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Coagulation and bleeding disorders
C H A P T E R
19 Coagulation and bleeding disorders Indu Singh1 and Raghu Inder Singh2 1
School of Medical Science, Griffith University, Southport, QLD, Australia 2RP WELLTAR Hospital, Bastara, Karnal, India
19.1 Introduction Thrombosis is a complex disorder involving interaction between many different types of genes with environment factors. Modification in genes coding for coagulation and regulatory proteins of hemostasis are important risk factors for thrombosis. Historically, the measurement of a clot endpoint has been the basis of coagulation testing mainly focused on bleeding disorders. Thrombosis diagnosis has slowly increased with most modern automated coagulation instruments utilizing optic, immunologic, and chromogenic methods. However, molecular diagnostics has added another dimension for the evaluation of clotting and bleeding disorders. DNA-based tests are commonly available for detection of the factor V Leiden (FVL) mutation, the prothrombin 20120A mutation, and the methyltetrahydrofolate reductase (MTHFR) mutation. Inherited disorders of primary hemostasis include von Willebrand disease (VWD), congenital thrombocytopenia, BernardSoulier syndrome, Glanzmann thrombasthenia, and storage pool deficiencies. Secondary hemostasis congenital disorders include hemophilia A (factor VIII deficiency), B (factor IX deficiency), C (factor XI deficiency), other factor deficiencies, and dysfibrinogenemia. Thrombus dislodged from the blood vessel and moving through vasculature as an embolus plays a crucial role in the pathogenesis of acute myocardial infarctions, stroke, and venous thrombosis often leading to fatality. For more than two decades now, FVL [1] and G20210A mutations in the nontranslational region 30 of the prothrombin gene [2] have been established as thrombotic risk factors in about 50% of the global population. However, in Asia and subcontinent of India, prevalence of these genetic aberrations seems to be much lower. It seems that single-nucleotide polymorphisms (SNPs) within or close to some hemostasis-associated candidate genes may be responsible for thrombosis and bleeding disorders. Some of the mutations, such as A384S in SERPINAC1 gene [3], coding for antithrombin (AT), and R67X mutation in SERPINC gene [4] coding for protein Z inhibitor [5], have also been associated with increased risk of thrombosis. It has also been shown that blood group A1 allele is also associated with increased risk of thrombotic events [68]. Homologous areas of the genomic DNA contain variations within their nucleotide sequence. When this sequence variation has a greater than 1% frequency in the population, it is referred to as a polymorphism [9]. These polymorphisms can occur in genes and gene-related sequences or noncoding extragenic DNA. Alleles represent polymorphisms within a single gene [10]. Fig. 19.1 shows various forms of changes in DNA sequences resulting in mutation. When the sequence variation has a population frequency of less than 1%, this might be referred to as a mutation; however, some mutations might be present with .1% frequency (e.g., FVL). Many different polymorphisms/mutations form the molecular basis of risk factors associated with coagulation disorders. With several genes of coagulation proteins now completely sequenced, several SNPs have been described, including point mutations in exons, introns, or regulatory regions [11]. These mutations can result in loss of function (e.g., protein C deficiency) or gain of function (e.g., prothrombin mutation 20210A) [11,12]. A single mutation in the F5 gene is
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FIGURE 19.1 The figure shows various forms of changes in DNA sequences resulting in mutation. Adapted from National Institutes of Health. National Human Genome Research Institute. Talking glossary of genetic terms. Retrieved August 30, 2018, from ,https://www.genome.gov/glossary/..
associated with resistance to activated protein C thus leading to thrombotic tendency. However, there is a remarkable difference in the prevalence of these mutations in individuals of distinct ethnic backgrounds. Different mutations and polymorphisms might affect the same gene; there might be silent mutations or frameshift mutations, the latter typically associated with a severe phenotypic abnormality [11]. Levels of coagulation factors might vary depending on the type of polymorphism or mutations and their interaction with each other [12,13]. This degree of genetic complexity remains a challenge for the molecular diagnostics laboratory. It has been established for a long time that hemophilia (bleeding disorders) has a genetic predisposition. Classic hemophilia (i.e., A and B) arises from hundreds of distinct mutations in the F8 genes, whereas 40% of severe hemophilia A results from a unique mutation characterized as F8 gene inversion. Genetic characterization of hemophilia A and B, the X-linked disorders, are now routinely incorporated into the standard of care, and genetic information is used for risk stratification of treatment complications. With electronic databases detailing .2100 unique mutations for hemophilia A and .1100 mutations for hemophilia B, these diseases are among the most extensively characterized inherited diseases in humans [14]. Mutations in some genes expressing coagulation proteins can lead to rare bleeding disorders including prothrombin, FV, FVII, FX, FXI, FXIII, combined FV and FVIII, or vitamin K-dependent factor deficiency. Molecular diagnostics plays an important role in the clinical management of inherited bleeding disorders. Identification of mutation in bleeding disorders, in particular hemophilia, is crucial for carrier and prenatal testing as well as prediction of risk for inhibitor formation or anaphylactic reaction after replacement therapy [15]. Molecular diagnostics is also important for differentiating the bleeding disorder with similar clinical presentations but different underlying genetic causes. Informed decision of treatment and clinical management requires correct diagnoses of any genetic condition resulting in bleeding. In most cases, initial diagnosis is made by traditional coagulation-based assays. Molecular testing provides additional details to aid in the future clinical management of these disorders. Hemophilia B Leiden phenotype has now been extensively characterized at the genetic level and found to be caused by a group of single-nucleotide substitutions clustered around the transcription start site of the F9 gene [16]. Mild or moderate hemophilia A has been sometimes underdiagnosed by traditional coagulation testing. It has been revealed that the cause of this phenotype is missense substitutions localized to the interfaces between the factor VIII “A” domains that result in increased instability of the molecule [17]. Conventional coagulation-based testing could be sometimes nonspecific and insensitive to mild factor deficiencies [18]. Tests of intermediate complexity, such as factor assays, platelet aggregometry, and flow cytometry, are dependent on preanalytical variables [19]. On the other hand, heterogeneity of molecular lesions makes it technically complex to use them as first-line diagnostic tests. Current applications of molecular testing in these disorders are therefore largely restricted to carrier detection and antenatal diagnosis [20]. Later generationsequencing techniques such as multiplexing have overcome many of the shortcomings of the conventional molecular assays with
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improved sensitivity and lower costs. Molecular testing provides additional details to aid in the future clinical management of these disorders. Molecular diagnostic testing routinely performed include in vitro testing for nucleic acids by polymerase chain reaction (PCR) and reverse transcriptase PCR (RT-PCR). DNA and RNA extractions followed by DNA amplification is then detected using either gel electrophoresis, restriction endonucleases, nucleic acid hybridization or southern blotting cleavage-based signal amplification and DNA sequencing. This chapter will introduce molecular systems underpinning various thrombotic and bleeding disorders in clinical setting with one case study description each for thrombosis and bleeding disorder.
19.2 Genetic basis of thrombosis 19.2.1 Case 1 A young lady of 25 years came to the doctor’s clinic with deep vein thrombosis (DVT) of left lower limb. She was treated for that and then she returned after 6 months with DVT symptoms in her right lower limb. Her coagulation profile was completed confirming antiphospholipid syndrome (APLS). She returned in her first trimester of pregnancy after 2 years with blue discoloration that seemed like thrombosis of left great toe. She was referred to a tertiary specialist institution, where she was started on low molecular weight heparin (LMWH). However, she aborted at about 20 weeks. Thereafter she had three pregnancies which all ended in abortion. About a year ago, she again came back with severe pain in her abdomen, which was finally diagnosed as superior mesenteric artery thrombosis. Is it possible that it is some gene mutation or inherited genetic abnormality? Could genetic testing have helped in this case?
19.2.2 Molecular genetics of APLS Human leukocyte antigen (HLA) class II antigens (DR, DP, DQ) loci are found on chromosome 6, and these molecules are highly polymorphic. HLA class II polymorphisms have been associated with autoimmune responses. Another gene implicated is β2-GPI gene located on chromosome 17 with at least 4 common SNPs in protein coding region. It has also been reported that APLS population or those with APL antibodies had higher incidence of FV mutation in patients presenting with venous thromboembolism (VTE). Another study reviewed by same authors reported an association between FVL, G20210A, in various combinations, with APLSpotentiated risk factor for venous thrombosis but had minimal risk of arterial thrombosis. It has also been demonstrated that antiphospholipid syndrome (APS) patients with MTHFR 677TT genotype had a lower mean age at first thrombotic event as was the situation with the patient in Case 1 earlier. On the contrary, other studies did not find significant association between VTE and APL. Since several genetic and ethnicity factors are involved in pathophysiology of APS, thrombosis and recurrent pregnancy loss, internationally collaborated multicenter case-control studies are required for better understanding of genetic predisposition to develop APLS and patient management [21]. Sharma et al. reported APC resistance (APCR), followed by PS, PC, and AT deficiency, to be most common in Indian women presenting with recurrent pregnancy loss. They found that APCR associated with FVL increased risk of pregnancy loss in the population they investigated [22]. However, they believe their study is the only one conducted in India at the time of this publication, so there is scant data to make a conclusive statement.
19.2.3 Molecular basis of other causes of thrombosis 19.2.3.1 Antithrombin deficiency ATIII is a protein expressed by SERPINC1 gene spanning 13.4 kb of genomic DNA on chromosome 1q2325. It inhibits activity of thrombin, factors IXa, Xa, and XIa. ATIII deficiency can be Type 1 where both activity and plasma antigen levels are attenuated or Type 2 with normal antigen levels but reduced activity [23]. More than 250 mutations and polymorphisms in protein coding region of SERPINC1 gene are known to cause DVT, but until 5 years ago, only one SNP (g.2085T . C) was identified in the SERPINC1 regulatory region. Recently, Bhakuni et al. identified two more SNPs (g.25G . A and g.-1A . T) and two previously known (g.67G . A and rs3138521) polymorphisms in Indian patients presenting with DVT for first time [23].
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19.2.3.2 Protein C and S deficiency and activated protein C (APC) resistance Protein C pathway and its cofactor protein S inactivates coagulation cofactors Va and VIIIa, which enhance the activity of serine protease coagulation factors VII, IX, X, XI, and XII, thus slowing down the clot-formation process. Protein C is a single-chain vitamin K-dependent protein synthesized by liver. Protein C gene is 11 kb of DNA on chromosome 2 with 9 exons and 8 introns. Protein C is activated by cleavage at Arg 169Leu 170 [24]. Mutations in PROS1 gene cause protein S deficiency. Like protein C-deficient individuals, protein S-deficient people cannot inactivate clotting proteins, resulting in the increased risk of developing abnormal blood clots. Protein S deficiency can be divided into Types 1, 2, and 3 based on how mutations in the PROS1 gene affect protein S levels and activity [24]. APCR is a common cause of hereditary thrombophilia. It results from a substitution of G to A base at nucleotide 1691 in exon 10 of F5 gene. APCR prolongs inactivation of activated FV allowing the activation of other downstream coagulation factors and continuing or enhancing clot formation with a risk of thrombophilia. APCR alone is not a significant risk factor but combined with other risk factors increases risk of thrombosis [24]. 19.2.3.2.1 Prothrombin allele G20210A
Factor II (prothrombin) G20210A is the third most common cause of cardiovascular disease (CVD) and the most common inherited coagulation disorder in the United States. With autosomal-dominant inheritance, this single point mutation (G to A at position 20210) in prothrombin gene affects 80 times increase in thrombosis in homozygous state. G to A translation at nucleotide 20210 in prothrombin gene leads to an increase in factor II (prothrombin) levels increasing the risk of venous thrombosis. The most common testing protocol to detect this mutation is PCR coupled with restriction-endonuclease digestion, gel electrophoresis, and RT-PCR [24]. Challenging clinical issues include the decisions regarding when to test for the mutation and how to manage individuals with the mutation, either in the setting of VTE or as an incidental finding. It has been used for patients with clinically suspected thrombophilia. There may be additional indications for direct PT G20210A mutation testing, such as in determining the duration of anticoagulation therapy of VTE patients and screening for women contemplating hormone therapy. 19.2.3.3 Factor V Leiden A specific mutation in the factor V gene is called FVL. This mutation causes factor V to be inactivated more slowly by APC, generating more thrombin and consequently increasing the potential for clot formation. It is inherited in an autosomal dominant manner. Factor V is a plasma glycoprotein of 330 kDa molecular weight with 2224 amino acids. It is coded by a complex 25 exons—80 kb gene on chromosome 1. It is converted to an active two-chain form by thrombin or factor Xa. Thrombin cleaves it at three separate sites. The APC cleavage sites are 306, 506, and 679 on an FVa heavy chain, which is hinged by Ca21 to the FVa light chain. Following the cleavage, 2 chains are linked via a divalent metal ion bridge. FV acts as a cofactor for APC and protein S in the inactivation of the procoagulant FVIIIa. A cleavage of FV at R506 by APC is required for this cofactor function. FVL, the mutant FV, is seen with APCR. In this mutant, R506 is replaced by a Q, which renders the 506-position insensitive to proteolysis by APC. This is referred to as Q506FV which does not show anticoagulant activity [25]. Inactivation of mutant FVa: Q506 site leads to FVL. FVL mutation accounts for about 90% of APCR and is prevalent in nearly 2%13% of general population. It is also seen in about 20%60% of VTE cases. Literature shows that a higher proportion of Caucasians are affected than other populations. This could also be relative, due to lack of studies and data from Asian and other populations. About 1 in 10 people carrying FVL heterozygotes develop VTE over their lifetime. Many patients with familial thrombophilia are found to have FVL. Other coexisting disorders and circumstantial risk factors also affect clinical expression of FVL. These include presence of prothrombin variant G20210A, obesity, age, immobility due to injury, surgery, oral contraception, hormone replacement therapy, pregnancy, and even air travel [26]. It has been suggested and is advisable to offer genetic testing and referral for consultation in first unprovoked VTE at any age, history of recurrent VTE, strong family history of thrombotic diseases, VTE during pregnancy or puerperium or when it is the first VTE incident and patient has a first-degree family member with a history of VTE under the age of 50. Case 1 in this chapter is a good example where offering genetic testing might be useful for patient and future generations. It has been suggested that 10% of FVL carriers develop VTE in their lifetime, and 25%40% are at higher risk where there is a family history of thrombophilia.
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One of the potential benefits of genetic testing is that asymptomatic FVL carriers could be educated about VTE high-risk circumstances, signs and symptoms, as well as possible potential need for prophylactic treatment in high-risk circumstances. However, genetic testing does not detect all possible thrombophilia conditions and has limited clinical utility. Further research is required to confidently improve surveillance and management guidelines as there is very limited and inconsistent data, if at all, in use currently. 19.2.3.4 Hyperhomocysteinemia MTHFR gene is responsible for production of enzyme MTHFR which has an important role in the homocysteine metabolism. MTHFR gene has at least two known polymorphisms, 677C . T and 1298A . C. These are associated with reduced enzyme activity, decreased folate concentration in blood, mildly increasing plasma, and total homocysteine concentration [24]. 19.2.3.5 Inherited deficiency of fibrinolysis Decreased fibrinolytic capacity due to increased plasminogen activator inhibitor-1 (PAI-1) activity and decreased tissue-type plasminogen activator (tPA) activity has been associated with thrombosis. A congenital gamma-chain molecular defect or gamma dysfibrinogenemia such as fibrinogen Dusard (Arg554Cys) results in impaired binding of tPA to fibrin-reducing plasminogen activation, impaired fibrinolysis, and tendency for thrombosis [27]. PAI-1 gene has also been shown to have multiple polymorphisms. However, only patients with 4G/4G genotype of the PAI-1 genetic polymorphism show an increased plasma PAI-1 concentration which is significantly associated with an increased risk of thrombosis [28]. Plasminogen is activated to plasmin by specific cleavage at the Arg560Val561 peptide bond.
19.3 Genetic basis of the bleeding disorders Bleeding disorders in men are well recognized due to a lot of research in X-linked recessive disorders, such as hemophilia, seen in young boys with prominent clinical significance. However, bleeding disorders in females are often missed due to milder presentation at later age. Some studies have focused on hemostatic disorders in menorrhagia [29,30]. Gupta et al. demonstrated that inherited bleeding disorders were present in most of the 200 women with various bleeding complaints in a tertiary hospital in India [31]. Current testing method, widely used by many accredited research-based facilities worldwide, includes Sanger or next-generation sequencing (NGS) for the testing of single-nucleotide variants particularly in hemophilia [32].
19.3.1 Case 2 A 14-year-old boy who had repeated episodes of hemarthrosis affecting his knees ultimately ended with flexion deformity of his right knee. The coagulation and factor testing reported factor VIII deficiency. It seems to be an obvious case of hemophilia, but do genetic and molecular studies on this patient have any role in patient care or future counseling? What other bleeding orders could be suspected? There is no clear discrimination between inherited or acquired mutations leading to bleeding disorders. The complexity of structure with variable gene expression as well as degree of consanguinity in certain populations, concentrating effect of certain mutations within community, indicate that there may be a benefit in investigating patient’s history, and molecular as well as cytogenetic evaluation to rule out coexisting recessively inherited risk factors.
19.3.2 Hemophilia A (factor VIII deficiency) Hemophilia A is an X-linked recessive disorder with factor VIII deficiency mostly in male population. Rarely females may be presented with this condition due to the inheritance of homozygous or compound heterozygous alleles or chromosomal abnormalities. Hemophilia A is caused by pathogenic variant in F8 gene encoding for FVIII protein. The F8 gene, that is 187 kb in size, comprises 25 introns and 26 exons, located on the long arm of the X-chromosome at the most distal band (Xq28) [33]. Ankala et al. found that intron 22 inversion in F8 gene is the most common cause of hemophilia A in Indian population followed by some cases of intron 1 inversion on the same gene [34].
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FIGURE 19.2 Factor VIII molecule and its cleavage sites.
Most pathogenic hemophilia A variants reduce the synthesis or secretion of FVIII or impair FVIII cofactor activity. A small subset of variants, localized to the A3, C1, and C2 domains (Fig. 19.2), are associated with impaired binding to the FVIII carrier molecule von Willebrand factor (VWF) [35], resulting in accelerated proteolysis and/or clearance of FVIII from the plasma. More than 2000 unique F8 gene variants have been reported in the FVIII-variant database [36]. The most common mutation in severe hemophilia A is inversion, producing abnormal or reduced amount of this protein. About half of the hemophilia A cases have reported an intrachromosomal inversion at intron 22 occurring spontaneously during meiosis in male germ cells [37] and a similar inversion has been reported within intron 1 accounting for about 1%2% cases only [38]. Most of the other heterogeneous cases are a result of nonsense/stop mutation preventing factor VIII production, missense mutations’ impact on activity, half-life or efficient factor VIII production, or may be due to some new mutations. Mutations in hemophilia A patients can be detected by chemical cleavage of mismatch method. Inversion mutations can be analyzed by restriction enzyme analysis and southern blotting. Molecular techniques including inverse-shifting PCR, long-range PCR, eight multiplex PCRs, and Sanger sequencing are also useful in diagnosis of these cases. Widely used FVIII-replacement therapy has the risk of FVIII-neutralizing antibodies or inhibitor development in patient. The genetic variability within the F8 gene as well as environmental and genetic risk factors may be able to regulate immune response to FVIII. Intron 22 inversion is associated with lower risk for inhibitors. FVIII mutation status can help predict patient’s response to immune tolerance induction (ITI) therapy for inhibitor eradication. Some large deletions have been shown to be associated with a decreased rate of ITI success [39].
19.3.3 Molecular basis of other inherited coagulopathies 19.3.3.1 von Willebrand disease VWD is the most prevalent of the congenital bleeding disorders affecting both sexes through autosomaldominant inheritance. It is a highly heterogeneous disorder due to the molecular mechanisms that produce various clinical and laboratory phenotypes. VWF gene is located on the short arm of chromosome 12 in the locus p13.3. It consists of 52 exons spanning 178 kilobase pairs [40]. VWF gene encodes for the VWF synthesized in endothelial cells and megakaryocytes. There are patients presented with excessive mucocutaneous bleeding caused by quantitative or qualitative abnormalities in VWF. VWF is a multimedia glycoprotein essential for the maintenance of hemostasis. Multidomain structure is composed of multiples of four domain types AD in the arrangement D1D2D0 D3A1A2A3D4B1B2B3C1C2 (Fig. 19.3). Different functions of VWF are assigned to different domains: A1 domain is involved in binding of VWF to platelet GP1b binding to fibrillar collagen, sulfatides, and heparin. D3 domain binds to factor VIII. C2 domain binds to GP11b-111a receptors. After initial synthesis, it is modified by posttranslational processing of dimerization and multimerization by cleavage of D1D2 domain of the polypeptide.
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FIGURE 19.3 VWF domain structure. VWF, von Willebrand factor.
FIGURE 19.4 Most type 1 VWD are due to missense mutations (dominant negative—interference with intracellular transport of dimeric pro-VWF). Some forms with incomplete penetrance require coinheritance of blood type O for expression (causes increased VWD proteolysis). Most type 3 VWD due to null alleles. VWD, von Willebrand disease; VWF, von Willebrand factor.
VWD is a result of either quantitative deficiency (Type 1), qualitative abnormality (Type 2), or complete absence of VWF (Type 3). Mutations giving rise to Type 2 are very well characterized, whereas those giving rise to Type 1 are not well characterized. Type 2A with point mutation in A2 structural domain of VWF renders VWF more susceptible to proteolysis leaving a lot of small molecular weight multimers in plasma. Type 2B is a rare mutation in A1 structural domain of VWF reducing large molecular weight multimers due to increased binding with resting platelets. Type 2M is a qualitative variant of VWF reducing normal multimers due to high platelet receptor binding. Type 2N results from auto recessive-translation or deletion mutations of VWF gene, where VWF and factor VIII are nearly absent (Fig. 19.4). People with VWF levels at upper or lower end of normal reference range are at risk of hemostatic disorders. Lower levels may lead to the common bleeding disorder Type 1 VWD, and high levels are associated with an increased risk for both venous and arterial thrombosis [41,42]. Most of the Type 1 VWD individuals with VWF levels under 40 have VWF missense gene mutation. Type 2 qualitative defect is often a result of missense mutation including Type 2A with mutations in either of the two domains involved in multimer formation or mutation in domain cleaved by ADAMTS-13. Type 2B shows gain of function mutation in platelet GP1b-binding domain, while Type 2M is a result of the loss of function mutation in GP1b-binding domain and Type 3 patients with severe deficiency are homozygous for null alleles. Results from genome-wide association studies accounted for common variants at ABO, VWF, and other loci in about 12% of the variance in plasma VWF levels [43]. The genetic defects responsible for VWD include a novel gene on chromosome 2 accounting for about 1 in 5 of VWF variations not identified by standard genetic approaches. Further characterization of this and other genes controlling VWF levels may lead to improved VWD diagnosis and prediction of bleeding and clotting risk. In Indian population, mutations c.2908delC and c.5335C . T (p.R1779 ) on VWF gene have been found to cause VWD [44].
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19.3.3.2 Hemophilia B (factor IX deficiency) Hemophilia B is a quantitative or qualitative defect of the coagulation zymogen factor IX presented as an X-linked recessive disorder. The incidence is lower than hemophilia A. Factor IX gene is located on the long arm of X-chromosome (Xq27) and has eight exons. The outcome of the review of literature by Ankala et al. suggested that mutation c.316G . A (p.G106S) is the most common mutation of F9 gene in Indian population [34]. Heterogenous pathogenic variants are found throughout the factor IX gene including the promoter and 30 UTR region. Over 1000 unique variants, majority of them being single-nucleotide substitutions, have been associated with hemophilia B [36]. Most of these variants are on exon 8, the largest exon encoding the functional serine protease domain of the active factor IX coagulation protein. Hemophilia B Leyden is found in about 2% cases presents with moderate to severe prepubertal factor IX deficiency, which eventually returns to normal. This is associated with variants in proximal promoter region of F9 gene and disrupts binding sites to one of the three transcription factors: hepatic nuclear factor 4a, CCAAT enhancer-binding protein (C/EBP), or ONECUT1/2 [4547]. Sequencing the promoter proximal region of F9 gene can be predictive of long-term normalization of factor IX levels. The incidence of inhibitors in these patients is significantly lower than hemophilia A cases. Chitlur et al. reported that about 60% of hemophilia B patients that develop inhibitors often experience an anaphylactic response to FIX concentrate at the time of inhibitor onset. They believe that this is more likely associated with large deletions or null mutations rather than single-nucleotide variants [48]. 19.3.3.3 Afibrinogenemia and dysfibrinogenemia Most cases of reduced or ineffective activity of fibrinogen have been reported from consanguineous parents. Inherited in recessive manner, these disorders are caused by more than 30 genetically heterogenous mutations. It results in moderate to severe bleeding, which could be due to low or zero synthesis of fibrinogen or problems with intracellular transport or secretion. Dysfibrinogenemia usually exhibits dominant inheritance caused by missense mutations affecting fibrin polymerization, fibrinopeptide cleavage, or fibrin stabilization by factor XIIIa. Depending on the mutation, the patient may have mild to severe bleeding tendency.
19.4 Structural defects of the vascular system 19.4.1 Hereditary hemorrhagic telangiectasia This condition has an autosomal dominant inheritance caused by mutation in endoglin gene that controls vascular remodeling. Molecular diagnosis is possible for this condition showing several small arteriovenous malformations in skin, mouth, GI tract, and lungs [24].
19.4.2 EhlersDanlos syndrome EhlersDanlos syndrome (EDS) is a defective collagen structure affecting connective tissues supporting the skin, bones, blood vessels, and many other organs and tissues. EDS is caused by the mutations in more than a dozen genes for various types of collagen with nine different variants, which leads to bruising due to thin, weak skin with poor healing and spontaneous joint dislocation due to hypermobile joints. Inheritance can be autosomal, dominant, recessive, or X-linked [24]. In 2017 EDS was classified describing 13 types. The specific gene affected determines the specific EDS in a patient. Till date, about 18 genes with mutations responsible for EDS have been identified. Some of these are fibrous protein genes, such as COLIA1, COLIA2, COL3A1, COL5A1, and TNXB, and enzymes including ADAMTS2, PLOD1, B4GALT7, DSE, and D4ST1/CHST14. Customized NGS is performed using a panel of various collagen genes. Analysis includes the coding exons of all genes in the panel plus 10 bases in the introns and untranslated regions (50 and 30 ). Sanger sequencing is performed to confirm variants suspected or confirmed to be pathogenic [49]. However, there is no known cure for any type of EDS, so treatment is supportive rather than therapeutic.
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19.5 Inherited defects of platelets 19.5.1 Inherited macrothrombocytopenia Giant platelets seen in peripheral blood could be due to genetic mutations or polymorphism leading to inherited platelet defects. Recent evidence from the literature and testing of different populations from various areas of India [50,51] focused on the presence of inherited disorder where patients have high number of giant platelets. They found an increased recognition of inherited macrothrombocytopenia in the country. Their findings suggest that about half of the patient population showed mutations of MYH9, GP1BB, GP1BA, GPIX, ABCG5, ABCG8, ACTN, FLI, TUBB, and RUNX1 [50], which are frequently seen in heterozygous state. Many of these asymptomatic patients do have mild-to-moderate bleeding history.
19.5.2 BernardSoulier syndrome BernardSoulier disease is caused due to reduced or absent levels of platelet glycoproteins GPIb/IX/V, which is one of the VWF receptors on platelets. This VWF receptor on platelets has four polypeptide chains: GP1b-α, GP1b-β, GPV, and GPIX. Mutations in GP1BA or GP1BB genes reduce expression of these genes. GP1BA insertion mutations (p.Met338fsX13) resulting in frameshift change and (p.Val485fsX13) a new termination codon were reported in people from India. Two missenses (p.Tyr95Asp and p.Cys32X), also resulting in frameshift, as well as one missense (p.Cys24Arg) in GPIX mutation, were also found in some Indian patients [52]. However, this study was conducted on a very small number of eight patients from seven unrelated families with six different detected mutations. This may not have the power to make a generalization about any set population, but it does show high variability, adding further to the complexity of using molecular diagnostics as a tool in many cases.
19.5.3 Glanzmann’s thrombasthenia Autosomal recessive inheritance with decreased platelet GPIIbIIIa expression resulting in defective platelet aggregation and moderate to severe bleeding. It is common in populations with high degree of consanguinity. More than 70 mutations in αIIb and β1 genes on chromosome 17 have been found to be responsible for this disorder [53].
19.5.4 Storage pool disease Dense and alpha granule deficiency is caused by variety of genetic abnormalities. Mutations in a gene for a nucleotide transporter MRP4 (ABCC4) have been suggested to affect nucleotide accumulation in dense granules [54].
19.5.5 MayHegglin anomaly It is a giant platelet syndrome associated with mutations in the nonmuscle myosin heavy chain gene MYH9 with autosomal dominant inheritance [24].
19.5.6 WiscottAldrich syndrome Mutation in WASP-signaling protein decreases secretion and aggregation of platelets with multiple agonists. It has X-linked inheritance. Current diagnosis is based on family history, clinical thrombocytopenia, small platelets with few granules, and genetic testing [24].
19.6 Conclusion Thrombophilia is a very complex multifactorial disorder. However, so far there are less than 10 welldocumented genetically associated risk factors of thrombosis. These defects account for a very low percentage of hereditary thrombophilia and thrombosis leading to a very common and much higher prevalence of
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thrombosis-related cardiovascular events worldwide. Considering these known genetic factors have low frequency particularly in Asia with highest population concentration, there is a need to identify new genetic factors contributing to variation as a collaborative effort amongst different ethnic groups instead of fragmented projects investigating random patient populations. This knowledge will help us not only to personalize the therapeutic algorithms but to propose preventive strategies. There are ever evolving methods, automation, and statistical tools for genetic testing and interpretation to guide effective diagnosis, prophylactic, and therapeutic strategies. Rapid advances in molecular genetics have informed current approaches to the diagnosis of many hemostatic and thrombotic disorders and have revolutionized the treatment of patients with hemophilia with the introduction of recombinant clotting proteins. However, curative gene therapy for inherited disorders of coagulation remains elusive, although continued improvements in the tools for effective and safe gene therapy suggest a bright future for genetic therapies of hemophilia. Dramatic advances in our understanding of the mechanisms of thrombosis and hemostasis have transformed the management of thrombotic and bleeding disorders through the introduction of new, effective, safer, and more convenient therapies. There are still lots of gaps with unknowns, and future studies need to identify directions of research and variation in different populations of world.
Conflict of interest There are no conflicts of interest to declare.
Author’s roles The chapter was written by IS in consultation with RIS and was reviewed by RIS.
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II. Molecular medicine in clinical practice
19 Coagulation and bleeding disorders Indu Singh1 and Raghu Inder Singh2 1
School of Medical Science, Griffith University, Southport, QLD, Australia 2RP WELLTAR Hospital, Bastara, Karnal, India
19.1 Introduction Thrombosis is a complex disorder involving interaction between many different types of genes with environment factors. Modification in genes coding for coagulation and regulatory proteins of hemostasis are important risk factors for thrombosis. Historically, the measurement of a clot endpoint has been the basis of coagulation testing mainly focused on bleeding disorders. Thrombosis diagnosis has slowly increased with most modern automated coagulation instruments utilizing optic, immunologic, and chromogenic methods. However, molecular diagnostics has added another dimension for the evaluation of clotting and bleeding disorders. DNA-based tests are commonly available for detection of the factor V Leiden (FVL) mutation, the prothrombin 20120A mutation, and the methyltetrahydrofolate reductase (MTHFR) mutation. Inherited disorders of primary hemostasis include von Willebrand disease (VWD), congenital thrombocytopenia, BernardSoulier syndrome, Glanzmann thrombasthenia, and storage pool deficiencies. Secondary hemostasis congenital disorders include hemophilia A (factor VIII deficiency), B (factor IX deficiency), C (factor XI deficiency), other factor deficiencies, and dysfibrinogenemia. Thrombus dislodged from the blood vessel and moving through vasculature as an embolus plays a crucial role in the pathogenesis of acute myocardial infarctions, stroke, and venous thrombosis often leading to fatality. For more than two decades now, FVL [1] and G20210A mutations in the nontranslational region 30 of the prothrombin gene [2] have been established as thrombotic risk factors in about 50% of the global population. However, in Asia and subcontinent of India, prevalence of these genetic aberrations seems to be much lower. It seems that single-nucleotide polymorphisms (SNPs) within or close to some hemostasis-associated candidate genes may be responsible for thrombosis and bleeding disorders. Some of the mutations, such as A384S in SERPINAC1 gene [3], coding for antithrombin (AT), and R67X mutation in SERPINC gene [4] coding for protein Z inhibitor [5], have also been associated with increased risk of thrombosis. It has also been shown that blood group A1 allele is also associated with increased risk of thrombotic events [68]. Homologous areas of the genomic DNA contain variations within their nucleotide sequence. When this sequence variation has a greater than 1% frequency in the population, it is referred to as a polymorphism [9]. These polymorphisms can occur in genes and gene-related sequences or noncoding extragenic DNA. Alleles represent polymorphisms within a single gene [10]. Fig. 19.1 shows various forms of changes in DNA sequences resulting in mutation. When the sequence variation has a population frequency of less than 1%, this might be referred to as a mutation; however, some mutations might be present with .1% frequency (e.g., FVL). Many different polymorphisms/mutations form the molecular basis of risk factors associated with coagulation disorders. With several genes of coagulation proteins now completely sequenced, several SNPs have been described, including point mutations in exons, introns, or regulatory regions [11]. These mutations can result in loss of function (e.g., protein C deficiency) or gain of function (e.g., prothrombin mutation 20210A) [11,12]. A single mutation in the F5 gene is
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FIGURE 19.1 The figure shows various forms of changes in DNA sequences resulting in mutation. Adapted from National Institutes of Health. National Human Genome Research Institute. Talking glossary of genetic terms. Retrieved August 30, 2018, from ,https://www.genome.gov/glossary/..
associated with resistance to activated protein C thus leading to thrombotic tendency. However, there is a remarkable difference in the prevalence of these mutations in individuals of distinct ethnic backgrounds. Different mutations and polymorphisms might affect the same gene; there might be silent mutations or frameshift mutations, the latter typically associated with a severe phenotypic abnormality [11]. Levels of coagulation factors might vary depending on the type of polymorphism or mutations and their interaction with each other [12,13]. This degree of genetic complexity remains a challenge for the molecular diagnostics laboratory. It has been established for a long time that hemophilia (bleeding disorders) has a genetic predisposition. Classic hemophilia (i.e., A and B) arises from hundreds of distinct mutations in the F8 genes, whereas 40% of severe hemophilia A results from a unique mutation characterized as F8 gene inversion. Genetic characterization of hemophilia A and B, the X-linked disorders, are now routinely incorporated into the standard of care, and genetic information is used for risk stratification of treatment complications. With electronic databases detailing .2100 unique mutations for hemophilia A and .1100 mutations for hemophilia B, these diseases are among the most extensively characterized inherited diseases in humans [14]. Mutations in some genes expressing coagulation proteins can lead to rare bleeding disorders including prothrombin, FV, FVII, FX, FXI, FXIII, combined FV and FVIII, or vitamin K-dependent factor deficiency. Molecular diagnostics plays an important role in the clinical management of inherited bleeding disorders. Identification of mutation in bleeding disorders, in particular hemophilia, is crucial for carrier and prenatal testing as well as prediction of risk for inhibitor formation or anaphylactic reaction after replacement therapy [15]. Molecular diagnostics is also important for differentiating the bleeding disorder with similar clinical presentations but different underlying genetic causes. Informed decision of treatment and clinical management requires correct diagnoses of any genetic condition resulting in bleeding. In most cases, initial diagnosis is made by traditional coagulation-based assays. Molecular testing provides additional details to aid in the future clinical management of these disorders. Hemophilia B Leiden phenotype has now been extensively characterized at the genetic level and found to be caused by a group of single-nucleotide substitutions clustered around the transcription start site of the F9 gene [16]. Mild or moderate hemophilia A has been sometimes underdiagnosed by traditional coagulation testing. It has been revealed that the cause of this phenotype is missense substitutions localized to the interfaces between the factor VIII “A” domains that result in increased instability of the molecule [17]. Conventional coagulation-based testing could be sometimes nonspecific and insensitive to mild factor deficiencies [18]. Tests of intermediate complexity, such as factor assays, platelet aggregometry, and flow cytometry, are dependent on preanalytical variables [19]. On the other hand, heterogeneity of molecular lesions makes it technically complex to use them as first-line diagnostic tests. Current applications of molecular testing in these disorders are therefore largely restricted to carrier detection and antenatal diagnosis [20]. Later generationsequencing techniques such as multiplexing have overcome many of the shortcomings of the conventional molecular assays with
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improved sensitivity and lower costs. Molecular testing provides additional details to aid in the future clinical management of these disorders. Molecular diagnostic testing routinely performed include in vitro testing for nucleic acids by polymerase chain reaction (PCR) and reverse transcriptase PCR (RT-PCR). DNA and RNA extractions followed by DNA amplification is then detected using either gel electrophoresis, restriction endonucleases, nucleic acid hybridization or southern blotting cleavage-based signal amplification and DNA sequencing. This chapter will introduce molecular systems underpinning various thrombotic and bleeding disorders in clinical setting with one case study description each for thrombosis and bleeding disorder.
19.2 Genetic basis of thrombosis 19.2.1 Case 1 A young lady of 25 years came to the doctor’s clinic with deep vein thrombosis (DVT) of left lower limb. She was treated for that and then she returned after 6 months with DVT symptoms in her right lower limb. Her coagulation profile was completed confirming antiphospholipid syndrome (APLS). She returned in her first trimester of pregnancy after 2 years with blue discoloration that seemed like thrombosis of left great toe. She was referred to a tertiary specialist institution, where she was started on low molecular weight heparin (LMWH). However, she aborted at about 20 weeks. Thereafter she had three pregnancies which all ended in abortion. About a year ago, she again came back with severe pain in her abdomen, which was finally diagnosed as superior mesenteric artery thrombosis. Is it possible that it is some gene mutation or inherited genetic abnormality? Could genetic testing have helped in this case?
19.2.2 Molecular genetics of APLS Human leukocyte antigen (HLA) class II antigens (DR, DP, DQ) loci are found on chromosome 6, and these molecules are highly polymorphic. HLA class II polymorphisms have been associated with autoimmune responses. Another gene implicated is β2-GPI gene located on chromosome 17 with at least 4 common SNPs in protein coding region. It has also been reported that APLS population or those with APL antibodies had higher incidence of FV mutation in patients presenting with venous thromboembolism (VTE). Another study reviewed by same authors reported an association between FVL, G20210A, in various combinations, with APLSpotentiated risk factor for venous thrombosis but had minimal risk of arterial thrombosis. It has also been demonstrated that antiphospholipid syndrome (APS) patients with MTHFR 677TT genotype had a lower mean age at first thrombotic event as was the situation with the patient in Case 1 earlier. On the contrary, other studies did not find significant association between VTE and APL. Since several genetic and ethnicity factors are involved in pathophysiology of APS, thrombosis and recurrent pregnancy loss, internationally collaborated multicenter case-control studies are required for better understanding of genetic predisposition to develop APLS and patient management [21]. Sharma et al. reported APC resistance (APCR), followed by PS, PC, and AT deficiency, to be most common in Indian women presenting with recurrent pregnancy loss. They found that APCR associated with FVL increased risk of pregnancy loss in the population they investigated [22]. However, they believe their study is the only one conducted in India at the time of this publication, so there is scant data to make a conclusive statement.
19.2.3 Molecular basis of other causes of thrombosis 19.2.3.1 Antithrombin deficiency ATIII is a protein expressed by SERPINC1 gene spanning 13.4 kb of genomic DNA on chromosome 1q2325. It inhibits activity of thrombin, factors IXa, Xa, and XIa. ATIII deficiency can be Type 1 where both activity and plasma antigen levels are attenuated or Type 2 with normal antigen levels but reduced activity [23]. More than 250 mutations and polymorphisms in protein coding region of SERPINC1 gene are known to cause DVT, but until 5 years ago, only one SNP (g.2085T . C) was identified in the SERPINC1 regulatory region. Recently, Bhakuni et al. identified two more SNPs (g.25G . A and g.-1A . T) and two previously known (g.67G . A and rs3138521) polymorphisms in Indian patients presenting with DVT for first time [23].
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19.2.3.2 Protein C and S deficiency and activated protein C (APC) resistance Protein C pathway and its cofactor protein S inactivates coagulation cofactors Va and VIIIa, which enhance the activity of serine protease coagulation factors VII, IX, X, XI, and XII, thus slowing down the clot-formation process. Protein C is a single-chain vitamin K-dependent protein synthesized by liver. Protein C gene is 11 kb of DNA on chromosome 2 with 9 exons and 8 introns. Protein C is activated by cleavage at Arg 169Leu 170 [24]. Mutations in PROS1 gene cause protein S deficiency. Like protein C-deficient individuals, protein S-deficient people cannot inactivate clotting proteins, resulting in the increased risk of developing abnormal blood clots. Protein S deficiency can be divided into Types 1, 2, and 3 based on how mutations in the PROS1 gene affect protein S levels and activity [24]. APCR is a common cause of hereditary thrombophilia. It results from a substitution of G to A base at nucleotide 1691 in exon 10 of F5 gene. APCR prolongs inactivation of activated FV allowing the activation of other downstream coagulation factors and continuing or enhancing clot formation with a risk of thrombophilia. APCR alone is not a significant risk factor but combined with other risk factors increases risk of thrombosis [24]. 19.2.3.2.1 Prothrombin allele G20210A
Factor II (prothrombin) G20210A is the third most common cause of cardiovascular disease (CVD) and the most common inherited coagulation disorder in the United States. With autosomal-dominant inheritance, this single point mutation (G to A at position 20210) in prothrombin gene affects 80 times increase in thrombosis in homozygous state. G to A translation at nucleotide 20210 in prothrombin gene leads to an increase in factor II (prothrombin) levels increasing the risk of venous thrombosis. The most common testing protocol to detect this mutation is PCR coupled with restriction-endonuclease digestion, gel electrophoresis, and RT-PCR [24]. Challenging clinical issues include the decisions regarding when to test for the mutation and how to manage individuals with the mutation, either in the setting of VTE or as an incidental finding. It has been used for patients with clinically suspected thrombophilia. There may be additional indications for direct PT G20210A mutation testing, such as in determining the duration of anticoagulation therapy of VTE patients and screening for women contemplating hormone therapy. 19.2.3.3 Factor V Leiden A specific mutation in the factor V gene is called FVL. This mutation causes factor V to be inactivated more slowly by APC, generating more thrombin and consequently increasing the potential for clot formation. It is inherited in an autosomal dominant manner. Factor V is a plasma glycoprotein of 330 kDa molecular weight with 2224 amino acids. It is coded by a complex 25 exons—80 kb gene on chromosome 1. It is converted to an active two-chain form by thrombin or factor Xa. Thrombin cleaves it at three separate sites. The APC cleavage sites are 306, 506, and 679 on an FVa heavy chain, which is hinged by Ca21 to the FVa light chain. Following the cleavage, 2 chains are linked via a divalent metal ion bridge. FV acts as a cofactor for APC and protein S in the inactivation of the procoagulant FVIIIa. A cleavage of FV at R506 by APC is required for this cofactor function. FVL, the mutant FV, is seen with APCR. In this mutant, R506 is replaced by a Q, which renders the 506-position insensitive to proteolysis by APC. This is referred to as Q506FV which does not show anticoagulant activity [25]. Inactivation of mutant FVa: Q506 site leads to FVL. FVL mutation accounts for about 90% of APCR and is prevalent in nearly 2%13% of general population. It is also seen in about 20%60% of VTE cases. Literature shows that a higher proportion of Caucasians are affected than other populations. This could also be relative, due to lack of studies and data from Asian and other populations. About 1 in 10 people carrying FVL heterozygotes develop VTE over their lifetime. Many patients with familial thrombophilia are found to have FVL. Other coexisting disorders and circumstantial risk factors also affect clinical expression of FVL. These include presence of prothrombin variant G20210A, obesity, age, immobility due to injury, surgery, oral contraception, hormone replacement therapy, pregnancy, and even air travel [26]. It has been suggested and is advisable to offer genetic testing and referral for consultation in first unprovoked VTE at any age, history of recurrent VTE, strong family history of thrombotic diseases, VTE during pregnancy or puerperium or when it is the first VTE incident and patient has a first-degree family member with a history of VTE under the age of 50. Case 1 in this chapter is a good example where offering genetic testing might be useful for patient and future generations. It has been suggested that 10% of FVL carriers develop VTE in their lifetime, and 25%40% are at higher risk where there is a family history of thrombophilia.
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One of the potential benefits of genetic testing is that asymptomatic FVL carriers could be educated about VTE high-risk circumstances, signs and symptoms, as well as possible potential need for prophylactic treatment in high-risk circumstances. However, genetic testing does not detect all possible thrombophilia conditions and has limited clinical utility. Further research is required to confidently improve surveillance and management guidelines as there is very limited and inconsistent data, if at all, in use currently. 19.2.3.4 Hyperhomocysteinemia MTHFR gene is responsible for production of enzyme MTHFR which has an important role in the homocysteine metabolism. MTHFR gene has at least two known polymorphisms, 677C . T and 1298A . C. These are associated with reduced enzyme activity, decreased folate concentration in blood, mildly increasing plasma, and total homocysteine concentration [24]. 19.2.3.5 Inherited deficiency of fibrinolysis Decreased fibrinolytic capacity due to increased plasminogen activator inhibitor-1 (PAI-1) activity and decreased tissue-type plasminogen activator (tPA) activity has been associated with thrombosis. A congenital gamma-chain molecular defect or gamma dysfibrinogenemia such as fibrinogen Dusard (Arg554Cys) results in impaired binding of tPA to fibrin-reducing plasminogen activation, impaired fibrinolysis, and tendency for thrombosis [27]. PAI-1 gene has also been shown to have multiple polymorphisms. However, only patients with 4G/4G genotype of the PAI-1 genetic polymorphism show an increased plasma PAI-1 concentration which is significantly associated with an increased risk of thrombosis [28]. Plasminogen is activated to plasmin by specific cleavage at the Arg560Val561 peptide bond.
19.3 Genetic basis of the bleeding disorders Bleeding disorders in men are well recognized due to a lot of research in X-linked recessive disorders, such as hemophilia, seen in young boys with prominent clinical significance. However, bleeding disorders in females are often missed due to milder presentation at later age. Some studies have focused on hemostatic disorders in menorrhagia [29,30]. Gupta et al. demonstrated that inherited bleeding disorders were present in most of the 200 women with various bleeding complaints in a tertiary hospital in India [31]. Current testing method, widely used by many accredited research-based facilities worldwide, includes Sanger or next-generation sequencing (NGS) for the testing of single-nucleotide variants particularly in hemophilia [32].
19.3.1 Case 2 A 14-year-old boy who had repeated episodes of hemarthrosis affecting his knees ultimately ended with flexion deformity of his right knee. The coagulation and factor testing reported factor VIII deficiency. It seems to be an obvious case of hemophilia, but do genetic and molecular studies on this patient have any role in patient care or future counseling? What other bleeding orders could be suspected? There is no clear discrimination between inherited or acquired mutations leading to bleeding disorders. The complexity of structure with variable gene expression as well as degree of consanguinity in certain populations, concentrating effect of certain mutations within community, indicate that there may be a benefit in investigating patient’s history, and molecular as well as cytogenetic evaluation to rule out coexisting recessively inherited risk factors.
19.3.2 Hemophilia A (factor VIII deficiency) Hemophilia A is an X-linked recessive disorder with factor VIII deficiency mostly in male population. Rarely females may be presented with this condition due to the inheritance of homozygous or compound heterozygous alleles or chromosomal abnormalities. Hemophilia A is caused by pathogenic variant in F8 gene encoding for FVIII protein. The F8 gene, that is 187 kb in size, comprises 25 introns and 26 exons, located on the long arm of the X-chromosome at the most distal band (Xq28) [33]. Ankala et al. found that intron 22 inversion in F8 gene is the most common cause of hemophilia A in Indian population followed by some cases of intron 1 inversion on the same gene [34].
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FIGURE 19.2 Factor VIII molecule and its cleavage sites.
Most pathogenic hemophilia A variants reduce the synthesis or secretion of FVIII or impair FVIII cofactor activity. A small subset of variants, localized to the A3, C1, and C2 domains (Fig. 19.2), are associated with impaired binding to the FVIII carrier molecule von Willebrand factor (VWF) [35], resulting in accelerated proteolysis and/or clearance of FVIII from the plasma. More than 2000 unique F8 gene variants have been reported in the FVIII-variant database [36]. The most common mutation in severe hemophilia A is inversion, producing abnormal or reduced amount of this protein. About half of the hemophilia A cases have reported an intrachromosomal inversion at intron 22 occurring spontaneously during meiosis in male germ cells [37] and a similar inversion has been reported within intron 1 accounting for about 1%2% cases only [38]. Most of the other heterogeneous cases are a result of nonsense/stop mutation preventing factor VIII production, missense mutations’ impact on activity, half-life or efficient factor VIII production, or may be due to some new mutations. Mutations in hemophilia A patients can be detected by chemical cleavage of mismatch method. Inversion mutations can be analyzed by restriction enzyme analysis and southern blotting. Molecular techniques including inverse-shifting PCR, long-range PCR, eight multiplex PCRs, and Sanger sequencing are also useful in diagnosis of these cases. Widely used FVIII-replacement therapy has the risk of FVIII-neutralizing antibodies or inhibitor development in patient. The genetic variability within the F8 gene as well as environmental and genetic risk factors may be able to regulate immune response to FVIII. Intron 22 inversion is associated with lower risk for inhibitors. FVIII mutation status can help predict patient’s response to immune tolerance induction (ITI) therapy for inhibitor eradication. Some large deletions have been shown to be associated with a decreased rate of ITI success [39].
19.3.3 Molecular basis of other inherited coagulopathies 19.3.3.1 von Willebrand disease VWD is the most prevalent of the congenital bleeding disorders affecting both sexes through autosomaldominant inheritance. It is a highly heterogeneous disorder due to the molecular mechanisms that produce various clinical and laboratory phenotypes. VWF gene is located on the short arm of chromosome 12 in the locus p13.3. It consists of 52 exons spanning 178 kilobase pairs [40]. VWF gene encodes for the VWF synthesized in endothelial cells and megakaryocytes. There are patients presented with excessive mucocutaneous bleeding caused by quantitative or qualitative abnormalities in VWF. VWF is a multimedia glycoprotein essential for the maintenance of hemostasis. Multidomain structure is composed of multiples of four domain types AD in the arrangement D1D2D0 D3A1A2A3D4B1B2B3C1C2 (Fig. 19.3). Different functions of VWF are assigned to different domains: A1 domain is involved in binding of VWF to platelet GP1b binding to fibrillar collagen, sulfatides, and heparin. D3 domain binds to factor VIII. C2 domain binds to GP11b-111a receptors. After initial synthesis, it is modified by posttranslational processing of dimerization and multimerization by cleavage of D1D2 domain of the polypeptide.
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FIGURE 19.3 VWF domain structure. VWF, von Willebrand factor.
FIGURE 19.4 Most type 1 VWD are due to missense mutations (dominant negative—interference with intracellular transport of dimeric pro-VWF). Some forms with incomplete penetrance require coinheritance of blood type O for expression (causes increased VWD proteolysis). Most type 3 VWD due to null alleles. VWD, von Willebrand disease; VWF, von Willebrand factor.
VWD is a result of either quantitative deficiency (Type 1), qualitative abnormality (Type 2), or complete absence of VWF (Type 3). Mutations giving rise to Type 2 are very well characterized, whereas those giving rise to Type 1 are not well characterized. Type 2A with point mutation in A2 structural domain of VWF renders VWF more susceptible to proteolysis leaving a lot of small molecular weight multimers in plasma. Type 2B is a rare mutation in A1 structural domain of VWF reducing large molecular weight multimers due to increased binding with resting platelets. Type 2M is a qualitative variant of VWF reducing normal multimers due to high platelet receptor binding. Type 2N results from auto recessive-translation or deletion mutations of VWF gene, where VWF and factor VIII are nearly absent (Fig. 19.4). People with VWF levels at upper or lower end of normal reference range are at risk of hemostatic disorders. Lower levels may lead to the common bleeding disorder Type 1 VWD, and high levels are associated with an increased risk for both venous and arterial thrombosis [41,42]. Most of the Type 1 VWD individuals with VWF levels under 40 have VWF missense gene mutation. Type 2 qualitative defect is often a result of missense mutation including Type 2A with mutations in either of the two domains involved in multimer formation or mutation in domain cleaved by ADAMTS-13. Type 2B shows gain of function mutation in platelet GP1b-binding domain, while Type 2M is a result of the loss of function mutation in GP1b-binding domain and Type 3 patients with severe deficiency are homozygous for null alleles. Results from genome-wide association studies accounted for common variants at ABO, VWF, and other loci in about 12% of the variance in plasma VWF levels [43]. The genetic defects responsible for VWD include a novel gene on chromosome 2 accounting for about 1 in 5 of VWF variations not identified by standard genetic approaches. Further characterization of this and other genes controlling VWF levels may lead to improved VWD diagnosis and prediction of bleeding and clotting risk. In Indian population, mutations c.2908delC and c.5335C . T (p.R1779 ) on VWF gene have been found to cause VWD [44].
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19.3.3.2 Hemophilia B (factor IX deficiency) Hemophilia B is a quantitative or qualitative defect of the coagulation zymogen factor IX presented as an X-linked recessive disorder. The incidence is lower than hemophilia A. Factor IX gene is located on the long arm of X-chromosome (Xq27) and has eight exons. The outcome of the review of literature by Ankala et al. suggested that mutation c.316G . A (p.G106S) is the most common mutation of F9 gene in Indian population [34]. Heterogenous pathogenic variants are found throughout the factor IX gene including the promoter and 30 UTR region. Over 1000 unique variants, majority of them being single-nucleotide substitutions, have been associated with hemophilia B [36]. Most of these variants are on exon 8, the largest exon encoding the functional serine protease domain of the active factor IX coagulation protein. Hemophilia B Leyden is found in about 2% cases presents with moderate to severe prepubertal factor IX deficiency, which eventually returns to normal. This is associated with variants in proximal promoter region of F9 gene and disrupts binding sites to one of the three transcription factors: hepatic nuclear factor 4a, CCAAT enhancer-binding protein (C/EBP), or ONECUT1/2 [4547]. Sequencing the promoter proximal region of F9 gene can be predictive of long-term normalization of factor IX levels. The incidence of inhibitors in these patients is significantly lower than hemophilia A cases. Chitlur et al. reported that about 60% of hemophilia B patients that develop inhibitors often experience an anaphylactic response to FIX concentrate at the time of inhibitor onset. They believe that this is more likely associated with large deletions or null mutations rather than single-nucleotide variants [48]. 19.3.3.3 Afibrinogenemia and dysfibrinogenemia Most cases of reduced or ineffective activity of fibrinogen have been reported from consanguineous parents. Inherited in recessive manner, these disorders are caused by more than 30 genetically heterogenous mutations. It results in moderate to severe bleeding, which could be due to low or zero synthesis of fibrinogen or problems with intracellular transport or secretion. Dysfibrinogenemia usually exhibits dominant inheritance caused by missense mutations affecting fibrin polymerization, fibrinopeptide cleavage, or fibrin stabilization by factor XIIIa. Depending on the mutation, the patient may have mild to severe bleeding tendency.
19.4 Structural defects of the vascular system 19.4.1 Hereditary hemorrhagic telangiectasia This condition has an autosomal dominant inheritance caused by mutation in endoglin gene that controls vascular remodeling. Molecular diagnosis is possible for this condition showing several small arteriovenous malformations in skin, mouth, GI tract, and lungs [24].
19.4.2 EhlersDanlos syndrome EhlersDanlos syndrome (EDS) is a defective collagen structure affecting connective tissues supporting the skin, bones, blood vessels, and many other organs and tissues. EDS is caused by the mutations in more than a dozen genes for various types of collagen with nine different variants, which leads to bruising due to thin, weak skin with poor healing and spontaneous joint dislocation due to hypermobile joints. Inheritance can be autosomal, dominant, recessive, or X-linked [24]. In 2017 EDS was classified describing 13 types. The specific gene affected determines the specific EDS in a patient. Till date, about 18 genes with mutations responsible for EDS have been identified. Some of these are fibrous protein genes, such as COLIA1, COLIA2, COL3A1, COL5A1, and TNXB, and enzymes including ADAMTS2, PLOD1, B4GALT7, DSE, and D4ST1/CHST14. Customized NGS is performed using a panel of various collagen genes. Analysis includes the coding exons of all genes in the panel plus 10 bases in the introns and untranslated regions (50 and 30 ). Sanger sequencing is performed to confirm variants suspected or confirmed to be pathogenic [49]. However, there is no known cure for any type of EDS, so treatment is supportive rather than therapeutic.
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19.5 Inherited defects of platelets 19.5.1 Inherited macrothrombocytopenia Giant platelets seen in peripheral blood could be due to genetic mutations or polymorphism leading to inherited platelet defects. Recent evidence from the literature and testing of different populations from various areas of India [50,51] focused on the presence of inherited disorder where patients have high number of giant platelets. They found an increased recognition of inherited macrothrombocytopenia in the country. Their findings suggest that about half of the patient population showed mutations of MYH9, GP1BB, GP1BA, GPIX, ABCG5, ABCG8, ACTN, FLI, TUBB, and RUNX1 [50], which are frequently seen in heterozygous state. Many of these asymptomatic patients do have mild-to-moderate bleeding history.
19.5.2 BernardSoulier syndrome BernardSoulier disease is caused due to reduced or absent levels of platelet glycoproteins GPIb/IX/V, which is one of the VWF receptors on platelets. This VWF receptor on platelets has four polypeptide chains: GP1b-α, GP1b-β, GPV, and GPIX. Mutations in GP1BA or GP1BB genes reduce expression of these genes. GP1BA insertion mutations (p.Met338fsX13) resulting in frameshift change and (p.Val485fsX13) a new termination codon were reported in people from India. Two missenses (p.Tyr95Asp and p.Cys32X), also resulting in frameshift, as well as one missense (p.Cys24Arg) in GPIX mutation, were also found in some Indian patients [52]. However, this study was conducted on a very small number of eight patients from seven unrelated families with six different detected mutations. This may not have the power to make a generalization about any set population, but it does show high variability, adding further to the complexity of using molecular diagnostics as a tool in many cases.
19.5.3 Glanzmann’s thrombasthenia Autosomal recessive inheritance with decreased platelet GPIIbIIIa expression resulting in defective platelet aggregation and moderate to severe bleeding. It is common in populations with high degree of consanguinity. More than 70 mutations in αIIb and β1 genes on chromosome 17 have been found to be responsible for this disorder [53].
19.5.4 Storage pool disease Dense and alpha granule deficiency is caused by variety of genetic abnormalities. Mutations in a gene for a nucleotide transporter MRP4 (ABCC4) have been suggested to affect nucleotide accumulation in dense granules [54].
19.5.5 MayHegglin anomaly It is a giant platelet syndrome associated with mutations in the nonmuscle myosin heavy chain gene MYH9 with autosomal dominant inheritance [24].
19.5.6 WiscottAldrich syndrome Mutation in WASP-signaling protein decreases secretion and aggregation of platelets with multiple agonists. It has X-linked inheritance. Current diagnosis is based on family history, clinical thrombocytopenia, small platelets with few granules, and genetic testing [24].
19.6 Conclusion Thrombophilia is a very complex multifactorial disorder. However, so far there are less than 10 welldocumented genetically associated risk factors of thrombosis. These defects account for a very low percentage of hereditary thrombophilia and thrombosis leading to a very common and much higher prevalence of
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thrombosis-related cardiovascular events worldwide. Considering these known genetic factors have low frequency particularly in Asia with highest population concentration, there is a need to identify new genetic factors contributing to variation as a collaborative effort amongst different ethnic groups instead of fragmented projects investigating random patient populations. This knowledge will help us not only to personalize the therapeutic algorithms but to propose preventive strategies. There are ever evolving methods, automation, and statistical tools for genetic testing and interpretation to guide effective diagnosis, prophylactic, and therapeutic strategies. Rapid advances in molecular genetics have informed current approaches to the diagnosis of many hemostatic and thrombotic disorders and have revolutionized the treatment of patients with hemophilia with the introduction of recombinant clotting proteins. However, curative gene therapy for inherited disorders of coagulation remains elusive, although continued improvements in the tools for effective and safe gene therapy suggest a bright future for genetic therapies of hemophilia. Dramatic advances in our understanding of the mechanisms of thrombosis and hemostasis have transformed the management of thrombotic and bleeding disorders through the introduction of new, effective, safer, and more convenient therapies. There are still lots of gaps with unknowns, and future studies need to identify directions of research and variation in different populations of world.
Conflict of interest There are no conflicts of interest to declare.
Author’s roles The chapter was written by IS in consultation with RIS and was reviewed by RIS.
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