Quantitative Fibrinogen Disorders Research Articles

Heterogeneity of Genotype-Phenotype in Congenital Hypofibrinogenemia-A Review of Case Reports Associated with Bleeding and Thrombosis.

Congenital fibrinogen disorders are diseases associated with a bleeding tendency; however, there are also reports of thrombotic events. Fibrinogen plays a role in the pathogenesis of thrombosis due to altered plasma concentrations or modifications to fibrinogen’s structural properties, which affect clot permeability, resistance to lysis, and its stiffness. Several distinct types of genetic change and pathogenetic mechanism have been described in patients with bleeding and a thrombotic phenotype, including mutations affecting synthesis or processing in three fibrinogen genes. In this paper, we focused on familial hypofibrinogenemia, a rare inherited quantitative fibrinogen disorder characterized by decreased fibrinogen levels with a high phenotypic heterogeneity. To begin, we briefly review the basic information regarding fibrinogen’s structure, its function, and the clinical consequences of low fibrinogen levels. Thereafter, we introduce 15 case reports with various gene mutations derived from the fibrinogen mutation database GFHT (French Study Group on Hemostasis and Thrombosis), which are associated with congenital hypofibrinogenemia with both bleeding and thrombosis. Predicting clinical presentations based on genotype data is difficult. Genotype–phenotype correlations would be of help to better understand the pathologic properties of this rare disease and to provide a valuable tool for the identification of patients who are not only at risk of bleeding, but also at risk of a thrombotic event.

Use of Fibrinogen Determination Methods in Differential Diagnosis of Hypofibrinogenemia and Dysfibrinogenemia.

Fibrinogen plays an important role in hemostasis. The normal concentration of fibrinogen in blood plasma is between 1.8 - 4.2 g/L. Decreased fibrinogen levels are observed in congenital afibrinogenemia, hypofibrinogenemia, dysfibrinogenemia, disseminated intravascular coagulation, fibrinolytic therapy, some more severe hepatic parenchymal disorders, and increased blood loss. Elevated fibrinogen levels occur in inflammatory diseases and neoplastic diseases, in pregnancy, and postoperative conditions. Functional fibrinogen measurement is also one of the basic coagulation screening tests. The fibrinogen antigen assay is used to distinguish between qualitative and quantitative fibrinogen disorders. The aim of the study was the use of fibrinogen determination methods in differential diagnosis of hypofibrinogenemia and dysfibrinogenemia, statistical evaluation and determine the relationship of fibrinogen Clauss assay, prothrombin time (PT) derived fibrinogen assay, and fibrinogen antigen in the group of 60 patients with congenital fibrinogen disorders (n = 40 dysfibrinogenemia; n = 20 hypofibrinogenemia). The results measured by the PT-derived fibrinogen assay were approximately four times higher compared to the fibrinogen Clauss assay in the group of patients with dysfibrinogenemia. In patients with hypofibrinogenemia, there is a correlation (r = 0.9016) between the fibrinogen Clauss assay and PT-derived fibrinogen assay with a statistical significance of p < 0.0001. Using a linear or quadratic interpolation function, we were able to determine the fibrinogen Clauss assay and the fibrinogen antigen assay before analysis. The higher level of the PT-derived fibrinogen assay compared to the fibrinogen Clauss assay in the group of patients with dysfibrinogenemia may pose a greater risk to asymptomatic patients who require diagnosis and treatment in case of bleeding. The fibrinogen value using the PT-derived fibrinogen assay could erroneously give a normal level. The use of the interpolation function is important to estimate the value of fibrinogen activity and antigen before the analysis itself by the Clauss assay or analysis by the fibrinogen antigen assay.

A Novel Nonsense Mutation in FGB (c.1421G>A; p.Trp474Ter) in the Beta Chain of Fibrinogen Causing Hypofibrinogenemia with Bleeding Phenotype.

Congenital hypofibrinogenemia is a rare bleeding disorder characterized by a proportional decrease of functional and antigenic fibrinogen levels. Hypofibrinogenemia can be considered the phenotypic expression of heterozygous loss of function mutations occurring within one of the three fibrinogen genes (FGA, FGB, and FGG). Clinical manifestations are highly variable; most patients are usually asymptomatic, but may appear with mild to severe bleeding or thrombotic complications. We have sequenced all exons of the FGA, FGB, and FGG genes using the DNA isolated from the peripheral blood in two unrelated probands with mild hypofibrinogenemia. Coagulation screening, global hemostasis, and functional analysis tests were performed. Molecular modeling was used to predict the defect of synthesis and structural changes of the identified mutation. DNA sequencing revealed a novel heterozygous variant c.1421G>A in exon 8 of the FGB gene encoding a Bβ chain (p.Trp474Ter) in both patients. Clinical data from patients showed bleeding episodes. Protein modelling confirmed changes in the secondary structure of the molecule, with the loss of three β sheet arrangements. As expected by the low fibrinogen levels, turbidity analyses showed a reduced fibrin polymerisation and imaging difference in thickness fibrin fibers. We have to emphasize that our patients have a quantitative fibrinogen disorder; therefore, the reduced function is due to the reduced concentration of fibrinogen, since the Bβ chains carrying the mutation predicted to be retained inside the cell. The study of fibrinogen molecules using protein modelling may help us to understand causality and effect of novel genetic mutations.

Genetic Variants in the FGB and FGG Genes Mapping in the Beta and Gamma Nodules of the Fibrinogen Molecule in Congenital Quantitative Fibrinogen Disorders Associated with a Thrombotic Phenotype

Fibrinogen is a hexameric plasmatic glycoprotein composed of pairs of three chains (Aα, Bβ, and γ), which play an essential role in hemostasis. Conversion of fibrinogen to insoluble polymer fibrin gives structural stability, strength, and adhesive surfaces for growing blood clots. Equally important, the exposure of its non-substrate thrombin-binding sites after fibrin clot formation promotes antithrombotic properties. Fibrinogen and fibrin have a major role in multiple biological processes in addition to hemostasis and thrombosis, i.e., fibrinolysis (during which the fibrin clot is broken down), matrix physiology (by interacting with factor XIII, plasminogen, vitronectin, and fibronectin), wound healing, inflammation, infection, cell interaction, angiogenesis, tumour growth, and metastasis. Congenital fibrinogen deficiencies are rare bleeding disorders, characterized by extensive genetic heterogeneity in all the three genes: FGA, FGB, and FGG (enconding the Aα, Bβ, and γ chain, respectively). Depending on the type and site of mutations, congenital defects of fibrinogen can result in variable clinical manifestations, which range from asymptomatic conditions to the life-threatening bleeds or even thromboembolic events. In this manuscript, we will briefly review the main pathogenic mechanisms and risk factors leading to thrombosis, and we will specifically focus on molecular mechanisms associated with mutations in the C-terminal end of the beta and gamma chains, which are often responsible for cases of congenital afibrinogenemia and hypofibrinogenemia associated with thrombotic manifestations.

Congenital structural and functional fibrinogen disorders: a primer for internists.

Congenital qualitative and quantitative fibrinogen disorders represent heterogeneous rare abnormalities caused by mutations in one of the 3 genes encoding individual fibrinogen polypeptide chains, located on chromosome 4q28. It is estimated that congenital fibrinogen disorder accounts for 8% of rare coagulation factor deficiencies. Most of congenital fibrinogen disorders are suspected in individuals with bleeding tendency or coincidentally discovered, for instance prior to surgery. Fibrinogen disorders could be also found in patients with thrombotic events, impaired wound healing, and recurrent spontaneous abortions. Afibrinogenemia manifests as mild to severe bleeding, while hypofibrinogenemia is often asymptomatic. Dysfibrinogenemia, a qualitative fibrinogen disorder, is associated with bleeding, thrombosis, or with no symptoms. Recent recommendations issued by the International Society on Thrombosis and Haemostasis in 2018 do not encourage routine evaluation of thrombin time or other coagulation tests in patients with suspected congenital fibrinogen disorders, highlighting the value of fibrinogen antigen measurement and genetic analysis, added to the key finding, that is, reduced fibrinogen concentration determined with a coagulometric assay. The current review summarizes practical issues in diagnostic workup and clinical management of patients with afibrinogenemia, hypofibrinogenemia, dysfibrinogenemia, and hypodysfibrinogenemia from a perspective of internists who may encounter patients with reduced fibrinogen concentration in everyday practice. Despite the fact that hematologists are in front line for the management of patients with bleeding tendency, internists should be aware of the clinical and laboratory findings in patients with inherited fibrinogen disorders including the risk of thromboembolism and management prior to invasive procedures.

Identification of Two Novel Fibrinogen Bβ Chain Mutations in Two Slovak Families with Quantitative Fibrinogen Disorders.

Congenital fibrinogen disorders are caused by mutations in one of the three fibrinogen genes that affect the synthesis, assembly, intracellular processing, stability or secretion of fibrinogen. Functional studies of mutant Bβ-chains revealed the importance of individual residues as well as three-dimensional structures for fibrinogen assembly and secretion. This study describes two novel homozygous fibrinogen Bβ chain mutations in two Slovak families with afibrinogenemia and hypofibrinogenemia. Peripheral blood samples were collected from all subjects with the aim of identifying the causative mutation. Coagulation-related tests and rotational thromboelastometry were performed. All exons and exon–intron boundaries of the fibrinogen genes (FGA, FGB and FGG) were amplified by PCR followed by direct sequencing. Sequence analysis of the three fibrinogen genes allowed us to identify two novel homozygous mutations in the FGB gene. A novel Bβ chain truncation (BβGln180Stop) was detected in a 28-year-old afibrinogenemic man with bleeding episodes including repeated haemorrhaging into muscles, joints, and soft tissues, and mucocutaneous bleeding and a novel Bβ missense mutation (BβTyr368His) was found in a 62-year-old hypofibrinogenemic man with recurrent deep and superficial venous thromboses of the lower extremities. The novel missense mutation was confirmed by molecular modelling. Both studying the molecular anomalies and the modelling of fibrinogenic mutants help us to understand the extremely complex machinery of fibrinogen biosynthesis and finally better assess its correlation with the patient’s clinical course.

Management of congenital quantitative fibrinogen disorders: a Delphi consensus.

No evidence-based guidelines for the management of patients suffering from afibrinogenaemia and hypofibrinogenaemia are available. The aim of this study was to harmonize patient's care among invited haemophilia experts from Belgium, France and Switzerland. A Delphi-like methodology was used to reach a consensus on: prophylaxis, bleeding, surgery, pregnancy and thrombosis management. The main final statements are as follows: (i) a secondary fibrinogen prophylaxis should be started after a first life-threatening bleeding in patients with afibrinogenaemia; (ii) during prophylaxis the target trough fibrinogen level should be 0.5 g L-1 ; (iii) if an adaptation of dosage is required, the frequency of infusions rather than the fibrinogen amount should be modified; (iv) afibrinogenaemic patients undergoing a surgery at high bleeding risk should receive fibrinogen concentrates regardless of the personal or family history of bleeding; (v) moderate hypofibrinogenaemic patients (i.e. ≥0.5 g L-1 ) without previous bleeding (despite haemostatic challenges) undergoing a surgery at low bleeding risk may not receive fibrinogen concentrates as prophylaxis; (vi) monitoring the trough fibrinogen levels should be performed at least once a month throughout the pregnancy and a foetal growth and placenta development close monitoring by ultrasound is recommended; (vii) fibrinogen replacement should be started concomitantly to the introduction of anticoagulation in afibrinogenaemic patients suffering from a venous thromboembolic event; and (viii) low-molecular-weight heparin is the anticoagulant of choice in case of venous thromboembolism. The results of this initiative should help clinicians in the difficult management of patients with congenital fibrinogen disorders.

Can the phenotype of inherited fibrinogen disorders be predicted?

Congenital fibrinogen disorders are rare diseases affecting either the quantity (afibrinogenaemia and hypofibrinogenaemia) or the quality (dysfibrinogenaemia) or both (hypodysfibrinogenaemia) of fibrinogen. In addition to bleeding, unexpected thrombosis, spontaneous spleen ruptures, painful bone cysts and intrahepatic inclusions can complicate the clinical course of patients with quantitative fibrinogen disorders. Clinical manifestations of dysfibrinogenaemia include absence of symptoms, major bleeding or thrombosis as well as systemic amyloidosis. Although the diagnosis of any type of congenital fibrinogen disorders is usually not too difficult with the help of conventional laboratory tests completed by genetic studies, the correlation between all available tests and the clinical manifestations is more problematic in many cases. Improving accuracy of diagnosis, performing genotype, analysing function of fibrinogen variants and carefully investigating the personal and familial histories may lead to a better assessment of patients' phenotype and therefore help in identifying patients at increased risk of adverse clinical outcomes. This review provides an update of various tests (conventional and global assays, molecular testing, fibrin clot analysis) and clinical features, which may help to better predict the phenotype of the different types of congenital fibrinogen disorders.

Clinical Features and Management of Congenital Fibrinogen Deficiencies.

Congenital fibrinogen disorders are rare diseases affecting either the quantity (afibrinogenemia and hypofibrinogenemia) or the quality (dysfibrinogenemia) or both (hypodysfibrinogenemia) of plasmatic fibrinogen. Afibrinogenemia is often diagnosed at birth following prolonged umbilical cord bleeding and is characterized by spontaneous bleeding in all tissues, while hypofibrinogenemic patients are more often asymptomatic. Spontaneous spleen ruptures, painful bone cysts, cardiovascular events, and intrahepatic inclusions can complicate the clinical course of patients with quantitative fibrinogen disorders. Clinical manifestations of dysfibrinogenemia are very heterogeneous, from absence of symptoms to major bleeding or thrombosis, chronic thromboembolic pulmonary hypertension, and renal amyloidosis. Hypodysfibrinogenemic patients can suffer from both major bleeding and recurrent thrombosis. Pregnancy of women with congenital fibrinogen disorders is a high-risk situation. Owing to the absence of controlled randomized studies, clinical management is mainly based on expert consensus. For the treatment and/or the prevention of bleeding, plasma-derived fibrinogen concentrates are the optimal choice. Treatment of thrombosis may be challenging. More specifically, management strategies should be tailored to each patient, taking the personal and familial history of bleeding and thrombosis, the genotype, and the specific clinical situation into account.

Molecular characterization of 7 patients affected by dys- or hypo-dysfibrinogenemia: Identification of a novel mutation in the fibrinogen Bbeta chain causing a gain of glycosylation

Fibrinogen is a hexameric glycoprotein consisting of two sets of three polypeptides (the Aα, Bβ, and γ chains, encoded by the three genes FGA, FGB, and FGG). It is involved in the final phase of the coagulation process, being the precursor of the fibrin monomers necessary for the formation of the hemostatic plug. Rare inherited fibrinogen disorders can manifest as quantitative deficiencies, qualitative defects, or both. In particular, dysfibrinogenemia and hypo-dysfibrinogenemia are characterized by reduced functional activity associated with normal or reduced antigen levels, and are usually determined by heterozygous mutations affecting any of the three fibrinogen genes.In this study, we investigated the genetic basis of dys- and hypo-dysfibrinogenemia in seven unrelated patients. Mutational screening disclosed six different variants, two of which novel (FGB-p.Asp185Asn and FGG-p.Asn230Lys). The molecular characterization of the FGG-p.Asn230Lys mutation, performed by transient expression experiments of the recombinant mutant protein, demonstrated that it induces an almost complete impairment in fibrinogen secretion, according to a molecular mechanism often associated with quantitative fibrinogen disorders. Conversely, the FGB-p.Asp185Asn variant was demonstrated to be a gain-of-glycosylation mutation leading to a hyperglycosylation of the Bβ chain, not affecting fibrinogen assembly and secretion. To our knowledge, this is the second gain-of-glycosylation mutation involving the FGB gene.

Clinical and molecular characterisation of 21 patients affected by quantitative fibrinogen deficiency.

Fibrinogen is a plasma glycoprotein mainly synthesised by hepatocytes and circulating as a 340-kDa hexamer consisting of two sets of three different polypeptide chains (Aα, Bβ, and γ, encoded by the FGA, FGB, and FGG gene, respectively). Congenital afibrinogenaemia and hypofibrinogenaemia are rare bleeding disorders characterised by abnormally low levels of functional and immunoreactive fibrinogen in plasma, associated with haemorrhagic manifestations of variable severity. While afibrinogenaemia is caused by mutations in the homozygous or compound heterozygous state in one of the three fibrinogen genes, hypofibrinogenaemia is generally due to heterozygous mutations, and is usually characterised by a milder phenotype. The mutational spectrum of these quantitative fibrinogen disorders includes large deletions, point mutations causing premature termination codons, and missense mutations often affecting fibrinogen assembly and/or secretion. Here we report the clinical and molecular characterisation of 13 unrelated afibrinogenaemic and eight hypofibrinogenaemic patients, leading to the identification of 17 different mutations (10 hitherto unknown). All the newly-identified missense and splicing mutations werein vitro expressed to verify their pathogenic role. Our data increase the number of mutations causing quantitative fibrinogen deficiencies by about 7 %. The high number of private mutations identified in the analysed probands indicates that the full mutational screening of the three fibrinogen genes is still required for molecular diagnosis.

Molecular basis of quantitative fibrinogen disorders in 27 patients from India

Congenital fibrinogen deficiency is an extremely rare (1:1 000 000) hereditary bleeding disorder caused by defects in genes coding for fibrinogen Aα-, Bβ- and γ-chains, respectively. We report here the molecular basis of fibrinogen deficiency in a large series of patients from India. Twenty-seven patients with clinical features suggestive of fibrinogen deficiency and with prolonged plasma clotting times and low fibrinogen levels were studied. Genomic DNA was screened for mutations in the fibrinogen alpha (FGA), beta (FGB), gamma (FGG) genes by PCR and conformation sensitive gel electrophoresis. Fourteen different disease-causing mutations including frameshifts (51.9%), splice site (22.2%), missense (18.5%) and nonsense mutation (7.4%) were identified in 27 patients. Thirteen of them were novel, including seven frameshifts (fibrinogen Aα: p.Asp296 fs*59, p.Thr466 fs*17 and p.Lys575 fs*74; fibrinogen Bβ: p.Gly414 fs*2 and fibrinogen γ: p.Ser81 fs*5, p.Lys185 fs*13 and p.Asp278_279 fs*17), three splice site mutations (FGA gene c.364+1G>A; c.510+2 T>G; FGB gene c.851+1G>A), two missense substitutions (fibrinogen Bβ: p.Gly288Ser; p.Arg445Thr) and a nonsense mutation in fibrinogen Aα (p.Tyr127*). Two common mutations (FGA: c.364+1G>A, n = 6, FGG: p.Lys185 fs*13, n = 7) affecting 13 patients were identified in this series, suggesting that these mutations could be screened first in Indian patients with fibrinogen deficiency. The molecular data presented here is the largest series of patients with fibrinogen deficiency reported so far, adding significantly to the mutation database of this condition. It also helps create an algorithm for its genetic diagnosis in India.

Congenital hypofibrinogenemia: Characterization of two missense mutations affecting fibrinogen assembly and secretion

Congenital hypofibrinogenemia is a rare bleeding disorder characterized by abnormally low levels of fibrinogen in plasma, generally due to heterozygous mutations in one of the three fibrinogen genes ( FGA, FGB, and FGG, coding for Aα, Bβ, and γ chain, respectively). Hypofibrinogenemic patients are usually asymptomatic, whereas individuals bearing similar mutations in the homozygous or compound heterozygous state develop a severe bleeding disorder: afibrinogenemia. The mutational spectrum of these quantitative fibrinogen disorders includes large deletions, point mutations causing premature termination codons, and missense mutations affecting fibrinogen assembly or secretion, distributed throughout the 50-kb fibrinogen gene cluster. In this study, we report the mutational screening of two unrelated hypofibrinogenemic patients leading to the identification of two missense mutations, one hitherto unknown (αCys45Phe), and one previously described (γAsn345Ser). The involvement of αCys45Phe and γAsn345Ser in the pathogenesis of hypofibrinogenemia was investigated by in-vitro expression experiments. Both mutations were demonstrated to cause a severe impairment of intracellular fibrinogen processing, either by affecting half-molecule dimerization (αCys45Phe) or by hampering hexamer secretion (γAsn345Ser).

The molecular basis of quantitative fibrinogen disorders.

Hereditary fibrinogen disorders include type I deficiencies (afibrinogenemia and hypofibrinogenemia, i.e. quantitative defects), with low or unmeasurable levels of immunoreactive protein; and type II deficiencies (dysfibrinogenemia and hypodysfibrinogenemia, i.e. qualitative defects), showing normal or altered antigen levels associated with reduced coagulant activity. While dysfibrinogenemias are in most cases autosomal dominant disorders, type I deficiencies are generally inherited as autosomal recessive traits. Patients affected by congenital afibrinogenemia or severe hypofibrinogenemia may experience bleeding manifestations varying from mild to severe. This review focuses on the genetic bases of type I fibrinogen deficiencies, which are invariantly represented by mutations within the three fibrinogen genes (FGA, FGB, and FGG) coding for the three polypeptide chains Aalpha, Bbeta, and gamma. From the inspection of the mutational spectrum of these disorders, some conclusions can be drawn: (i) genetic defects are scattered throughout the three fibrinogen genes, with only few sites appearing to represent relative mutational hot spots; (ii) several different types of genetic lesions and pathogenic mechanisms have been described in affected individuals (including gross deletions, point mutations causing premature termination codons, missense mutations affecting fibrinogen assembly/secretion, and uniparental isodisomy associated with a large deletion); (iii) the possibility to express recombinant fibrinogen mutants in eukaryotic cells is rapidly shedding light into the molecular mechanisms responsible for physiologic and pathologic properties of the molecule; (iv) though mutation analysis of the fibrinogen cluster does not yield precise information for predicting genotype/phenotype correlations, it still provides a valuable tool for diagnosis confirmation, identification of potential carriers, and prenatal diagnosis.