Fibrinogen as a Pleiotropic Protein Causing Human Diseases: The Mutational Burden of Aα, Bß, and γ Chains.

Fibrinogen is a highly pleiotropic protein that is involved in the final step of the coagulation cascade, wound healing, inflammation, and angiogenesis. Heterozygous mutations in Aα, Bß, or γ fibrinogen-chain genes (FGA, FGB, FGG) have been described as being responsible for fibrinogen deficiencies (hypofibrinogenemia, hypo-dysfibrinogenemia, dysfibrinogenemia) and for more rare conditions, such as fibrinogen storage disease and hereditary renal amyloidosis. Instead, biallelic mutations have been associated with afibrinogenemia/severe hypofibrinogenemia, i.e., the severest forms of fibrinogen deficiency, affecting approximately 1-2 cases per million people. However, the "true" prevalence for these conditions on a global scale is currently not available. Here, we defined the mutational burden of the FGA, FGB, and FGG genes, and estimated the prevalence of inherited fibrinogen disorders through a systematic analysis of exome/genome data from ~140,000 individuals belonging to the genome Aggregation Database. Our analysis showed that the world-wide prevalence for recessively-inherited fibrinogen deficiencies could be 10-fold higher than that reported so far (prevalence rates vary from 1 in 106 in East Asians to 24.5 in 106 in non-Finnish Europeans). The global prevalence for autosomal-dominant fibrinogen disorders was estimated to be ~11 in 1000 individuals, with heterozygous carriers present at a frequency varying from 3 every 1000 individuals in Finns, to 1-2 every 100 individuals among non-Finnish Europeans and Africans/African Americans. Our analysis also allowed for the identification of recurrent (i.e., FGG-p.Ala108Gly, FGG-Thr47Ile) or ethnic-specific mutations (e.g., FGB-p.Gly103Arg in Admixed Americans, FGG-p.Ser245Phe in Africans/African Americans).

Novel fibrinogen mutations (Aα17Gly→Cys and Aα381Ser→Phe) occurring with a 312Thr→Ala polymorphism: allelic phase assigned by direct mass measurement.

The aim of the study was to determine the molecular cause of dysfibrinogenaemia in a woman with a prolonged thrombin time. Functional fibrinogen abnormalities can be benign or may lead to bleeding or thrombotic conditions. In complex cases, phenotypes may be acquired or involve interplay between several coinherited mutations. The authors developed a new whole-protein time-of-flight mass spectrometry (TOF MS) approach to direct targeted DNA sequencing of the fibrinogen genes and determine the phase of multiple substitutions in a single individual. TOF MS analysis of the individual's fibrinogen indicated normal Bß, γ, and alternately transcribed γ' chain isoforms, but aberrant Aα chain masses. Subsequent fibrinogen Aα gene (FGA) sequencing indicated the presence of three different mutations. Two of the substitutions, Aα17Gly→Cys (at the thrombin cleavage site) and Aα381Ser→Phe (in the αC connector) were novel and the third, Aα312Thr→Ala, was a known benign polymorphism. Accurate mass measurements of isolated control Aα chains showed the predicted Aα polypeptide at 66 132 Da and additional phosphorylated species at + 80 and + 160 Da. Patient's Aα chains on the other hand had masses of 66 103 and 66 241 Da indicating that she had one 312Ala allele (-30 Da) and one 312Thr allele which carried both the 17Gly→Cys (+ 46 Da) and 381Ser→Phe (+ 60) Da mutations. Cotransmission of these new mutations was confirmed by Aα chain TOF MS of plasma fibrinogen and targeted FGA nucleotide sequencing for 10 additional family members.

Novel mutations (γTrp208Leu and γLys232Thr) leading to congenital hypofibrinogenemia in two unrelated Chinese families.

Congenital hypofibrinogenemia is a rare disorder caused by heterozygous mutations in the fibrinogen genes. The aim of this study was to elucidate the molecular defects in two unrelated families with hypofibrinogenemia. The proband from family A was a 19-year-old Chinese boy who was suffering from cervical lymphadenitis. A low plasma fibrinogen concentration (0.63 g/l by Clauss method and 0.77 g/l by immunoturbidimetry) was found in routine clotting tests. Further gene analysis revealed a heterozygous g.5792 G>T mutation in exon 7 of the FGG, leading to a novel Trp208Leu change in the γ D domain. This mutation was also found in other family members with low fibrinogen levels. The proposita from family B was a 37-year-old female who suffered from recurrent shoulder pain for 7 years. Routine clotting studies revealed that her prothrombin time was 15.5 s (normal range: 11.8-14.8 s) and thrombin time was 22.8 s (normal range: 14.0-20.0 s), and the fibrinogen concentration in her plasma was only 0.64 g/l by Clauss method and 0.79 g/l by immunoturbidimetry. A heterozygous A>C transition at nucleotide 5864 of FGG was found in the γ chain, causing a Lys232Thr substitution in the fibrinogen. Further sequencing established that her mother, son, brother and nephew were also heterozygous for the mutation.

Expression and analysis of a split premature termination codon in FGG responsible for congenital afibrinogenemia: escape from RNA surveillance mechanisms in transfected cells.

Congenital afibrinogenemia, the most severe form of fibrinogen deficiency, is characterized by the complete absence of fibrinogen. The disease is caused by mutations in 1 of the 3 fibrinogen genes FGG, FGA, and FGB, clustered on the long arm of human chromosome 4. The majority of cases are due to null mutations in the FGA gene although one would expect the 3 genes to be equally implicated. However, most patients studied so far are white, and therefore the identification of causative mutations in non-European families is necessary to establish if this finding holds true in all ethnic groups. In this study, we report the identification of a novel nonsense mutation (Arg134Xaa) in the FGG gene responsible for congenital afibrinogenemia in 10 patients from Lebanon. Expression studies in COS-7 cells demonstrated that the Arg134Xaa codon, which is encoded by adjacent exons (TG-intron 4-A) affected neither mRNA splicing nor stability, but led to the production of an unstable, severely truncated fibrinogen gamma chain that is not incorporated into a functional fibrinogen hexamer.

Analysis of Iranian patients allowed the identification of the first truncating mutation in the fibrinogen Bbeta-chain gene causing afibrinogenemia.

BACKGROUND AND OBJECTIVES: Congenital afibrinogenemia is a rare coagulation disorder whose molecular basis is still poorly characterized. Most mutations have been identified in the fibrinogen Aalpha- and gamma-chain genes, whereas only two missense mutations have been reported in the Bbeta-chain gene. The aim of this work was to widen knowledge about the mutational spectrum of this disease by analyzing the molecular bases of congenital afibrinogenemia in three unrelated Iranian patients. DESIGN AND METHODS: All patients showed unmeasurable levels of clottable fibrinogen in plasma. Mutational screening was performed by sequencing the whole coding region, including exon-intron boundaries and part of the promoter region of the three fibrinogen genes. RESULTS: Sequencing in one patient revealed the presence of a novel nonsense mutation (3282C-->T) in exon 2 of the fibrinogen Bbeta-chain gene, causing a severe truncation of the corresponding polypeptide (R17X). In the remaining probands, two already known small deletions (4209delA and 4220delT), both located in exon 5 of the fibrinogen Aalpha-chain gene, were identified, and their effect at the protein level explored by computer-assisted analysis. INTERPRETATION AND CONCLUSIONS: The identification of the first truncating mutation in the fibrinogen Bbeta-chain gene confirms the involvement of all three fibrinogen genes in the pathogenesis of congenital afibrinogenemia and widens the mutational spectrum of the disease. This knowledge is clinically essential in order to carry out prenatal diagnosis in families at risk.