[Show abstract][Hide abstract] ABSTRACT: Methods 7 patients with CTEPH, 17 controls (8 healthy and 9 under VKA therapy), two dysfibrinogenemic patients with known fibrinolysis resistance (Chapel-Hill/Dusart fibrinogen due to a missense mutation of the fibrinogen alpha-chain (FGA exon 5: c.1717G>C, p.Arg573Cys or Arg554Cys without the signal peptide) were included. Turbidimetric analysis (wavelength: 405 nm) was performed to define the global fibrinolytic profile. The clot formation was triggered by tissue-factor (TF) and the lysis by exogenous t-PA in two conditions: final concentrations were : 5 pM of TF, 4 uM of phospholipids (PPP reagent, Stago) and 400 ng/mL or 200 ng/mL of t-PA (Metalyse®, Boehringer Ingelheim). The plasma fibrinolytic potential was assessed by the clot lysis time (CTL), defined as the time from the midpoint of the baseline to maximum turbid transition, to the final plateau phase. All tests were performed in triplicate. All exons and intron-exon junctions of the fibrinogen genes were amplified by polymerase chain reaction and sequenced. All results are presented as median (Q1 – Q3).
[Show abstract][Hide abstract] ABSTRACT: Congenital dysfibrinogenemia is a qualitative congenital fibrinogen disorder characterized by normal antigen levels of a dysfunctional fibrinogen. Diagnosis is usually based on discrepancy between fibrinogen activity and antigen levels but could require more specialized techniques for the assessment of fibrinogen function due to some limitations in routine assays. Molecular abnormalities, frequently heterozygous missense mutations localized in exon 2 of FGA and exon 8 of FGG, lead to defects in one or more phases of fibrinogen to fibrin conversion, fibrin network formation and other important functions of fibrinogen. The clinical phenotype is highly heterogeneous, from no manifestations to bleeding and/or thrombotic events. Asymptomatic propositi and relatives with the predisposing genotype are at risk to develop adverse outcomes during the natural course of the disease. Correlations between genotype and phenotype have not yet been clearly established, with the exception of some abnormal fibrinogens that severely increase the risk of thrombosis. Functional analysis of polymerisation and fibrinolysis, structural studies of the fibrin network and viscoelastic properties of fibrin clot could help to predict the phenotype of congenital dysfibrinogenemia but have not yet been evaluated in detail. The management is essentially based on personal and familial history; however, even individuals still asymptomatic and without family history should be carefully assessed and monitored. Particular situations, such as pregnancy, delivery and surgery require a multidisciplinary approach. This article is protected by copyright. All rights reserved.
This article is protected by copyright. All rights reserved.
Journal of Thrombosis and Haemostasis 03/2015; 13(6). DOI:10.1111/jth.12916 · 5.72 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We conducted a multicentre study of 101 subjects with Congenital Dysfibrinogenemia (CD) to characterize the incidence of hemorrhagic and thrombotic events as well as complications of pregnancy and surgery. At the time of diagnosis, 10.9% and 13.9% had experienced major bleeding and thrombotic events, respectively. During a mean follow-up of 8.8 years after CD diagnosis, the incidence of major bleeding and of thrombotic events was 2.5 and 18.7 per 1000 patient-years respectively, with estimated cumulative incidences at an age of 50 years of 19.2% and 30.1%. We identified 111 pregnancies with an overall incidence of spontaneous abortions and post-partum hemorrhage of 19.8% and 21.4%, respectively. The risk of post-partum hemorrhage was associated with a previously identified bleeding phenotype (OR 5.8; 95%CI 1.2-28.0). Among 137 surgical procedures analyzed, 9 (6.5%) were complicated by abnormal bleeding. Propositi versus relatives, sex, mutation hotspots, fibrinogen levels and activity:antigen ratios were not associated with the risk of thrombotic or bleeding outcomes. In conclusion, the results of our study, the largest in genotyped CD and the first including long term history, indicate that propositi with CD and their relatives carry not only a high risk of major bleeding, including post-partum hemorrhage, but also of thrombotic event.
[Show abstract][Hide abstract] ABSTRACT: Introduction
Thromboelastography (TEG), a widely used clinical point of care coagulation test, is poorly understood. To investigate its fibrin determinants we used normal and variant fibrinogen isolates.
Materials and Methods
We focused mainly on the TEG maximum signal amplitude (MA), a shear modulus and clot stiffness indicator. Isolates included normal des-αC, cord, and abnormal congenital variants with amino acid substitutions or deletions that impaired fibrin polymerization. Heterophenotypic congenital isolates were from cryoprecipitate-depleted plasma owing to their more diminished clot MA than their cryoprecipitate counterparts. By colorimetric assay, the amount of fibrinogen adsorbed by untreated TEG cups was 83.5 ± 12.4 pM/cm2, n = 18. Thrombin-induced clots were obtained at pH 6.4 or 7.4, the latter containing 8 mM CaCl2, and 14% afibrinogenemic plasma with and without gel-sieved platelets.
Results and Conclusions
Measured by the water droplet contact angle, > 90% reduction of surface hydrophobicity by exposure of TEG cup and pin to ozone plasma decreased MA by 74%. Increasing normal fibrinogen or thrombin concentrations progressively increased MA. Platelets increased MA further ~ 2 fold, except for ≥ 10 fold for des-αC clots. Examined in the absence of platelets, MA of heterophenotypic fibrin variants averaged 21%, n = 15. The results imply that essential MA determinants include hydrophobic fibrinogen/fibrin adsorption and each polymerization contact site, with substantial enhancement by platelets. Also, cryoprecipitate-harvested soluble fibrinogen/fibrin complexes contained mostly normal molecules, while cryoprecipitate-depleted plasma contained mostly variant molecules. Moreover, significantly decreased MA by fibrinogen anomalies and/or low level thrombin generation can potentially impact clinical interpretation of MA.
Thrombosis Research 06/2014; 133(6). DOI:10.1016/j.thromres.2014.03.026 · 2.45 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Introduction
Causative mutations leading to congenital quantitative fibrinogen are frequently clustered in FGA encoding the fibrinogen Aα-chain. Mutations of FGB encoding the Bβ-chain are less common and of interest since the Bβ chain is considered the rate-limiting factor in the hepatic production of the fibrinogen hexamer.
Four novel FGB mutations were identified in two afibrinogenemic (one new-born and one 30 years old male) and hypofibrinogenemic (a 49 years old female) patient, with heterogeneous thrombotic and bleeding phenotype. The human fibrinogen beta chain precursor protein sequence (P02675) was obtained from the UniProt database. The resulting models were analysed in SwissPdbViewer 4.1 and POV-Ray 3.7.
The FGB c.895 T > C p.Y299H (numbering from the initiator Met) and the FGB c.1415G > T p.G472V were predicted to be deleterious by SIFT analysis. The first replaces an uncharged aromatic amino acid side chain by a positively charged side chain modifying the balance in the distribution of hydrophobic and hydrophilic of the 10 Å neighbourhood residues. The second replaces one non-charged aliphatic side chain by another without any changes for the 10 Å surrounding region. The FGB c.352C > T p.Q118X leads to a severe premature termination codon and the FGB intron 4: IVS4-1G > C (c719-1G > C) leads to skipping of exon 5 or usage of a cryptic acceptor site located upstream or downstream of the normal site.
The continuous characterization of novel molecular defects responsible for fibrinogen deficiency combined with modelling of mutant proteins will continue to provide a better comprehension of the complexity of fibrinogen synthesis and physiology.
Thrombosis Research 05/2014; 133(5). DOI:10.1016/j.thromres.2014.01.022 · 2.45 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Mutations in the human fibrinogen genes can lead to the absence of circulating fibrinogen and cause congenital afibrinogenemia. This rare bleeding disorder is associated with a variable phenotype, which may be influenced by environment and genotype. Here we present a zebrafish model of afibrinogenemia. We introduced targeted mutations into the zebrafish fga gene using zinc finger nuclease technology. Animals carrying three distinct frameshift mutations in fga were raised and bred to produce homozygous mutants. Using a panel of anti-zebrafish fibrinogen antibodies, fibrinogen was undetectable in plasma preparations from homozygous mutant fish. We observed hemorrhaging in fga mutants and reduced survival compared to control animals. This model will now serve in the search for afibrinogenemia modifying genes or agents and, to our knowledge, is the first transmissible zebrafish model of a defined human bleeding disorder.
[Show abstract][Hide abstract] ABSTRACT: Vertebrate genomes contain around 20,000 protein-encoding genes, of which a large fraction is still not associated with specific functions. A major task in future genomics will thus be to assign physiological roles to all open reading frames revealed by genome sequencing. Here we show that C2orf62, a highly conserved protein with little homology to characterized proteins, is strongly expressed in testis in zebrafish and mammals, and in various types of ciliated cells during zebrafish development. By yeast two hybrid and GST pull-down, C2orf62 was shown to interact with TTC17, another uncharacterized protein. Depletion of either C2orf62 or TTC17 in human ciliated cells interferes with actin polymerization and reduces the number of primary cilia without changing their length. Zebrafish embryos injected with morpholinos against C2orf62 or TTC17, or with mRNA coding for the C2orf62 C-terminal part containing a RII dimerization/docking (R2D2) - like domain show morphological defects consistent with imperfect ciliogenesis. We provide here the first evidence for a C2orf62-TTC17 axis that would regulate actin polymerization and ciliogenesis.
PLoS ONE 01/2014; 9(1):e86476. DOI:10.1371/journal.pone.0086476 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Congenital dysfibrinogenemias are characterized by biosynthesis of a structurally abnormal fibrinogen molecule that exhibits reduced functional levels compared with the level of fibrinogen antigen. To date a large number of mutations have been identified in patients with dysfibrinogenemia. Mutations occurring at the thrombin cleavage site (Arg16-Gly17 in the mature alpha-chain) at the amino-terminal end of the fibrinogen alpha chain are a common cause of the disease. These mutations causing abnormal fibrin polymerization are associated with different phenotypes. Here, we report the identification of a novel heterozygous missense mutation of Glycine 17 (Gly17Asp) in a female patient with mild bleeding manifestations, and compare it with other previously reported mutations also resulting in abnormal knob A.
Blood coagulation & fibrinolysis: an international journal in haemostasis and thrombosis 12/2013; 25(3). DOI:10.1097/MBC.0000000000000039 · 1.40 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The fibrinogen genes FGA, FGB and FGG show coordinated expression in hepatocytes. Understanding the underlying transcriptional regulation may elucidate how their tissue-specific expression is maintained and explain the high variability in fibrinogen blood levels. DNA methylation of CpG-poor gene promoters is dynamic with low methylation correlating with tissue-specific gene expression but its direct effect on gene regulation as well as implications of non-promoter CpG methylation are not clear. Here we compared methylation of CpG sites throughout the fibrinogen gene cluster in human cells and mouse and zebrafish tissues. We observed low DNA methylation of the CpG-poor fibrinogen promoters and of additional regulatory elements (the liver enhancers CNC12 and PFE2) in fibrinogen-expressing samples. In a gene reporter assay, CpG-methylation in the FGA promoter reduced promoter activity, suggesting a repressive function for DNA methylation in the fibrinogen locus. In mouse and zebrafish livers we measured reductions in DNA methylation around fibrinogen genes during development that were preceded by increased fibrinogen expression and tri-methylation of Histone3 lysine4 (H3K4me3) in fibrinogen promoters. Our data support a model where changes in hepatic transcription factor expression and histone modification provide the switch for increased fibrinogen gene expression in the developing liver which is followed by reduction of CpG methylation.
PLoS ONE 08/2013; 8(8):e73089. DOI:10.1371/journal.pone.0073089 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Hereditary fibrinogen abnormalities comprise two classes of plasma fibrinogen defects: Type I, afibrinogenemia or hypofibrinogenemia, which has absent or low plasma fibrinogen antigen levels (quantitative fibrinogen deficiencies), and Type II, dysfibrinogenemia or hypodysfibrinogenemia, which shows normal or reduced antigen levels associated with disproportionately low functional activity (qualitative fibrinogen deficiencies). In afibrinogenemia and hypofibrinogenemia, most mutations of the FGA, FGB, or FGG fibrinogen encoding genes are null mutations. In some cases, missense or late truncating nonsense mutations allow synthesis of the corresponding fibrinogen chain but intracellular fibrinogen assembly and/or secretion are impaired. Afibrinogenemia is associated with mild-to-severe bleeding, whereas hypofibrinogenemia is most often asymptomatic. Thromboembolism may occur either spontaneously or in association with fibrinogen substitution therapy. Women with afibrinogenemia suffer from recurrent pregnancy loss but this can also occur in women with hypofibrinogenemia. Dysfibrinogenemia, caused mainly by missense mutations, is commonly associated with bleeding, thrombophilia, or both; however, most individuals are asymptomatic. Hypodysfibrinogenemia is a subcategory of this disorder. Even in specialized laboratories, the precise diagnosis of some fibrinogen disorders may be difficult. Determination of the molecular defects is important because it gives the possibility to confirm the diagnosis, to elaborate a diagnostic strategy, to distinguish in some cases that the patient is at risk of thrombosis rather than bleeding, and to enable prenatal diagnosis. However, genotype-phenotype correlations are not easy to establish. Replacement therapy is effective in treating bleeding episodes, but because the pharmacokinetics of fibrinogen after replacement therapy is highly variable among patients, it is important to adjust the treatment individually.
Seminars in Thrombosis and Hemostasis 07/2013; 39(6). DOI:10.1055/s-0033-1349222 · 3.88 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The Aα, Bβ and γ polypeptide chains of fibrinogen are encoded by a three gene cluster on human chromosome four. The fibrinogen genes ( FGB-FGA-FGG ) are expressed almost exclusively in hepatocytes where their output is coordinated to ensure a sufficient mRNA pool for each chain and maintain an abundant plasma fibrinogen protein level. Fibrinogen gene expression is controlled by the activity of proximal promoters which contain binding sites for hepatocyte transcription factors, including proteins which influence fibrinogen transcription in response to acute-phase inflammatory stimuli. The fibrinogen gene cluster also contains cis regulatory elements; enhancer sequences with liver activities identified by sequence conservation and functional genomics. While the transcriptional control of this gene cluster is fascinating biology, the medical impetus to understand fibrinogen gene regulation stems from the association of cardiovascular disease risk with high level circulating fibrinogen. In the general population this level varies from about 1.5 to 3.5 g/l. This variation between individuals is influenced by genotype, suggesting there are genetic variants contributing to fibrinogen levels which reside in fibrinogen regulatory loci. A complete picture of how fibrinogen genes are regulated will therefore point towards novel sources of regulatory variants. In this review we discuss regulation of the fibrinogen genes from proximal promoters and enhancers, the influence of acute-phase stimulation, post-transcriptional regulation by miRNAs and functional regulatory variants identified in genetic studies. Finally, we discuss the fibrinogen locus in light of recent advances in understanding chromosomal architecture and suggest future directions for researching the mechanisms that control fibrinogen expression.
Thrombosis and Haemostasis 07/2012; 108(3):419-26. DOI:10.1160/TH12-04-0273 · 4.98 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: High circulating fibrinogen levels correlate with cardiovascular disease (CVD) risk. Fibrinogen levels vary between people and also change in response to physiological and environmental stimuli. A modest proportion of the variation in fibrinogen levels can be explained by genotype, inferring that variation in genomic sequences that regulate the fibrinogen genes ( FGA , FGB and FGG ) may affect hepatic fibrinogen production and perhaps CVD risk. We previously identified a conserved liver enhancer in the fibrinogen gene cluster (CNC12), between FGB and FGA . Genome-wide Chromatin immunoprecipitation-sequencing (ChIP-seq) demonstrated that transcription factors which bind fibrinogen gene promoters also interact with CNC12, as well as two potential fibrinogen enhancers (PFE), between FGA and FGG . Here we show that one of the PFE sequences has potent hepatocyte enhancer activity. Using a luciferase reporter gene system, we found that PFE2 enhances minimal promoter- and FGA promoter-driven gene expression in hepatoma cells, regardless of its orientation with respect to the promoters. A region within PFE2 bears a short series of conserved nucleotides which maintain enhancer activity without flanking sequence. We also demonstrate that PFE2 is a liver enhancer in vivo , driving enhanced green fluorescent protein expression in transgenic zebrafish larval livers. Our study shows that combining public domain ChIP-seq data with in vitro and in vivo functional tests can identify novel fibrinogen gene cluster regulatory sequences. Variation in such elements could affect fibrinogen production and influence CVD risk.
Thrombosis and Haemostasis 07/2012; 108(3):427-34. DOI:10.1160/TH12-04-0274 · 4.98 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Congenital afibrinogenemia is characterized by the complete absence of fibrinogen, the precursor of the major protein constituent of the blood clot, fibrin. Extensive allelic heterogeneity has been found for this disorder and more than 40 mutations, the majority in FGA, have been identified in homozygosity or in compound heterozygosity. However, the continuous genetic analysis of additional patients still allows the identification of novel mutations and thus the greater understanding of fibrinogen structure and function. Here we report the identification of a novel missense mutation in FGA exon 1 affecting the translation initiation codon: c.1 A>T (ATG>TTG) M1L, identified in a young boy from Madagascar in compound heterozygosity with a second mutation in FGA exon 4: c.385 C>T (CGA>TGA) R129X. The patient suffered from occasional severe arthralgias (shoulder, knee) most likely caused by intra-articular bleeding with subsequent inflammation.
Blood coagulation & fibrinolysis: an international journal in haemostasis and thrombosis 06/2012; 23(6):556-8. DOI:10.1097/MBC.0b013e328355a76e · 1.40 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We investigated an asymptomatic 19-year-old patient with factor XI deficiency diagnosed in the context of presurgical laboratory screening. The F11 gene was analyzed and a novel missense mutation I463S in exon 12 was identified in heterozygosity in the proband. His mother, also diagnosed with asymptomatic factor XI deficiency, was found to be heterozygous for the same mutation. This novel amino acid substitution in the serine protease catalytic domain appears to be responsible for the low factor XI levels in both individuals.
Blood coagulation & fibrinolysis: an international journal in haemostasis and thrombosis 02/2012; 23(3):251-2. DOI:10.1097/MBC.0b013e32834ea02a · 1.40 Impact Factor