Hypofibrinogenaemia caused by a novel FGG missense mutation (W253C) in the chain globular domain impairing fibrinogen secretion (original) (raw)
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The molecular basis of quantitative fibrinogen disorders
Journal of Thrombosis and Haemostasis, 2006
Summary. 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 Aα, Bβ, and γ. 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.
Thrombosis and Haemostasis, 2010
SummaryInherited 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 CO...
International Journal of Molecular Sciences
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.
A novel missense mutation in the FGB g. 3354 T>A (p. Y41N), fibrinogen Caracas VIII
Thrombosis and haemostasis, 2011
A novel dysfibrinogenaemia with a replacement of Tyr by Asn at Bβ41 has been discovered (fibrinogen Caracas VIII). An asymptomatic 39-year-old male was diagnosed as having dysfibrinogenaemia due to a mildly prolonged thrombin time (+ 5.8 seconds); his fibrinogen concentration was in the low normal range, both by Clauss and gravimetric determination, 1.9 g/l and 2.1 g/l, respectively. The plasma polymerization process was slightly impaired, characterised by a mildly prolonged lag time and a slightly increased final turbidity. Permeation through the patients' clots was dramatically increased, with the Darcy constant around four times greater than that of the control (22 ± 2 x 10(-9) cm² compared to 6 ± 0.5 x 10(-9) cm² in controls). The plasma fibrin structure of the patient, by scanning electron microscopy, featured a mesh composed of thick fibres (148 ± 50 nm vs. 120 ± 31 nm in controls, p<0.05) and larger pores than those of the control fibrin clot. The viscoelastic properti...
Journal of Blood Disorders & Transfusion, 2014
Inherited fibrinogen disorders affect either the quantity (hypofibrinogenemia, fibrinogen levels <150 mg/dL) and afibrinogenemia, characterized by the complete deficiency of fibrinogen or the quality of the circulating fibrinogen (dysfibrinogenemia) or both (hypo-dysfibrinogenemia) [5]. Up to date, approximately 115 mutations have been reported that cause dysfibrinogenemia, 67 hypofibrinogenemia, 75 afibrinogenemia, and 13 hypodysfibrinogenemia; 101 in the Aα, 63 in the Bβ and 93 in the γ chain. About 50% of approximately more than 600 cases reported in the literature are silent [6,7]. Thrombin binds to its substrate, fibrinogen, and remains bound to the product, fibrin, after fibrinopeptides are removed [8,9]. Different studies have established that Asp7 to Val20, particularly residues on the N-terminal side P1 to P10 (nomenclature is that suggested by Abramovitz [10]) are required for the binding of fibrinogen´s fibrinopeptides to thrombin [11]. Within the sequence of fibrinopeptide A there are both critical (nonvariable) residues and those that can be modified without impairs thrombin catalytic activity [12]. The amino acid sequence of FpA between Asp7 and Arg16 is highly conserved among mammalian species, suggesting that this region is critical for thrombin binding [13,14]. Several abnormal fibrinogens
Quality control of fibrinogen secretion in the molecular pathogenesis of congenital afibrinogenemia
Human Molecular Genetics, 2005
Congenital afibrinogenemia is a rare bleeding disorder characterized by the absence in circulation of fibrinogen, a hexamer composed of two sets of three polypeptides (Aa, Bb and g). Each polypeptide is encoded by a distinct gene, FGA, FGB and FGG, all three clustered in a region of 50 kb on 4q31. A subset of afibrinogenemia mutations has been shown to specifically impair fibrinogen secretion, but the underlying molecular mechanisms remained to be elucidated. Here, we show that truncation of the seven most C-terminal residues (R455-Q461) of the Bb chain specifically inhibits fibrinogen secretion. Expression of additional mutants and structural modelling suggests that neither the last six residues nor R455 is crucial per se for secretion, but prevent protein misfolding by protecting hydrophobic residues in the bC core. Immunofluorescence and immuno-electron microscopy studies indicate that secretion-impaired mutants are retained in a pre-Golgi compartment. In addition, expression of Bb, g and angiopoietin-2 chimeric molecules demonstrated that the bC domain prevents the secretion of single chains and complexes, whereas the gC domain allows their secretion. Our data provide new insight into the mechanisms accounting for the quality control of fibrinogen secretion and confirm that mutant fibrinogen retention is one of the pathological mechanisms responsible for congenital afibrinogenemia.
Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease, 2007
Congenital afibrinogenemia is a rare coagulopathy characterized by extremely low levels of functional and immunoreactive fibrinogen in plasma, associated with a hemorrhagic phenotype of variable severity. It is transmitted as an autosomal recessive trait and is invariantly associated with mutations affecting 1 of the 3 fibrinogen genes (FGA, FGB, and FGG, coding for Aα, Bβ, and γ chain, respectively). Most genetic defects causing afibrinogenemia are truncating mutations, whereas only few missense mutations (6) have been identified so far, all located in FGB. In this study, the mutational screening of an afibrinogenemic Italian male identified the first missense mutation (Met51Arg) in FGA leading to afibrinogenemia. The patient was a compound heterozygote for a previously described frameshift mutation (1215delT) in the same gene. Met51Arg involves a residue located at the very beginning of the coiled-coil domain, in a region demonstrated to play a pivotal role in hexamer formation. In-vitro expression experiments showed that Met51Arg strongly reduces secretion of hexameric fibrinogen, whereas traces of not completely assembled trimeric intermediate were found in conditioned media. Western blot analysis on the proband's plasma confirmed the presence in vivo of the trimeric fibrinogen, supporting the hypothesis that Met51Arg prevents the final step of fibrinogen assembly.
Biomedicines, 2020
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.
Thrombosis and Haemostasis, 2014
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.