Afibrinogenemia Research Articles

Identification and functional characterization of a novel nonsense mutation in FGA accounting for congenital afibrinogenemia in six Egyptian patients

Congenital afibrinogenemia is a rare coagulation disorder attributed to over forty mutations found either in homozygosity or in compound heterozygosity, the majority localized in FGA encoding the fibrinogen Aalpha-chain. Despite the number of genetic analyses performed the study of additional patients still allows the identification of novel mutations and a better understanding of fibrinogen structure and function. Here we report the identification and functional analysis of a novel nonsense mutation in FGA exon 5: c.718C>T (CAG>TAG) p.Q240X (Q221X in the mature chain lacking the signal peptide), accounting for fibrinogen deficiency in six Egyptian patients. Expression of the mutant Aalpha-chain cDNA in combination with wild-type Bbeta-chain and gamma-chain cDNAs demonstrated that although the mutant chain could be detected in the cell media of transfected COS-7 cells it was less secreted in comparison to the wild-type Aalpha-chain. Our patients were all homozygous for p.Q240X(Q221X) yet their clinical spectrum varied considerably in their onset of presentation or severity, with bleeding ranging from moderate mucous membrane bleeds in adolescence to life threatening intracranial hemorrhage in infancy.

Severe Elbow Arthropathy in a Patient with Congenital Afibrinogenemia

Afibrinogenemia is a rare coagulation deficiency, inherited as an autosomal recessive trait. It can be manifested in homozygotes or compound heterozygotes by a severe bleeding tendency caused by deficiency of fibrinogen (factor I). The estimated prevalence in Italy is about 1:1,000,000, and 0.2% of the patients with a coagulation deficiency in the United Kingdom have afibrinogenemia1,2. In Switzerland, nine patients are listed in the national hemophilia registry. Among severe bleeding symptoms, umbilical cord bleeding is the most frequent and life-endangering event3. Intracranial hemorrhage is the major cause of death and is the primary reason for prophylaxis. Less severe findings, such as spontaneous muscle hematomas, epistaxis, and menorrhagia, are also frequent3. Patients with afibrinogenemia have remarkably fewer spontaneous hemarthroses in contrast to better known bleeding disorders such as hemophilia A and hemophilia B3-6. We report the case of a patient with congenital afibrinogenemia who had severe elbow arthropathy and was treated with a total elbow arthroplasty. The patient was informed that data concerning the case would be submitted for publication, and he consented. A forty-seven-year-old man was referred to our institution with a severe arthropathy of the left, nondominant elbow. Afibrinogenemia was diagnosed at birth. For many years, he was administered 4 g of Haemocomplettan HS (human fibrinogen; CSL Behring, Bern, Switzerland) intravenously every two weeks. The causative mutation in his family (the parents were nonconsanguineous, and his brother and two cousins were also affected) was identified as a homozygous deletion of approximately 11 kb of the fibrinogen alpha chain gene7. Over the years, the left elbow became increasingly symptomatic secondary to recurrent hemarthroses. The elbow was always the primary joint involved, although mild osteoarthritic changes were noted in the ankles. In 1991, at the age of …

Congenital Afibrinogenemia presenting as antenatal intracranial bleed: a case report

Congenital afibrinogenemia is a very rare inherited coagulation disorder, characterized by virtual absence of plasma fibrinogen (factor I). There are only about 250 cases reported in the world literature [1]. We describe a case of congenital afibrinogenemia which presented as an antenatally detected intracranial bleed.

Hypodysfibrinogenaemia due to production of mutant fibrinogen alpha-chains lacking fibrinopeptide A and polymerisation knob ‘A’

Inherited disorders of fibrinogen are rare and affect either the quantity (hypofibrinogenaemia and afibrinogenaemia) or the quality of the circulating fibrinogen (dysfibrinogenaemia) or both (hypodysfibrinogenaemia). Extensive allelic heterogeneity has been found for all these disorders: in congenital afibrinogenaemia for example more than 40 mutations, the majority in FGA , have been identified in homozygosity or in compound heterozygosity. Numerous mutations have also been identified in patients with hypofibrinogenaemia, many of these patients are in fact heterozygous carriers of afibrinogenaemia mutations. Despite the number of genetic analyses performed, the study of additional patients still allows the identification of novel mutations. Here we describe the characterization of a novel FGA intron 2 donor splice-site mutation (Fibrinogen Montpellier II) identified in three siblings with hypodysfibrinogenaemia. Functional analysis of RNA produced by the mutant minigene in COS-7 cells revealed that the mutation led to the in-frame skipping of exon 2. Western blot analysis of COS-7 cells expressing an exon 2 deleted FGA cDNA revealed that an alpha-chain lacking exon 2, which codes in particular for fibrinopeptide A and polymerisation knob 'A', has the potential to be assembled into a hexamer and secreted. Analysis of precipitated fibrinogen from patient plasma showed that the defect leads to the presence in the circulation of alpha-chains lacking knob 'A' which is essential for the early stages of fibrin polymerisation. Fibrin made from purified patient fibrinogen clotted with thrombin displayed thinner fibers with frequent ends and large pores.

Intracranial Haemorrhage in Patients with An Inherited Bleeding Disorder: A Review of 10 Years Experience in a UK Haemophilia Comprehensive Centre.

Abstract 3489Poster Board III-426 Introduction-Inherited bleeding disorders comprise a heterogeneous group of diseases that reflect abnormalities of coagulation proteins, platelets and blood vessels. Intracranial haemorrhage (ICH) is an unusual but life-threatening complication of such disorders. A 10-year retrospective study was conducted to investigate the episodes of ICH in patients with an inherited bleeding disorder who where followed by a regional haemophilia comprehensive care center in the UK. Methods-All cases of ICH occurring in patients with an inherited bleeding disorder which presented between 1999 and 2009 were retrospectively identified using the haemophilia centre database. Case notes were reviewed and aetiology, clinical features, radiological findings, management and outcome of ICH in this population were studied. Results-Of 640 patients registered with an inherited bleeding disorder in haemophilia centre database, a total of 10 ICHs in 9 patients were identified. Age of patients ranged from 5 months to 72 years with a higher number of males (M:F = 7:2). Diagnosis included: Severe haemophilia A (3 patients); Mild haemophilia A (1 patient); Severe haemophilia B (1 patient); Type 2A von Willebrand disease (VWD) (1 patient); Type 3 VWD (1 patient); Congenital afibrinogenemia (1 patient); Severe factor V deficiency (1 patient). Two of the patients with severe haemophilia A had inhibitors, one of whom was receiving immune tolerance therapy at the time of the ICH. 7 of ICHs were spontaneous and 3 were trauma-related. ICH was the first significant bleeding event in 2 patients- one with severe haemophilia A, aged 5 months, and one with severe haemophilia B, aged 6 months. Type of ICHs were 7 subdural, 1 subarachnoid and 2 subdural with intracerebral. All patients were symptomatic. Common presenting features were headache (6 patients), vomiting (5 patients), limb paralysis (3 patients), cranial nerve palsy (3 patients) and seizures (2 patients). None had systemic bleeding concurrent to the ICH. Initial diagnostic modality was CT scan without contrast for all patients. The two patients with severe haemophilia A and inhibitors were initially treated with recombinant factor VIIa. The remaining patients with haemophilia were treated with factor VIII/IX concentrate. The patient with severe haemophilia B required surgical evacuation. The patients with severe haemophilia A/B without inhibitors were commenced on long-term prophylaxis with factor replacement therapy following their ICH. The patient with congenital afibrinogenemia was initially treated with fibrinogen concentrate, repeated CT scan on day ten showed significant expansion of hematoma, surgical evacuation was done and he was commenced on long-term prophylaxis. The patient with factor V deficiency received prothrombin complex concentrate (octaplex). The two patients with VWD were treated with haemate P, the patient with type 2A VWD received prophylaxis for 4 weeks. The patient with type 3 VWD died within an hour of admission despite haemate P treatment. Six patients had long-term morbidity including limb weakness, seizure, ventriculoperitoneal shunt and developmental delay. These results are summarized in Table 1. Conclusions-ICH can occur in patients with a severe inherited bleeding disorder and can result in substantial morbidity and mortality. ICH can also occur in patients with a mild inherited bleeding disorder following trauma. There is no reliable predictor for intracranial bleeding which could aid decision-making in terms of prophylactic therapy to prevent ICH. CT scan is an appropriate initial diagnostic modality. It is important to educate patients and parents about symptoms to allow early diagnosis. Treatment should be administered rapidly to control bleeding in order to limit adverse outcomes.Table 1AgeSexDiagnosisType of ICHSpontaneous or traumaticLong term morbidity/mortality5MoMSevere Haemophilia ASDHSpontaneousDevelopmental delay, VP shunt3YMSevere Haemophilia ASAHSpontaneousLimb weakness48YMSevere Haemophilia ASDHSpontaneousSeizure20YMMild Haemophilia ASDH and IntracerebralTraumaticNil6MoMSevere Haemophilia BSDHSpontaneousDevelopmental delay, Seizure, VP shunt37YMCongenital afibrinogenemiaSDHSpontaneousLimb weakness72YMType 2A VWDSDH and IntracerebralSpontaneousNil19YFType 3 VWDSDHTraumaticDied53 YFSevere factor V deficiencySDHTraumaticLimb weakness Disclosures:No relevant conflicts of interest to declare.

A novel frameshift mutation in FGA accounting for congenital afibrinogenemia predicted to encode an aberrant peptide terminating 158 amino acids downstream

Congenital afibrinogenemia is a rare autosomal recessive disorder characterized by complete absence of detectable fibrinogen and bleeding symptoms. Many causative mutations have been described to date in all three fibrinogen genes, most of them in the fibrinogen A alpha-chain gene (FGA), but also in the fibrinogen B beta-chain gene (FGB) and the fibrinogen gamma-chain gene (FGG). We report here a novel frameshift mutation (p.Glu262AspfsX158) in FGA exon 5 predicted to lead to a truncated polypeptide with an exceptionally long stretch of abnormal residues identified in homozygosity in a patient with congenital afibrinogenemia. Interestingly, five other frameshift mutations predicted to truncate at the same stop codon have already been described in FGA exon 5.

Spinal Cord Infarction in Congenital Afibrinogenemia: A Case Report and Review of the Literature

A 22-year-old woman with congenital afibrinogenemia presented with acute-onset rapidly progressive quadriparesis as a result of spinal cord infarction caused by vertebral artery dissection. Magnetic resonance imaging scans showed microhemorrhages in addition to edematous swelling suggesting acute ischemia throughout cervical and upper thoracic portions of the spinal cord. Fat-saturated T1-weighted magnetic resonance examination and digital subtraction angiography studies demonstrated cervical vertebral artery dissection on the right. This case provides an example of how a primary bleeding disorder could result in a severe ischemic complication caused by an occlusive vessel wall hematoma. Along with other reports, diagnostic and therapeutic aspects of this paradoxical situation were discussed in the particular setting of acute spinal cord ischemia.

The Evaluation of a Prothrombotic Risk in a Human Fibrinogen Concentrate

Fibrinogen is a circulating coagulant protein which is converted to polymerizable fibrin by thrombin. The human fibrinogen concentrate (FC) (CSL Behring) has been used for the treatment of bleeding in fibrinogen deficiency. In this investigation we investigated the prothrombotic potential of FC using the in-vivo, Wessler venous stasis assay in rabbits Thrombogenicity in clinical use was assessed in a postmarketing drug surveillance review. Forty-five rabbits were randomized to receive either placebo or 40 mg/kg of one of four different lots of FC, injected into a marginal ear vein. Thirty seconds thereafter, a segment of the right jugular vein (1.5 – 2.0 cm) was ligated. After 15 minutes the segment was removed and evaluated for thrombus formation according to a score system from 0 –3. Thrombogenic materials, such as activated prothrombin complexes, have consistently shown high scores of thrombus formation in this assay. The mean score in the FC treated rabbits (0.85 ± 1.04) was not different from that in the placebo-treated rabbits (0.8 ± 0.84). Postmarketing data was comprised of spontaneous reports to a postmarketing surveillance system for Adverse Drug Reports (ADR). The number of ADR reports was expressed relative to the number of estimated single standard doses. The estimated dose per patient was adjusted to the degree of bleeding (between one to eight gram). Between January 01, 1986 and April 30, 2008 in total 983,195 g of Haemocomplettan® P had been distributed. Very few reports (n=9) of thromboembolic events have been reported throughout postmarketing surveillance, corresponding to one case report for every 13,655 treatments of 8 grams FC. The reported thromboembolic events occurred in patients with congenital afibrinogenemia or acquired hypofibrinogenemia. In most cases, patients had additional risk factors (e.g. concomitant administration of blood products prothrombin complex concentrate, platelet concentrates, activated factor VII, etc.). Although a causal relationship to administration of FC could not be definitely excluded, the occurrence of thromboembolic events is very rare. From congruent results of both a classical preclinical model of thrombogenicity and analysis of postmarketing data, FC appears to have a very low risk of thrombogenicity.

Capsule endoscopy findings in congenital afibrinogenemia-associated angiopathy

Congenital afibrinogenemia is a rare genetic disorder characterized by the complete absence of functional fibrinogen. We report a 22-year-old female who developed nephrogenic arterial hypertension and intestinal ischemia due to congenital afibrinogenemia-associated angiopathy of large abdominal arteries. We describe, for the first time, the capsule findings and discuss the pathophysiology of this unusual condition.

Interstitial pregnancy in a woman with congenital afibrinogenemia

Without fibrinogen replacement, pregnancies in patients with congenital afibrinogenemia end in miscarriage between 6 and 8 weeks of gestation. Studies in animal models suggest that disturbance of the embryo's decidual attachment contributes to miscarriage in afibrinogenemia. Ectopic pregnancy in human congenital afibrinogenemia has not been previously documented. We present a case of right interstitial pregnancy in a woman with congenital afibrinogenemia, who had not received the recommended fibrinogen replacement for the first 6 weeks of pregnancy. The pregnancy followed a right salpingo-oophorectomy for ovarian hemorrhage. Therefore the embryo most likely entered the uterine cavity via the left tube only to move upstream into the lumen of the interstitial portion of the right tube. Our experience suggests that disturbance of the embryo's decidual attachment may have contributed to the development of an ectopic pregnancy. Therefore, fibrinogen replacement early in the first trimester is of utmost importance.

Pharmacokinetics and pharmacodynamics of a new highly secured fibrinogen concentrate.

Inherited afibrinogenemia is a rare autosomal recessive disorder characterized by the absence or trace amounts of plasma fibrinogen inducing varying bleeding tendencies. Little is known about the pharmacokinetics of plasma-derived fibrinogen concentrates used in the treatment of afibrinogenemic patients. This open, prospective, multicenter study assessed the pharmacokinetic and pharmacodynamic profiles of FIBRINOGENE T1 (FGT1; LFB, Les Ulis, France), a human fibrinogen concentrate treated with three specific biological safety steps. Five adult patients with congenital afibrinogenemia received a single infusion of 0.06 g kg(-1) of FGT1. Plasma samples drawn up to day 14 were assayed for fibrinogen antigen and activity and for coagulation parameters in a central laboratory. Fibrinogen antigen and activity were similar and highly correlated, with very low between-patient variability for pharmacokinetic parameters. Fibrinogen levels increased rapidly and significantly, with a mean plasma concentration of 1.39 g L(-1) being achieved 1 h after the end of the infusion, leading to almost complete in vivo recovery (94%). The mean half-life was 3.4 days, with slow linear elimination, and the distribution was mainly restricted to the vascular compartment. Coagulation parameters were normalized after the infusion and during the following 6-10 days. FGT1 was well tolerated overall. FGT1 behaves like natural functional fibrinogen, and its pharmacokinetic properties are in line with those expected from a fibrinogen concentrate. Our findings suggest that FGT1 can restore efficient hemostasis in afibrinogenemic patients, and predict good clinical efficacy.

Bilateral leukocoria in infant with afibrinogenemia

To report a bilateral leukocoria case in a patient suffering from afibrinogenemia. An observational case where congenital afibrinogenemia was presented with bilateral retinal and vitreous hemorrhages that proceeded to vitreoretinal surgery was presented. In addition, complete ophthalmic and radiological examinations and vitreoretinal surgery were performed. Right eye had a complete recovery while the left eye showed serious proliferative vitreoretinopathy and shortened retina. Three years after the surgery clinical examination showed that the right eye was aphacic with an attached retina and clear ocular media while the left eye was phtysic. We recommend broad clotting profile for infants suffering from vitreous or retinal hemorrhages with no obvious physical abuse. Our present case furthermore implies that afibrinogenemia can lie beneath the pathogenesis of bilateral leukocoria and should alert physician for the presence of an afibrinogenemia among several types of bleeding predispositions.

Treatment of Congenital Afibrinogenemia in a Premature Neonate

Treatment of Congenital Afibrinogenemia in a Premature Neonate

Afibrinogenemia resulting from homozygous nonsense mutation in A alpha chain gene associated with multiple thrombotic episodes

Congenital afibrinogenemia is a rare disorder characterized by the absence in circulating fibrinogen, a hexamer composed of two sets of three polypeptides (Aalpha, Bbeta and gamma). Although predisposition to thrombosis is a well known feature of dysfibrinogenemia, the relatively frequent thrombotic manifestations seen in congenital afibrinogenemia are puzzling. We herein report a mutational analysis of a young afibrinogenemic man from Turkey with multiple thrombo-embolic events involving both arteries and veins. Purified DNAs of the propositus was used for amplification by polymerase chain reaction of all the exons of the A subunit gene with primers allowing the analysis of the intron-exon boundaries. Analysis of the genes coding for the three fibrinogen chains of the propositus found a homozygous G to A transition in the exon 5 of the A alpha chain gene (g.g4277a; access number gi458553). The TGG to TGA codon change predicts a homozygous W315X in the A alpha chain (p.W334X when referring to the translation initiation codon). Both parents and his brother were found to carry this heterozygous mutation. This is the first report of a patient homozygous for this rare mutation associated with afibrinogenemia. Our patient was free of known risk factors as well as diseases associated with thrombosis including atherosclerosis, vasculitis, Buerger's disease, and it seems therefore probable that afibrinogenemia itself might have contributed to both arterial and venous thrombosis.

Spontaneous extra-axial intracranial hemorrhage followed by thrombosis in congenital afibrinogenemia: perioperative management of this rare combination

Background Although congenital afibrinogenemia can commonly present with hemorrhage from the umbilical cord at birth, or with spontaneous mucosal or intracranial hemorrhage in the neonatal period, life-threatening intracerebral hemorrhage in adults is infrequent. Case Description We report a 32-year-old woman with congenital afibrinogenemia. Postoperatively, she developed bilateral pulmonary emboli despite the fact that her INR was elevated to 2.3. Highly purified fibrinogen concentrate infusion may have partly contributed to this complication. An inferior vena caval filter was used successfully to prevent further pulmonary emboli. Conclusions Spontaneous intracerebral hemorrhage must be included in the differential diagnosis in patients with known afibrinogenemia presenting with symptoms suggesting raised intracranial pressure. Immediately after surgery, intracranial pressure monitoring of patients is mandatory to pick up further intracranial bleeding early. Fibrinogen replacement therapy is recommended before surgery, but its use as a long-term prophylaxis against hemorrhage should be weighed against the risk of thrombosis.

Commentary

Fibrinogen is a complex protein playing a major role in coagulation. Congenital afibrinogenemia, characterized by the complete absence of fibrinogen, is associated with major hemostatic defects. Even though the clinical course is unpredictable and can be completely different among patients, severe bleeding is the prominent symptom. Patients are also at increased risk of thrombosis and sometimes suffer from spontaneous spleen rupture, bone cysts and defective wound healing. Due to the relative rarity of afibrinogenemia, there are no evidence-based strategies for helping physicians in care of these patients. Fibrinogen supplementation is the keystone to prevent or treat bleeding events. In addition, fibrinogen, a pleiotropic protein with numerous physiological roles in immunity, angiogenesis and tissue repair, is involved in many diseases. Indeed, depletion of fibrinogen in animal models of infections, tumors and neurological diseases has an effect on the clinical course. The consequences for patients with afibrinogenemia still need to be investigated.

Postoperative deep venous thrombosis in a woman with congenital afibrinogenaemia treated with fibrinogen concentrates

Postoperative deep venous thrombosis in a woman with congenital afibrinogenaemia treated with fibrinogen concentrates

Manipulating the quality control pathway in transfected cells: low temperature allows rescue of secretion-defective fibrinogen mutants

Congenital afibrinogenemia is characterized by the absence of fibrinogen, a hexamer composed of two copies of three polypeptides, Aalpha. Bbeta and gamma. The disease is caused by mutations in one of the three fibrinogen-encoding genes, FGA, FGB and FGG. Among these, several mutations have been reported to specifically impair fibrinogen secretion. We previously showed that secretion-defective fibrinogen mutants are retained in a pre-Golgi compartment and demonstrated the importance of the homologous betaC and gammaC domains in secretion. Here our aim was to restore the secretion of these mutants and study the properties of the rescued mutant molecules. COS-7 cells were transfected and incubated with chemical chaperones or at low temperature. Clotting assays and plasmin digestion studies were performed to characterize secreted fibrinogen molecules. The secretion defect of two missense mutants but not that of late-truncating mutants could be partially corrected by incubating cells at 27 degrees C. By contrast, exposure of cells to chemical chaperones i.e. 4-phenylbutyrate, dimethyl sulfoxide or trimethylamine N-oxide had no effect. The mutants rescued at 27 degrees C were incorporated into fibrin clots and formed factor XIII-mediated gamma-gamma dimers in contrast to the dysfibrinogenemia Vlissingen/Frankfurt IV mutant, a negative control for these assays. However, plasmin digestion analyses revealed aberrant patterns for the mutants compared to normal fibrinogen. Low temperature can restore the secretion of a subset of mutant fibrinogen molecules demonstrating that therapeutic manipulation of the quality control pathway is feasible for afibrinogenemia even though functional assays suggested a non-native conformation for the mutant molecules analyzed.

Pseudo‐exon activation caused by a deep‐intronic mutation in the fibrinogen γ‐chain gene as a novel mechanism for congenital afibrinogenaemia

Congenital afibrinogenaemia, characterized by severe fibrinogen deficiency, is caused by mutations within FGA, FGB or FGG. Conventional sequencing of coding regions and splice signals of these three genes did not reveal any mutation in an afibrinogenaemic proband. After confirming disease co-segregation with the fibrinogen cluster, full intron sequencing was tackled leading to the identification of a novel transvertion within FGG intron 6 (IVS6-320A-->T). Its effect on mRNA processing was evaluated in-vitro: the in-frame inclusion of a 75-bp pseudo-exon carrying a premature stop was found, representing the first report of pseudo-exon activation as a mechanism leading to afibrinogenaemia.

Molecular mechanisms accounting for fibrinogen deficiency: from large deletions to intracellular retention of misfolded proteins.

Fibrinogen, the soluble precursor of fibrin, which is the main protein constituent of the blood clot, is synthesized in hepatocytes in the form of a hexamer composed of two sets of three polypeptides (Aalpha, Bbeta, and gamma). Each polypeptide is encoded by a distinct gene, FGA, FGB and FGG, all three clustered in a region of 50 kb on 4q32. Congenital afibrinogenemia is characterized by the complete absence of fibrinogen. The first causative mutation for this disease was identified in Geneva in a non-consanguineous Swiss family in 1999: the four patients were homozygous for a large deletion in the fibrinogen cluster, which eliminated almost the entire FGA genomic sequence. Mutations in the fibrinogen genes may lead to deficiency of fibrinogen by several mechanisms: acting at the DNA level, at the RNA level by affecting mRNA splicing or stability, or at the protein level by affecting protein synthesis, assembly or secretion. Recent reviews have provided helpful updates for the rapidly growing number of causative mutations identified in patients with fibrinogen deficiencies, either afibrinogenemia or hypofibrinogenemia. The aim of this review is to highlight specifically the subset of mutations that allow fibrinogen chain synthesis and hexamer assembly but impair secretion. Indeed, functional studies of these mutations have shed light on the specific sequences and structures in the fibrinogen molecule involved in the quality control of fibrinogen secretion.