DEFINITION, HISTORY, AND EPIDEMIOLOGY
Type I disorders (afibrinogenemia and hypofibrinogenemia) affect the quantity of fibrinogen in circulation. Type II disorders (dysfibrinogenemia and hypodysfibrinogenemia) affect the quality of circulating fibrinogen.1 While the first dysfibrinogenemia mutation was identified as early as 1968,34 the molecular basis of afibrinogenemia was elucidated much later.35 This disorder is characterized by autosomal recessive inheritance and the complete absence of fibrinogen in plasma.
The disease, originally described in 1920,36 has an estimated prevalence of approximately one in 1,000,000. In populations where consanguineous marriages are common, the prevalence of afibrinogenemia, is increased.37 Because hypofibrinogenemia (fibrinogen levels below 1.5 g L−1) is often caused by heterozygosity for a fibrinogen gene mutation, this is much more frequent than afibrinogenemia. If one applies the Hardy Weinberg binomial distribution of alleles in the population to afibrinogenemia, carriers of fibrinogen deficiency causing mutations could be as frequent as 1 in 500.
ETIOLOGY AND PATHOGENESIS
Since the identification of the first causative mutation for congenital afibrinogenemia in 1999,35 approximately 100 distinct mutations, the majority in FGA, have been identified in patients with afibrinogenemia (in homozygosity or in compound heterozygosity) or in hypofibrinogenemia. Causative mutations can be divided into two main classes: null mutations with no protein production at all and mutations producing abnormal protein chains which are retained inside the cell.1
The first causative mutation for afibrinogenemia was identified in a nonconsanguineous Swiss family with two pairs of afibrinogenemic brothers.35 In a first step toward establishing whether or not the disease was linked to the fibrinogen gene cluster on chromosome 4, haplotype data were obtained for five microsatellite markers surrounding this locus. One of these, FGAi3, a (TCTT)n polymorphic marker located in intron 3 of the FGA gene, was found to be deleted in all four affected individuals and was hemizygous in the obligate carriers, implying that homozygous deletion of at least part of the FGA gene was responsible for the congenital afibrinogenemia in this family. Indeed, the genetic defect was found to be a recurrent deletion of approximately 11 kb of DNA, with breakpoints in FGA intron 1 and the FGA–FGB intergenic region, resulting in an absence of fibrinogen.
Three other large deletions in the fibrinogen gene cluster have been identified, all involving part of the FGA gene. These are: a deletion of 1.2 kb eliminating the entire FGA exon 4 in a Japanese patient38; a deletion of 15 kb, with breakpoints situated in FGA intron 4 and in the FGA–FGB intergenic region in a Thai patient39; and a 4.1-kb deletion encompassing FGA exon 1 in an Italian patient.40 All patients were homozygous for the identified deletions except for the Thai patient, for whom complete maternal uniparental disomy was confirmed for the deleted chromosome 4.39
Several splice-site mutations have been identified in all three fibrinogen genes. In afibrinogenemic patients of European origin, the most common mutation is a donor splice mutation in intron 4, c.510+1G→T (previously described as IVS4+1 G→T).1,41 Haplotype data suggest that this mutation, like the FGA 11-kb deletion, is also recurrent, or a very ancient mutation, because the c.510+1G→T mutation is found on multiple discrete haplotypes.
Frameshift mutations have been identified in all three fibrinogen genes. FGA exon 5, the largest fibrinogen-coding exon has the most frameshift mutations. Interestingly, seven single base-pair deletions in FGA exon 5 result in usage of the same new reading frame. All seven mutations are predicted to encode a long stretch of aberrant amino acids before terminating at the same premature stop codon, 69 to 158 codons downstream.42 The aberrant amino acid sequence (if the abnormal protein is synthesized and stable, which remains to be determined) may lead to abnormal folding of the Aα chain, thus affecting fibrinogen chain assembly or secretion.
Many nonsense mutations accounting for afibrinogenemia and hypofibrinogenemia have been identified. Of the nine nonsense mutations identified in FGB, four are located in FGB exon 8.43 In particular, two FGB nonsense mutations—W467X (W437X) and W470X (W440X)—are localized very close to the β-chain C-terminus and are expected to cause the synthesis of βC chains truncated of only 25 and 22 residues, respectively.44,45 Expression studies in transfected COS cells performed for both mutations showed that the mutations allowed individual chain synthesis and intracellular assembly of the hexamer but impaired secretion, suggesting that an intact FGB C-terminal domain is necessary for fibrinogen secretion into the circulation.46
Null mutations, that is, large deletions, frameshift, early truncating nonsense, and splice-site mutations account for the majority of afibrinogenemia alleles, as expected. Missense mutations leading to complete fibrinogen deficiency are therefore particularly interesting, revealing the functional importance of individual residues or three-dimensional structures. Missense mutations are clustered in the highly conserved C-terminal globular domains of the γ and Bβ chains.1,43 Expression studies in transfected cells for five FGB missense mutations, all identified in homozygosity or compound heterozygosity in afibrinogenemic patients, showed that these mutations, like the late-truncating nonsense mutations discussed previously, allowed individual chain synthesis and intracellular assembly of the hexamer but again impaired secretion.47,48,49,50 Further characterization of the FGB G444S (G414S) mutant using immunostaining for fibrinogen and visualization by confocal microscopy revealed that the secretion-impaired mutant was retained in the ER proving the existence of an efficient quality control mechanism for fibrinogen secretion.46
Several missense mutations have been identified in FGG in heterozygosity in patients with hypofibrinogenemia. For the majority of these mutations, analysis of patient plasma fibrinogen by mass spectrometry confirmed absence of the mutant γ chain in the circulation. Others have been studied at the functional level in transfected cells: fibrinogen Matsumoto IV C179R (C153R) was found to impair intracellular hexamer assembly,51 whereas fibrinogen Bratislava W253C (W227C) was found to impair fibrinogen secretion.52
Mutations Causing Hepatic Endoplasmic Reticulum Retention and Hypofibrinogenemia
In the majority of patients with afibrinogenemia or hypofibrinogenemia there is no evidence of intracellular accumulation of the mutant fibrinogen chain. This implies the existence of an efficient degradation pathway for fibrinogen mutants that allow individual chain synthesis and assembly but not secretion. Four mutations, all in FGG, are known to cause hypofibrinogenemia accompanied by hepatic storage disease. These are three missense mutations (fibrinogen Brescia, Aguadilla, and Al duPont,53,54,55 and a 15-bp deletion at the end of FGG exon 8 (fibrinogen Angers),56 which creates a new FGG exon 8–intron 8 junction and donor splice site. All four mutations cause fibrinogen deficiency in the heterozygous state because of the absence of the mutant γ chain in patient plasma, but also progressive liver disease associated with hepatocellular cytoplasmic inclusions. The molecular mechanism by which these mutations, localized in the five-stranded β-sheet of γC and hole “a,” which are crucial for fibrin polymerization, leads to impaired secretion, retention in the ER, and formation of aggregates remains to be determined.
Bleeding because of afibrinogenemia usually manifests in the neonatal period, with 85 percent of cases presenting umbilical cord bleeding, but a later age of onset is not unusual. Bleeding may occur in the skin, gastrointestinal tract, genitourinary tract, or the central nervous system with intracranial hemorrhage being the major cause of death. Joint bleeding, which is common in patients with severe hemophilia, is less frequent: in a series of 72 patients with severe fibrinogen deficiency, hemarthrosis was observed in 25 percent of cases.57 There is an intriguing susceptibility of spontaneous rupture of the spleen in afibrinogenemic patients. Bone cysts have also been described as a rare complication of afibrinogenemia and appear to benefit from prophylactic therapy with fibrinogen concentrate.58
Menstruating women may experience menometrorrhagia but some have normal menses. First trimester abortion is usual in afibrinogenemic women. The importance of fibrinogen in pregnancy was demonstrated in studies with fibrinogen knockout mice that cannot carry fetuses to term.59 Women may also have antepartum and postpartum hemorrhage. Hemoperitoneum after rupture of the corpus luteum has also been observed.
Paradoxically both arterial and venous thromboembolic complications are observed in afibrinogenemic patients. These complications can occur in the presence of concomitant risk factors such as a coinherited thrombophilic risk factor or after replacement therapy. However, in many patients, no known risk factors are present. Many hypotheses have been put forward to explain this predisposition to thrombosis. One explanation is that even in the absence of fibrinogen platelet aggregation is possible because of the action of von Willebrand factor and, in contrast to patients with severe hemophilia, afibrinogenemic patients are able to generate thrombin, both in the initial phase of limited production and also in the secondary burst of thrombin generation. In some patients, an increase of prothrombin activation fragments or thrombin–antithrombin complexes has been observed, which may reflect enhanced thrombin generation.60 These abnormal levels can be normalized by fibrinogen infusions.
As previously mentioned, fibrin also acts as antithrombin I by both sequestering and downregulating thrombin activity.6 Thrombin which is not trapped by the clot is available for platelet activation and smooth muscle cell migration and proliferation, particularly in the arterial vessel wall. Thrombus formation is maintained in fibrinogen-deficient mice,61 and in fibrinogen-deficient zebrafish,62 but the thrombus is unstable and has a tendency to embolize. Similarly, the absence of fibrinogen in human plasma results in large but loosely packed thrombi under flow conditions.63
Hypofibrinogenemia patients are very often heterozygous carriers of afibrinogenemia mutations.1 These patients are usually asymptomatic with fibrinogen levels of approximately 1.0 g L−1, levels which are in theory high enough to protect against bleeding and maintain pregnancy. However they can bleed when exposed to trauma, or if they have a second associated hemostatic abnormality. Hypofibrinogenemic women may also suffer from pregnancy loss.
The clinical diagnosis is established by functional and immunologic measurements of fibrinogen concentration backed by genetic analyses.
Absence of immunoreactive fibrinogen is essential for the diagnosis of congenital afibrinogenemia. All coagulation tests that depend on the formation of fibrin as the end point—that is, prothrombin time (PT), partial thromboplastin time (PTT), or thrombin time (TT)—are infinitely prolonged. Plasma activity of all other clotting factors is usually normal. Some abnormalities in platelet functions tests can be observed which can be reversed upon addition of fibrinogen. Because fibrinogen is one of the main determinants of erythrocyte sedimentation, it is not surprising that afibrinogenemic patients have very low erythrocyte sedimentation rates. When skin testing is performed for delayed hypersensitivity, there is no induration because of the lack of fibrin deposition.
Hypofibrinogenemia is defined as a proportional decrease of functional and immunoreactive fibrinogen. Coagulation tests depending on the formation of fibrin as well as the assays used are variably prolonged, the most sensitive assay being the TT.
The large number of mutations identified in patients with afibrinogenemia allows the design of an efficient flow-chart for mutation detection in new cases.64 Two common mutations are found in individuals of European origin, both in FGA: the c.510+1G→T intron 4 donor splice-site mutation and the FGA 11-kb deletion, both found on multiple haplotypes. In all new patients of European origin, the FGA c.510+1G→ T should be the first mutation to be screened. Southern blot or polymerase chain reaction (PCR) analysis of the FGA 11-kb deletion should also be performed, because it is the second most common mutation in patients of European origin and because of the risk of diagnostic error: a nonconsanguineous patient who appears to be homozygous for a mutation in FGA exons 2 to 6 may in reality be a heterozygous carrier of the large 11-kb deletion.65 Given the high frequency of mutations in FGA, the other FGA exons (starting with exon 5) should then be studied for mutations before screening FGB (starting with exon 8) and FGG (starting with exons 7 and 8). The same strategy can also be applied to afibrinogenemic patients of non-European origin for whom recurrent mutations have yet to be identified. If the patient comes from a geographical region or population in which a mutation has already been identified, that mutation should be the first to be screened for. Screening of patients with hypofibrinogenemia can follow the same strategy apart from patients with ER fibrinogen-positive liver inclusions, for which four mutations in FGG are known so far to cause hepatic storage disease.
Prenatal diagnosis has been performed in a few cases.66 This is important for families with afibrinogenemia and access to adequate treatment because the prenatal diagnosis of an affected infant allows initiation of treatment immediately after birth before the first bleeding manifestation.
Genotype–Phenotype Correlations: Potential Importance of Global Assays
Current diagnostic tests are appropriate for establishing the diagnosis but clearly additional tests are required for a more accurate prediction of the clinical phenotype of a patient and consequently the appropriate treatment. Indeed, although in afibrinogenemia all patients have unmeasurable functional fibrinogen, the severity of bleeding is highly variable amongst patients, even amongst those with the same genotype. Similarly, there is no clear relationship between the molecular defect and the risk of thrombosis.
One possible explanation for the observed variability of clinical manifestations is the existence of modifier genes/alleles: some variants may increase the severity of bleeding while others may ameliorate the phenotype. Such modifiers have yet to be identified. However, the common thrombophilias (e.g., factor V Leiden) most certainly play a role in decreasing the severity of bleeding. The existence of modifying genes/polymorphisms is also strongly suspected in the previously discussed cases of hypofibrinogenemia associated with fibrinogen inclusion bodies in hepatocytes. Indeed, all individuals heterozygous for one of the four FGG causative mutations have hypofibrinogenemia, but not all have fibrinogen aggregates and associated liver disease.
Global assays, such as thromboelastography and thrombin generation test, may provide a complementary and in some cases a better evaluation of an individual’s hemostatic state. Such global assays could be useful for the design of individual therapeutic strategies.67
Inherited afibrinogenemia and hypofibrinogenemia have to be distinguished from acquired disorders. These include disseminated intravascular coagulation, primary fibrinolysis, liver disease, and can be caused by certain drugs (e.g., thrombolytic agents and l-asparaginase). In addition, one should be aware that artifactually low levels of fibrinogen can be observed with samples that have clotted as a result of improper collection. In most cases, the clinical context as well as the association with other laboratory abnormalities will allow differentiation of inherited from acquired disorders. Identification of a causative mutation in one of the three fibrinogen genes will confirm the diagnosis.
Available Treatments and Modalities
Replacement therapy is effective in treating bleeding episodes in congenital fibrinogen disorders. Depending on the country of residence, patients receive fresh-frozen plasma (FFP), cryoprecipitate, or fibrinogen concentrates.64 Fibrinogen concentrate preparations include safety steps for inactivation or removal of viruses, which make them safer than cryoprecipitate or FFP. Furthermore, more precise dosing can be accomplished with fibrinogen concentrates because their potency is known, in contrast to FFP or cryoprecipitates.
The conventional treatment is on demand, in which fibrinogen is administered as soon as possible after onset of bleeding. Another approach is primary prophylaxis that includes administration of fibrinogen concentrates from an early age to prevent bleeding and, in the case of pregnancy, to prevent miscarriage. Effective long-term secondary prophylaxis with administration of fibrinogen every 7 to 14 days (particularly after central nervous system bleeds) has been advocated. The frequency and dose of fibrinogen concentrates should be adjusted to maintain a level above 0.5 g L−1.64
The United Kingdom guidelines on therapeutic products for coagulation disorders68 provide recommendations about the best treatment options (dosage, management of bleeding, surgery and pregnancy as well as prophylaxis). According to these guidelines, in case of bleeding fibrinogen levels should be increased to 1.0 g L−1 and maintained above this threshold until hemostasis is secured, and above 0.5 g L−1 until wound healing is complete. To increase the fibrinogen concentration of 1 g L−1, a dose of approximately 50 mg/kg is required. The doses and duration of treatment also vary depending on the type of injury or operative procedure and on the patient’s personal and familial history of bleeding and thrombosis.
Women with congenital afibrinogenemia are able to conceive and embryonic implantation is normal, but the pregnancy usually results in spontaneous abortion at 5 to 8 weeks of gestation unless fibrinogen replacement is given.69 Maintaining the fibrinogen level above 0.6 g L−1 and if possible higher than 1.0 g L−1 is recommended. Lower fibrinogen concentrations (<0.4 g L−1) have proven adequate to maintain pregnancy but not to avoid hemorrhagic complications. Continuous infusion of fibrinogen concentrate should be performed during labor to maintain fibrinogen higher than 1.5 g L−1 (ideally greater than 2.0 g L−1).70 Thromboembolic events can occur, particularly with the use of cryoprecipitates that contain appreciable quantities of factor VIII and von Willebrand factor in addition to fibrinogen.
In addition to fibrinogen substitution, antifibrinolytic agents may be given, particularly to treat mucosal bleeding or to prevent bleeding following procedures such as dental extraction. Fibrin glue is useful to treat superficial wounds or following dental extractions. Oral contraceptive preparations are useful in case of menorrhagia. Oral iron preparations can be given in cases with associated iron-deficiency anemia. Routine vaccination against hepatitis, as well as a regular surveillance for both the disease and treatment-related complications in a comprehensive care setting, is highly recommended.64
Finally, orthotopic liver transplantation is a possible rescue treatment for failure of fibrinogen replacement therapy. This procedure successfully restored normal hemostasis in an afibrinogenemic patient with severe Budd-Chiari syndrome and inferior cava vein thrombosis71 and in one of the four afibrinogenemic patients homozygous for the 11-kb FGA mutation.35,72
In many countries only FFP or cryoprecipitate are available, which is problematic because the viral inactivation process is in general not as efficient as it is for fibrinogen concentrates (although emerging nonviral pathogens such as the prion responsible for variant Creutzfeldt-Jacob disease must be considered, even for concentrates). Even if viral inactivation steps are performed, these preparations (particularly FFP) can induce volume overload. There is also a risk of transfusion-related acute lung injury, because of the presence of cytotoxic antibodies in the infused plasma.
Acquired inhibitors to fibrinogen after replacement therapy have been reported in only two cases. It is not clear why afibrinogenemic patients do not develop inhibitors more frequently. One explanation for some cases is that minute amounts of fibrinogen, which can only be detected by highly sensitive immunoassays, are present in the circulation.
One of the major complications in afibrinogenemic patients is thrombosis, which can occur spontaneously following blood component therapy. Some clinicians give small doses of heparin or low-molecular-weight heparin (LMWH) during administration of fibrinogen. Before surgery, patients with a thrombotic phenotype should be treated with compression stockings and LMWH. Successful use of lepirudin has been reported for an afibrinogenemic patient who suffered recurrent arterial thrombosis despite treatment with heparin and aspirin.73 Thromboembolic complications are difficult to manage because both anticoagulants and fibrinogen preparations have to be administered.
The increasing need for fibrinogen preparations in congenital but also in acquired deficiencies has stimulated some companies to improve existing preparations or to develop new ones. A recombinant fibrinogen molecule is also under development.74