling server (http:// including two similar nonsense mutations in siblings zhanglab.ccmb.med.umich.edu/I-TASSER/; accessed on and one frameshift mutation in unrelated proband in 12th November 2014). The model for this region was then different exons of FGG gene (Table 1). joined to the remaining beta chain for which the structure All patients had markedly absent fibrinogen levels (0 g/l) has already been determined and submitted in the protein and prolonged PT >120 s and APTT >180 s (Table 2). structure database (PDB file ID: 3GHG; 2.9 Å resolution). Model joining was performed by replacing the last two Structural analysis of novel missense mutations using amino acid residues common to the model and the crystal molecular modeling structure (PDB file ID: 3GHG; chain A) (PDB: Protein A) alpha chain missense mutations Data Base, ID: Identity, 3ghg is a 4-character unique iden- All four novel missense mutations from the α-chain re- tifier of every entry in the Protein Data Bank) downloaded ported in this study were present in a region (residues from the protein structure database (http://rcsb.org/pdb/ 220–860) of the α-chain, which had no resolved/known home/home.do;) accessed 20th November 2014) to main- crystal structure. The region surrounding the reported tain the dihedral angles for the full model at the point of mutations (residues 300–400) was relatively poorly con- joining the same. The complete model was refined by a served with most of it missing from some fibrinogen short solvated simulation lasting 500 ps as described in homologues. Krieger et al., 2004 (Force field: Yamber3, periodic boun- Among the mutated residues, p.Pro302 was present in dary conditions, temperature: 298 K, water density: all homologues, which contained this part. The p.Ser325 0.997 g/L, pH: 7.4). The local neighborhood of the wild residue was also conserved in all homologues with the type residue corresponding to the reported mutation was exception of Musmusculus, where it was substituted by investigated to establish a logical hypothesis for the effect an Asn. The two Thr residues, p.Thr302 and p.Thr331, of the mutation. An additional one missense mutations were relatively variable and substituted by Ser or Asp in (p.Trp432Arg) in the beta chain lies on the structurally a few homologues. Only in the homologue from Canis resolved region of the PDB file 3GHG; chain B). Simi- lupus familiaris was one of the Thr residues (p.Thr302) larly the local molecular environment for this wild-type observed to be substituted by an Ala residue, which has residue was also inspected. All structural analysis and been reported as a mutated residue for both Thr Naz et al. Thrombosis Journal (2017) 15:24 Page 4 of 8

Table 1 Genotypic expression of mutations in fibrinogen gene (FGA, FGB & FGG) IP # Gene Exon Mutation Amino Acid change Zygosity Mutation type Reported/Novel C1 FGA 1 c.24C > A p.Cys8a Homozygous Nonsense Ref [23] € C2 2 c.143_144 del AA p.Lys(AAA)48Arg fs9a Compound Heterozygous Frame shift Novel mutation C3 5 c.846delG p.Gln282Thr fsx83a Compound Heterozygous Frame shift Novel mutation 4 c.385C > T p.Arg129a Homozygous Nonsense Ref [24] € C4 4 c.385 C > T p.Arg129a Homozygous Nonsense Ref [24] € C5 5 c.598C > T p.Gln183a Homozygous Nonsense Novel mutation C6 5 c.904C > G p.Pro302Ala Homozygous Missense Novel mutation C7 5 c.913A > G p.Thr 305 Ala Homozygous Missense Novel mutation C8 5 c.992A > G p.Thr331Ala Homozygous Missense Novel mutation C9 5 c.992A > G p.Thr331Ala Homozygous Missense Novel mutation C10 5 c.974A > G p.Ser325Gly Homozygous Missense Novel mutation C11A FGB 2 c.141 > T p.Arg47a Homozygous Nonsense Ref [25] € C11B 2 c.141C > T p.Arg47a Homozygous Nonsense Ref [25] € C9 8 c.1294 T > A p.Trp 432Arg Homozygous Missense Novel mutation C12 FGG 2 c.120_126dupTTCTTCA TTCTTCA Homozygous Frame shift Novel mutation C13A 4 c.361A > T p.Lys121a Homozygous Nonsense Novel mutation C13B 4 c.361A > T Lys121a Homozygous Nonsense Novel mutation Identified novel and reported mutations in three genes of fibrinogen. The letter A and B with patient code designate the sibling status. € (reported mutation,) c (complimentary deoxyribonucleic acid), A (adenine), T (thymine), C (cytosine), G (guanine), Lys (lysine), Arg (arginine), Tyr (tyrosine), Pro (proline), Trp (tryptophan), Thr (threonine), Gln (glycine), Cys = cystine, fs = frame shift, a stop codon number, FGA (fibrinogen Aα-chain gene), FGB (fibrinogen Bβ-chain gene), FGG (fibrinogen GƔ-chain gene

Table 2 Assessment of coagulation markers and bleeding scores with consanguinity/ethnicity IP# Fibrinogen Thrombin Prothrombin Activated partial thromboplastin Bleeding Consanguinity Interfamilial Ethnic Level (g/l) Time (Sec) Time (Sec) Time (aPTT)(Sec) Score Relation Origin C1 0.01 23 >120 >180 20 positive Unrelated Urdu Speaking C2 0.02 24 >120 >180 21 positive Unrelated Punjabi C3 0 33 >120 >180 22 positive Unrelated Punjabi C4 0.1 24 >120 >180 17 positive Unrelated Urdu Speaking C5 0.02 31 >120 >180 20 positive Unrelated Sindhi C6 0.01 25 >120 >180 20 positive Unrelated Urdu speaking C7 0.02 29 >120 >180 22 positive Unrelated Sindhi C8 0.0 30 >120 >180 20 positive Unrelated Sindhi C9 0.0 32 >120 >180 22 positive Unrelated Punjabi C10 0.01 25 >120 >180 16 positive Unrelated Punjabi C11A 0.02 28 >120 >180 18 positive ** Punjabi C11B 0.01 24 >120 >180 16 positive Punjabi C12 0.0 30 >120 >180 21 positive Unrelated Punjabi C13 0.01 24 >120 >180 20 positive Unrelated Punjabi C14 0.0 26 >120 >180 21 positive Unrelated Punjabi C15A 0.02 24 >120 >180 20 positive ** Punjabi C15B 0.01 25 >120 >180 21 positive Punjabi Shows the individual test values of PT, aPTT and fibrinogen (Claus Method), consanguinity and the relationship status. Bleeding score calculated, Tosetto et al. [26]. ** Siblings, NA not available, s (seconds). The fibrinogen levels in all patients were found to be equal to or lower than 0.1 g/l (Normal Range 2-4 g/dl), PT more than 120 s (Normal Range 9–11 s) aPTT more than 180 s (Normal Range 24–27 s) and prolonged thrombin time (normal range 10–13 s). Ethnicity explains the frequency of majorly affected, thickly populated and largest province of Pakistan (Punjab) Naz et al. Thrombosis Journal (2017) 15:24 Page 5 of 8

residues in our study. Modeling of this region showed chain. This densely packed hydrophobic core consists that this region could be split into two central cores, of a number of other aromatic acids, which are in close each of which is organized as a beta sheet surrounded proximity to p.Trp432 (p.His400 and p.His438, by flexible coils (Fig. 1). The two cores are connected by p.Trp433 and p.Tyr434). The p.Trp432 residue hydro- a central long helix. The first core, apart from being sur- gen bond contacts with p.Tyr434 and p.Ser406. rounded by flexible coils, also contains a few short helices. Three of the four reported mutated residues were Pathogenecity score located on these short helices with the exception that Pathogenecity scoring of six novel missense mutations p.Ser325 is located on a short loop connecting one of identified in FGA and FGB was done on five different the short helices to the central core. The residues pathogenicity scoring software (Table 3). Out of five mis- p.Pro302, p.Thr305 and p.Thr331 are partially buried, sense mutations of FGA, two mutations were found to with the p.Pro302 and p.Thr305 side chains oriented to- have damaging effect and decreased protein stability cal- ward the central core beta sheets. The residues p.Pro302 culated by two different softwares (MUPRO and Provean). and p.Thr305 participate in intra-helical hydrogen bonds Other software didn’t show the deleterious effect for the with each other and with p.Ser399 and p.Arg308, re- same two mutations identified in two unrelated proband. spectively. The residue p.Thr331 lies at the edge of a In FGB gene the missense mutation was found to be short helix and also participates in intra-helical hydrogen damaging or deleterious and showed decreased structure bonding (p.Gly327). Interestingly, within the fold on stability. The damaging effect and lack of protein stability which all four of these mutations reside, lysine residue in structure may lead to the bleeding manifestations in p.Lys322 is known to be cross-linked to ∝−2 antiplas- patients which can vary from mild to severe bleeding. min proteins and a glutamine residue, p.Gln347, which participates in inter-chain cross-links during clot Discussion formation. Fibrinogen deficiency is a rare inherited bleeding disorder that is characterized by two subtypes of either reduced or B) Beta chain missense mutations completely absent levels of fibrinogen in the blood [18]. The one novel missense mutations (p.Trp432Arg) reported FGA is documented as the most affected gene in literature in the chain occurs in a highly conserved region. (Fig. 2). [19, 20]. We have found the larger chunk of mutations in The residues are completely conserved in homo- FGA gene in our set of data. A total of 169 mutations in fi- logues that have been used for the present alignment. brinogen are reported on the Human Gene Mutation The p.Trp432 residue lies completely in the densely Database (http://www.hgmd.cf.ac.uk/ac/index.php) date packed hydrophobic core of the C-terminal region of accessed August 12, 2014). Consanguinity involving

Fig. 1 Molecular Remodeling of a missense mutation in FGA Naz et al. Thrombosis Journal (2017) 15:24 Page 6 of 8

Fig. 2 Molecular Remodeling of a missense mutation in FGB

first and second cousin marriages is accelerating the mutations in our study results. A frame shift mutation spread of disease in areas such as Pakistan, Iran, the (p.Glu262AspfsX158) in FGA exon 5 reported in one Middle East, China and the far Middle East, including study is predicted as truncated polypeptide. It is as- Turkey, in societies where consanguinity is frequent. sociated with exceptionally long stretch of abnormal The spectrum of causative mutations for afibrinoge- residues in homozygous patient with congenital afi- nemia is interesting as FGA appears to stand out from brinogenemia [22]. Frameshift mutation (p.Gln282Thr the two other fibrinogen genes [21]. The predominant fsx83*) and (p. Lys (AAA) 48Arg fs9*) are the novel inheritance pattern was homozygous with a high pro- compound heterozygous mutations which have mani- portion of nonsense mutations followed by missense fested deletions along with frameshift defects. The

Table 3 Pathogenicity score of missense mutations Missense Polyphen- 2 Provean MUpro SNP&GO Sift Mutations Score prediction Score prediction SVM score Protein structure stability Score Prediction Score Prediction p.Pro302Ala 0.028 Benign −4.257 Deleterious −0.797 Decrease stability (0.4) Neutral 0.00 Benign p.Thr 305 Ala 0.00 Benign −0.387 Neutral 0.134 Increase stability (0.05) Neutral 0.00 Benign p.Thr331Ala 0.025 Benign −1.100 Neutral 0.122 Increase stability (0.03) Neutral 0.00 Benign p.Thr331Ala 0.025 Benign −1.100 Neutral 0.122 Increase stability (0.03) Neutral 0.00 Benign p.Ser325Gly 0.014 Benign −2.331 Neutral −0.063 Decrease stability (0.1) Neutral 0.00 Benign p.Trp 432Arg 1.00 Damaging −12.18 Deleterious −0.411 Decrease stability (0.8) Disease Na Na Pathogenecity of missense mutations was calculated by five different softwares to check for the protein structure stability and deleterious effects. Na not available Naz et al. Thrombosis Journal (2017) 15:24 Page 7 of 8

bleeding phenotype is severe as these mutations worsen and their impact on the clinical manifestation of pa- the symptoms due to combined effect of compound tients. In this way the genotype well correlated with mutation and truncation of polypeptide chain. phenotype of these patients. Three missense mutations (Pro302Ala, Thr305Ala and Abbreviations Thr331Ala) in the alpha chain reside on short helices Ala: Alanine; ALT: Alanine transaminase; Anti-HCV: Anti hepatitis C antibodies; surrounding a central beta sheet core. All these muta- aPTT: Activated partial thromboplastin time; Arg: Arginine; Asn: Asparagine; tions are non-conservative in nature, i.e., the Pro302Ala AST: Aspartate transaminase; C score: Confidence score; EDTA: Ethylenediaminetetraacetic acid; FGA: Fibrinogen gene alpha; substitution results in the replacement of a rigid imino FGB: Fibrinogen gene beta; FGG: Fibrinogen gene gamma; HBsAG: Hepatitis group with a smaller, more flexible residue, and the B surface antigen; HIV: Human immunodeficiency virus; ITASSER: Iterative Thr305Ala and Thr302Ala substitutions result in the re- threading assembly refinement; KDa: Kilo daltons; Poly-phen2: Polymorphism phenotyping v2; Pro: Proline; Provean: Protein variation effect analyzer; placement of polar side chains by smaller but hydropho- PT: Prothrombin time; SIFT: Sorting intolerant from tolerant; SNP: Single bic side chains. In addition, the introduction of alanine nucleotide polymorphism; SNP&GO: Single nucleotide polymorphism and in these regions will most likely disrupt some of the GO terms; SVM: Support vector machine; Trp: Tryptophan; TT: Thrombin time; UniProt: Universal protein resource; YASARA: Yet another scientific artificial intra-helical hydrogen bonds, thereby breaking the he- reality application lical structure surrounding the central core. Because these short helices provide order and stability around an Acknowledgements otherwise disordered coiled-coil region, their disruption We thank all the patients and their families who participated in this study. We acknowledge Marguerite Neerman-Arbez (Professor, Department of might result in a loss of stability for this region and the Genetic Medicine and Development University Medical Centre) Switzerland alpha chain. The third mutation in this chain, Ser325Gly, for generously providing primer sequences and help with validation of the is also non-conservative, i.e., it results in the substitution results. We would also like to acknowledge the contribution of Dr. Philipe de Moerloose (Hôpitaux Universitaires de Genève HUG) for their unconditional of a polar residue to a very small and flexible Gly resi- support in establishing the technique. We would like to thank Dr.Shahla Tariq due. Because the wild-type residue already lies on the and Dr. Ayisha Imran from Chughtai’s Lab, Lahore for their contribution in flexible loop, the introduction of a small residue will providing samples for this study. make this region more disordered and therefore un- Funding stable. Moreover, because all four missense mutations This entire project had been funded by Novo Nordisk Hemophilia Foundation belong to a fold of the alpha chain that might be inter- (NNHF) as PK-4 funding. All expenditures inclusive of collection of samples till their processing and sequencing were carried out under the PK-4 funding. The acting with Factor XIII (this fold also contains the Lys processing of all samples from CBC, DNA extraction, PCR and Direct sequencing and Gln residues that participate in interchain cross was conducted in our institution under PK4 project. linking and cross linking to alpha 2- antiplasmin), con- We sent extracted DNAs of four samples to University of Geneva, Switzerland, for sequencing and validated their results and interpretations in our institution formational changes induced by these mutations on this as well and the entire portion of molecular modeling was done in Bonn, fold might interfere with the interaction of fibrinogen Germany. The manuscript writing, Study design and interpretation of the alpha chain with Factor XIII. The one beta chain mis- data were completely independent of the role of funding body and done sense mutation resides on a highly conserved region of without any monetary expenditure. the beta chain, most likely because many of the residues Availability of data and materials of this region contribute to the stability of its densely This will be provided once the paper will get your kind approval of acceptance packed hydrophobic core. The p.Trp432Arg substitution for publication. occurs in the middle of the hydrophobic core. The intro- Consent duction of a large polar, positively charged residue ins- All participant/guardians in case of minors has given their consent by signing tead of a hydrophobic aromatic one would destabilize the consent forms in English and local language after proper explanation of the procedure and purpose. the hydrophobic core of this region. Thus, the mutation affects the stability of the beta chain by disrupting its Authors’ contributions C-terminal hydrophobic core. AN designed and supervised the research. AB reviewed the data, performed molecular remodeling and edited the text. TNK wrote the paper and analyzed the samples. Conclusions AG reviewed the paper, edited the text and contributed to the mutation Rare inherited bleeding disorder specifically congenital nomenclature and other necessary changes in the text. ’ afibrinogenemia has a growing incidence especially in NA provides samples from the Children s Hospital Lahore. NS Helped in making diagnosis and sample collection from the Children’s regions like Pakistan where consanguinity factor is Hospital Lahore. strongly present. Our study is purely based on Pakistani SA DNA extraction, PCR and gene sequencing of samples. Result analyses. patients of congenital afibrinogenemia. It has shown the IDU reviewed paper. TSS project approval and secured funding for this study. frequently affected gene FGA in our set of patients. We JO Supported in establishing the technique and validation of results along have documented the pathogenicity scores for missense with data review. All authors read and approved the final manuscript mutations as a description for protein molecule stability Ethics approval and consent to participate and functional defects. We have also performed molecu- The Ethics Committee of National Institute of blood diseases and bone marrow lar modeling to see the structural defects and damages transplantation has approved this study. Naz et al. Thrombosis Journal (2017) 15:24 Page 8 of 8

Consent for publication 13. Asselta R, Spena S, Duga S, Peyv IF, Malcovati M, Mannucci P, Tenchini M. ‘Written informed consent for publication of their clinical details was obtained Analysis of Iranian patients allowed the identification of the first truncating from the patient/parent/guardian/relative of the patient. mutation in the fibrinogen Bbeta-chain gene causing afibrinogenemia. Haematologica. 2002;87:855–9. Competing interests 14. Wu S, Wang Z, Dong N, Bai X, Ruan C. A novel nonsense mutation in the The authors declare that they have no competing interests. FGA gene in a Chinese family with congenital afibrinogenaemia. Blood coagulation & fibrinolysis. 2005;16(3):221–6. ’ 15. Sumitha E, Jayandharan G, Arora N, Abraham A, David S, Devi GS, Publisher sNote Shenbagapriya P, Nair SC, George B, Mathews V, Chandy M, Viswabandya A, Springer Nature remains neutral with regard to jurisdictional claims in published Srivastava A. Molecular basis of quantitative fibrinogen disorders in 27 maps and institutional affiliations. patients from India. Haemophilia. 2013;19:611–8. 16. Tyrrell DAJ. Polymerase Chain Reaction. BMJ. 1997;314(7073):5–5. Author details 1 17. Sanger Sequencing Method | Thermo Fisher Scientific [Internet]. National Institute of Blood Diseases and Bone Marrow Transplantation, Thermofisher.com. 2017 [cited 2 Febuary 2017]. Available from: https:// Karachi University of Bonn, ST 2/A, Block-17, Gulshan-e-Iqbal KDA scheme, 24, www.thermofisher.com/pk/en/home/lifescience/sequencing/sanger- Karachi, Pakistan. 2Institute of Experimental Hematology and Transfusion 3 sequencing/sanger_sequencing_method.html Medicine, Bonn, Germany. University of Shieffield, Shiefield, United 18. Korte W, Poon M, Iorio A, Makris M. Thrombosis in Inherited Fibrinogen 4 ’ Kingdom. Children s Hospital, Resident, Paediatric hematology, Main Disorders. Transfusion Medicine and Hemotherapy. 2017;44(2):70–6. Ferozpur Road, Lahore, Pakistan. 5Liaquat university of medical and health 6 19. Neerman-Arbez M. Prenatal diagnosis for congenital afibrinogenemia sciences, Jamshoro, Pakistan. Institute of Experimental Hematology and caused by a novel nonsense mutation in the FGB gene in a Palestinian Transfusion Medicine, AG, FXIII Room No. 2.308 Sigmund Freud Street-25, – 7 family. Blood. 2003;101(9):3492 4. 53127 Bonn, Germany. National Institute of blood diseases and bone 20. VU D, NEERMAN-ARBEZ M. Molecular mechanisms accounting for marrow transplantation, ST 2/A, Block-17, Gulshan-e-Iqbal KDA scheme, 24, 8 fibrinogen deficiency: from large deletions to intracellular retention of Karachi, Pakistan. Clinical Scientist and Professor of Molecular Medicine, misfolded proteins. Journal of Thrombosis and Haemostasis. 2007;5:125–31. Sheffield Diagnostic Genetics Service, Sheffield Children’s NHS Foundation 9 21. Robert-Ebadi H, De Moerloose P, El Khorassani M, El Khattab M, Neerman- Trust, Western Bank, Sheffield S10 2TH, UK. Institute of Experimental Arbez M. A novel frameshift mutation in FGA accounting for congenital Hematology and Transfusion Medicine, Sigmund Freud Street-25, 53127 afibrinogenemia predicted to encode an aberrant peptide terminating 158 Bonn, Germany. amino acids downstream. Blood Coagulation & Fibrinolysis. 2009;20(5):385–7. 22. Levrat E, Aboukhamis I, de Moerloose P, Farho J, Chamaa S, Reber G, et al. A Received: 9 November 2016 Accepted: 28 June 2017 novel frameshift mutation in FGA (c.1846 del A) leading to congenital afibrinogenemia in a consanguineous Syrian family. Blood Coagulation & Fibrinolysis. 2011;22(2):148–50. References 23. Asselta R, Platè M, Robusto M, Borhany M, Guella I, Soldà G, et al. Clinical 1. Peyvandi F, Haertel S, Knaub S, Mannucci P. Incidence of bleeding symptoms and molecular characterisation of 21 patients affected by quantitative in 100 patients with inherited afibrinogenemia or hypofibrinogenemia. Journal fibrinogen deficiency. Thrombosis and Haemostasis. 2014;113(3):567–76. – of Thrombosis and Haemostasis. 2006;4:1634 7. 24. Palermo P, Barbados B, Asselta R, Duga S, Tenchini M. The molecular basis 2. Acharya S, Dimichele D. Rare inherited disorders of fibrinogen. Haemophilia. of quantitative fibrinogen disorders. Journal of Thrombosis and – 2008;14:1151 8. Haemostasis. 2006;4:2115–29. 3. Neerman-Arbez M, De Moerloose P, Bridel C, Honsberger A, Sconborner A, 25. Sheen C, Brennan S, Jabado N, George P. Fibrinogen Montreal: a novel Rossier C, Peerlinck K, Claeyssens S, Di Michele D, D'oiron R, Dreyfus M, missense mutation (A alpha D496N) associated with hypofibrinogenemia. Laubriat-Bianchin M, Dieval J, Antonarakis SE, Morris MA. Mutations in the Thrombosis and haemostasis. 2006;96:231–2. fibrinogen Aa gene account for the majority of cases of congenital 26. Tosetto A, Rodeghiero F, Castaman G, Goodeve A, Federici A, Batlle J, Meyer – afibrinogenemia. Blood. 2000;96:149 52. D, Fressinaud E, Mazurier C, Goudemand J, Eikenboom J, Schneppenheim R, 4. Janciauskiene S, Welte T, Mahadev R. Acute Phase Proteins: Structure and Budde U, Ingerslev J, Vorlova Z, Habart D, Holmberg L, Lethagen S, Pasi J, Function Relationship. Acute Phase Proteins - Regulation and Functions of Hill F, Peake I. A quantitative analysis of bleeding symptoms in type 1 von Acute Phase Proteins. 2011;. Willebrand disease: results from a multicenter European study (MCMDM-1 5. Neerman-Arbez M, De Moerloose P, Honsberger A, Parlier G, Arnuti B, Biron VWD). J Thromb Haemost. 2006;4:766–73. C, Borg J, Eber S, Meili E, Peter-Salonen K, Ripoll L, Vervel C, d'Oiron R, Staeger P, Antonarakis SE, Morris MA. Molecular analysis of the fibrinogen gene cluster in 16 patients with congenital afibrinogenemia: novel truncating mutations in the FGA and FGG genes. Human genetics. 2001; 108:237–40. 6. Anwar M, Iqbal H, Gul M, Saeed N, Ayyub M. Congenital afibrinogenemia: report of three cases. Journal of Thrombosis and Haemostasis. 2005;3:407–9. 7. Neerman-Arbez M, De Moerloose P. Mutations in the fibrinogen gene cluster accounting for congenital afibrinogenemia: an update and report of 10 novel mutations. Human Mutation. 2007;28:540–53. 8. Asselta R, Duga S, Tenchini M. The molecular basis of quantitative fibrinogen Submit your next manuscript to BioMed Central disorders. Journal of Thrombosis and Haemostasis. 2006;4:2115–29. 9. Grieninger G, Lu X, Cao Y, Fu Y, Kudryk BJ, Galanakis DK, Hertzberg KM. and we will help you at every step: Fib420, the novel fibrinogen subclass: newborn levels are higher than adult. Blood. 1997;90:2609–14. • We accept pre-submission inquiries 10. Fang Y, DAI B, WANG X, FU Q, Dai J, Xie F, CAI X, WANG H, WANG Z. • Our selector tool helps you to ﬁnd the most relevant journal Identification of three FGA mutations in two Chinese families with • We provide round the clock customer support congenital afibrinogenaemia. Haemophilia. 2006;12:615–20. 11. Asselta R, Duga S, Simonic T, Malcovati M, Santagostino E, Giangr EP, • Convenient online submission Mannucci P, Tenchini M. Afibrinogenemia: first identification of a splicing • Thorough peer review mutation in the fibrinogen gamma chain gene leading to a major gamma • Inclusion in PubMed and all major indexing services chain truncation. Blood. 2000;96:2496–500. 12. Casini A, Neerman-Arbez M, Ariëns R, de Moerloose P. Dysfibrinogenemia: • Maximum visibility for your research from molecular anomalies to clinical manifestations and management. Journal of Thrombosis and Haemostasis. 2015;13(6):909–19. Submit your manuscript at www.biomedcentral.com/submit Naz et al. Thrombosis Journal (2019) 17:5 https://doi.org/10.1186/s12959-019-0193-9

CORRECTION Open Access Correction to: Identification of novel mutations in congenital afibrinogenemia patients and molecular modeling of missense mutations in Pakistani population Arshi Naz1* , Arijit Biswas2, Tehmina Nafees Khan1, Anne Goodeve3, Nisar Ahmed4, Nazish Saqlain4, Shariq Ahmed1, Ikram Din Ujjan5, Tahir S. Shamsi1 and Johannes Oldenburg2

Correction to: Thromb J (2017) 15:24 https://doi.org/10.1186/s12959-017-0143-3

Following the publication of this article [1], the authors including two similar nonsense mutations in siblings noted the following typographical errors: and one frameshift mutation in unrelated proband in different exons of FGG gene (Table 1).” 1) Affiliation 3 should read “University of Sheffield, Should be: Sheffield, United Kingdom” and Affiliations 6, 7, 8 “In FGA gene, seven mutations were identified as and 9 were unnecessary duplicates novel and the remaining three were reported mutations. 2) In the abstract the sentence “Ten patients had Seven novel mutations include five missense and two mutations in FGA followed by three mutations in frameshift mutations including homozygous and a com- FGB and three mutations in FGG, respectively” pound heterozygous frameshift mutation. The three should be “Ten patients had mutations in FGA nonsense mutations in FGA are reported in literature. followed by four mutations in FGB and two There is one more mutation with reported status in pro- mutations in FGG, respectively.” band (C3). This patient had compound heterozygous 3) In the Results section the following three sentences: mutation with frameshift as novel mutation and nonsense as reported. We identified four mutations in FGB “In FGA gene, eight mutations were identified as novel including one novel missense mutation (C9), two homo- and the remaining two were reported mutations. Eight zygous nonsense mutations reported in siblings and one novel mutations include five missense, one nonsense frameshift mutation(C12). The FGG gene mutations are and two frameshift mutations including homozygous the rarest of all three fibrinogen genes. We detected two and a compound heterozygous frameshift mutation. The novel similar nonsense mutations in siblings (Table 1).” two nonsense mutations in FGA are reported in literature. There is one more mutation with reported status in 4) There are a number of errors in Tables 1 and 2.The proband (C3). This patient had compound heterozygous corrected versions are provided in this Correction mutation with frameshift as novel mutation and non- article with the corrections given in bold. sense as reported. We identified three mutations in FGB 5) Frameshift mutation (p.Gln282Thr fsx83*) and including one novel missense mutation (C9) and two (p. Lys (AAA) 48Arg fs9*) are the novel compound homozygous nonsense mutations reported in siblings. heterozygous mutations which have manifested The FGG gene mutations are the rarest of all three fi- deletions along with frameshift defects” should in brinogen genes. We detected three novel mutations fact read “Frameshift mutations (p.Thr283Arg fs138*) and (p. Lys (AAA) 48Arg fs9*) are the novel * Correspondence: [email protected]; [email protected] compound heterozygous mutations which have 1 National Institute of Blood Diseases and Bone Marrow Transplantation, manifested deletions along with frameshift defects. Karachi, Pakistan Full list of author information is available at the end of the article

© The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Naz et al. Thrombosis Journal (2019) 17:5 Page 2 of 3

Table 1 Genotypic expression of mutations in fibrinogen gene (FGA, FGB & FGG) IP # Gene Exon Mutation Amino Acid change Zygosity Mutation type Reported/Novel C1 FGA 1 c.24C > A p.Cys8* Homozygous Nonsense Ref #23 € C2 2 c.143_144 del AA p.Lys (AAA)48Arg fs9* Compound Heterozygous Frame shift Novel mutation C3 5 c.846delG p.Thr 283Arg fs138* Compound Heterozygous Frame shift Novel mutation 4 c.385C > T p.Arg129* Homozygous Nonsense Ref #24 € C4 4 c.385 C > T p.Arg129* Homozygous Nonsense Ref #24 € C5 5 c.598C > T p.Gln200* Homozygous Nonsense Ref 27* C6 5 c.904C > G p.Pro302Ala Homozygous Missense Novel mutation C7 5 c.913A > G p.Thr 305 Ala Homozygous Missense Novel mutation C8 5 c.992A > G p.Thr331Ala Homozygous Missense Novel mutation C9i 5 c.992A > G p.Thr331Ala Homozygous Missense Novel mutation C10 5 c.973A > G p.Ser325Gly Homozygous Missense Novel mutation C11A FGB 2 c.141 > T p.Arg47* Homozygous Nonsense Ref # 25 € C11B 2 c.141C > T p.Arg47* Homozygous Nonsense Ref # 25 € C9 ii 8 c.1294T > A p.Trp 432Arg Homozygous Missense Novel mutation C12 2 c.118_124dupTTCTTCA TTCTTCA Homozygous Frame shift Novel mutation C13A FGG 4 c.361A > T p.Lys121* Homozygous Nonsense Novel mutation C13B 4 c.361A > T Lys121* Homozygous Nonsense Novel mutation Identified novel and reported mutations in three genes of fibrinogen. The letter A and B with patient code designate the sibling status, i & ii shows mutation identified in same patient but in different genes, € (repor`ted mutation) c (complimentary deoxyribonucleic acid), A (adenine), T (thymine), C (cytosine), G (guanine), Lys (lysine), Arg (arginine), Tyr (tyrosine), Pro (proline), Trp (tryptophan), Thr (threonine), Gln (glycine), Cys = cystine, fs = frame shift, * stop codon number, FGA (fibrinogen Aα-chain gene), FGB (fibrinogen Bβ-chain gene), FGG (fibrinogen GƔ-chain gene.

Table 2 Assessment of coagulation markers and bleeding scores with consanguinity/ethnicity IP# Fibrinogen Thrombin Prothrombin Activated partial thromboplastin Time Bleeding Consanguinity Interfamilial Ethnic Level Time Time (aPTT) (Sec) Score Relation Origin (g/l) (Sec) (Sec) *C1 0.01 23 > 120 > 180 20 positive Unrelated NA C2 0.02 24 > 120 > 180 21 positive Unrelated Punjabi C3 0 33 > 120 > 180 22 positive Unrelated Punjabi C4 0.1 24 > 120 > 180 17 positive Unrelated Urdu Speaking C5 0.02 31 > 120 > 180 20 positive Unrelated Sindhi C6 0.01 25 > 120 > 180 20 positive Unrelated Urdu speaking C7 0.02 29 > 120 > 180 22 positive Unrelated Sindhi C8 0.0 30 > 120 > 180 20 positive Unrelated Sindhi C9 0.0 32 > 120 > 180 22 positive Unrelated Punjabi C10 0.01 25 > 120 > 180 16 positive Unrelated Punjabi C11A 0.02 28 > 120 > 180 18 positive ** Punjabi C11B 0.01 24 > 120 > 180 16 positive Punjabi C12 0.0 30 > 120 > 180 21 positive Unrelated Punjabi C13A 0.02 24 > 120 > 180 20 positive ** Punjabi C13B 0.01 25 > 120 > 180 21 positive Punjabi Shows the individual test values of PT, aPTT and fibrinogen (Clauss Method), consanguinity and the relationship status. Bleeding score calculated, Tosetto et al [26]. ** Siblings, NA = not available, s (seconds). The fibrinogen levels in all patients were found to be equal to or lower than 0 .1g/l (Normal Range 2-4 g/dl), PT more than 120 s (Normal Range 9–11 s) aPTT more than 180 s (Normal Range 24–27 s) and prolonged thrombin time (normal range 10–13 s). Ethnicity explains the frequency of majorly affected, thickly populated and largest province of Pakistan (Punjab). Naz et al. Thrombosis Journal (2019) 17:5 Page 3 of 3

Author details 1National Institute of Blood Diseases and Bone Marrow Transplantation, Karachi, Pakistan. 2Institute of Experimental Hematology and Transfusion Medicine, Bonn, Germany. 3University of Sheffield, Sheffield, UK. 4Children’s Hospital, Lahore, Pakistan. 5Liaquat university of medical and health sciences, Jamshoro, Pakistan.

Received: 6 March 2019 Accepted: 6 March 2019

Reference 1. Naz A, et al. Identification of novel mutations in congenital afibrinogenemia patients and molecular modeling of missense mutations in Pakistani population. Thromb J. 2017;15:24. https://doi.org/10.1186/s12959-017-0143-3. GLOBAL CAPACITY-BUILDING SHOWCASE

Establishment of diagnostic facilities for autosomal recessive bleeding disorders in Pakistan

Arshi Naz, Humayun Patel, Shariq Ahmed, Tariq Masood, Tehmina Nafees, Younus Jamal, Munira Borhany, Saqib Ansari, Mehwesh Taj, Tasneem Farzana, and Tahir S. Shamsi

Department of Pathology and Clinical Hematology, National Institute of Blood Disease and Bone Marrow Transplantation, Karachi, Pakistan

Background

Pakistan has a population of 200 million, and 40% of the people live in rural areas. Autosomal recessive bleeding disorders (ARBDs) are not rare because of consanguinity. Only basic hemostatic testing was available before 2001. The ability to diagnose hemophilia A and B was available in only 2 to 3 tertiary care hospitals, but they did not have the expertise to measure hemophilia inhibitors. Very little literature was published regarding our population because of the lack of expertise and facilities. All congenital bleeders were considered as having hemophilia A or B. The capacity-building project of hemophilia and bleeding disorders was planned on the principles of build, operate, and transfer in 2007. Objectives

1. To produce master trainers to teach local health care providers for the diagnosis of ARBDs. 2. To provide materials for labelling of actual bleeding disorders rather than hemophilia. 3. To screen all registered participants according to the planned protocol. 4. To explore rare congenital bleeding disorders rather than those that are common. 5. To provide genetic analysis for all inherited bleeding disorders. 6. To share patient data according to the project regulations. 7. To determine the prevalence of multiple clotting defects according to the regulations. 8. To publish and share information and data regarding the project. Hypotheses

A. Collaborative research-based investigations of locally important health conditions are an effective method of capacity building with sustainable goals. B. The prevalence of von Willebrand disease (VWD) will be higher than that of hemophilia A and B in Pakistan. C. Rare bleeding disorders will not be so rare in Pakistan. D. Novel mutations will be identified. Methods The study was approved by the ethics committee of the National Institute of Blood Diseases and Bone Marrow Transplantation (NIBD), Karachi, Pakistan, in accordance with the Declaration of Helsinki. It was a descriptive study with a cross-sectional time prospect and was conducted from March 2007 to December 2014. The study was divided into 2 phases. In the first phase, only patients from Karachi or Sindh were screened. In phase 2, patients were selected from all areas of Pakistan. A doctor at each corresponding recruitment center filled out a general questionnaire with basic demographic details and clinical and family histories and also completed the Tosetto bleeding score questionnaire for each patient. To identify the prevalence period for various ARBDs, records from the current study were merged with those from all the studies reported in the last 12 years. For this purpose, the common national (PakMedinet) and international (PubMed, Google Scholar, ISI Web of Science, EMBASE, and SCOPUS) databases were searched for studies on ARBDs in the Pakistani population.

30 NOVEMBER 2018 • VOLUME 2, SUPPLEMENT 1 GLOBAL CAPACITY-BUILDING SHOWCASE 35

From www.bloodadvances.org by guest on April 24, 2019. For personal use only. Capacity building

Figure 1. Capacity building for the diagnosis of ARBDs across Pakistan. PCR, polymerase chain reaction.

Figure 2. (A) Samples were sent to the central reference laboratory at NIBD where the tests were repeated to establish reliability. (B) The following tertiary health care centers (from left to right) are shown: Hayatabad Medical Complex (HMCP); Lady Reading Hospital (LRHP); Khyber Pakhtunkhwa (KP); Fatimid Foundation Karachi (FFK); National Institute of Blood Diseases (NIBD); Chughtai’s Laboratory (CL); Children’s Hospital Lahore (CHL); and Pakistan Atomic Energy Commission (PAEC). For each tertiary health care center, “N” indicates the number of patients with ARBDs and the number in parentheses indicates the total number of patients initially recruited from each center. Prothrombin time (PT), activated partial thromboplastin time (APTT), bleeding time, and fibrinogen levels were measured. Patients with isolated prolonged APTT were tested for factor VIII (FVIII) and FIX using factor assays This was followed by FXI:C-level assessment for patients with normal FVIII and FIX levels. Samples were reanalyzed except platelet aggregation studies. All rare clotting factor defects such as FII, VII, X, XI , and XIII were tested in a reference laboratory (NIBD) because of the unavailability of testing facilities. Platelet aggregation studies were repeated in PAEC, CL, CHL, and NIBD. Patients with low FVIII levels were screened for VWD. Patients with simultaneous prolongation of PT and APTT were tested for FII, FV, and FX. Peripheral blood film examination and platelet aggregation studies were performed to assess platelet disorders. Urea clot solubility testing was performed to detect FXIII levels if platelet function tests were normal. Sanger sequencing was performed for genetic analysis of patient samples. Descriptive analysis was performed using SPSS version 16software.

36 GLOBAL CAPACITY-BUILDING SHOWCASE 30 NOVEMBER 2018 • VOLUME 2, SUPPLEMENT 1

From www.bloodadvances.org by guest on April 24, 2019. For personal use only. Results the cohort of patients included gum bleeding (57%) and easy bruising (39%). Spontaneous epistaxis and gum bleeding were The study cohort consisted of 429 patients (250 males and 179 females) with a male:female ratio of 1.3:1. The median age of foundin6%,andmenorrhagiawasreportedin19%oftheadult patients was 11 6 5 years. A history of consanguinity was present female patients. Anemia was found in 48% of the patients. Life- in 89% of the patients. The most common symptoms reported by threatening intracranial hemorrhage affected 4% of the patients.

Figure 3. Comparative studies of patients of different nationalities who have ARBDs. BSS, Bernard-Soulier syndrome; GT, Glanzmann thrombasthenia.

Table 1. Frequency of ARBDs in different provinces of Pakistan

Federal Previously reported cases Local prevalence International prevalence ARBD Sindh Punjab capital KPK Total Percentage from Pakistan* Total per million per million†

Type 3 VWD disorder 05 62 21 7 95 33.8 61 156 1.0 0.5 Fibrinogen deficiency 11 20 3 0 34 12 9 43 0.3 0.5 Glanzmann thrombasthenia 18 9 0 0 27 9.6 50 77 0.5 1

FXIII deficiency 7 2 4 0 13 4.6 29 42 0.3 0.5 FVII deficiency 4 6 1 1 12 4.3 84 96 0.6 2

FV deficiency 0 9 0 0 9 3.2 28 37 0.2 1 Vitamin K–dependent clotting 0 7 0 1 8 2.8 0 8 0.04 1 factor deficiency Bernard-Soulier syndrome 3 0 4 0 7 2.5 5 12 0.07 1

FX deficiency 1 1 0 0 2 0.7 41 43 0.3 1 FII deficiency 0 2 0 0 2 0.7 10 12 0.07 0.5

FXI deficiency 1 0 0 0 1 0.4 1 2 0.01 ;1 Combined FV and FVIII 1 0 0 0 1 0.4 0 1 0.006 1 deficiency

*There were no patients from the provinces of Baluchistan, Gilgit-Baltistan, Azad Jammu, and Kashmir because of a lack of health and diagnostic facilities. †International prevalence data from world hemophilia database and Orphanet Journal of Rare Diseases.

30 NOVEMBER 2018 • VOLUME 2, SUPPLEMENT 1 GLOBAL CAPACITY-BUILDING SHOWCASE 37

From www.bloodadvances.org by guest on April 24, 2019. For personal use only. Table 2. Genetic analysis of all clotting factor defects Acknowledgments Clotting factor defect Reported Novel Total No. of patients screened The authors thank Shehla Tariq, Nauman Malik, Hafiz Rafiq, Nazish FI 15 18 30 Saqlain, and Salwa Paracha (Lahore); Shahtaj Masood (HMCP), FII 02 02 04 Samina Amanat (Islamabad); and Children’s Hospital (Lahore) for FV 03 02 05 providing patient data and samples; and Abdul Malik Khan and Asif Khan for providing computer data input from NIBD. They extend FVII 08 07 15 special thanks to their international collaborators (University of Bonn, FVIII 08 02 10 Bonn, Germany; Debrecen University, Budapest, Hungary; FX 03 02 05 M. Neerman-Arbez, University of Geneva, Geneva, Switzerland; and FXI 02 — 02 Royal Free Hospital, London, United Kingdom) for supporting them VWF type 3 25 23 50 unconditionally for the development of genetic analysis in Pakistan. GT 05 05 20 This work was supported by Novo Nordisk Foundation and the International Society of Thrombosis and Hemostasis. BSS 02 03 05

VWF, von Willibrand factor. Authorship Conflict-of-interest disclosure: The authors declare no competing financial interests. Correspondence: Arshi Naz, Department of Pathology, National Conclusion Institute of Blood Disease and Bone Marrow Transplantation, Karachi, These data have shown that VWD type 3 has the highest incidence Pakistan; e-mail: [email protected] or [email protected]. among the ARBDs in this study cohort, followed by fibrinogen deficiency. Glanzmann thrombasthenia (GT) was found to be the DOI 10.1182/bloodadvances.2018GS110924 third most common disorder. The incidence of ARBDs in this region is higher than previously thought. © 2018 by The American Society of Hematology

38 GLOBAL CAPACITY-BUILDING SHOWCASE 30 NOVEMBER 2018 • VOLUME 2, SUPPLEMENT 1

From www.bloodadvances.org by guest on April 24, 2019. For personal use only.