Hypofibrinogenaemia caused by a novel FGG missense mutation (W253C) in the   chain globular domain impairing fibrinogen secretion

Abstract

Background: Inherited disorders of fibrinogen are rare and affect either the quantity (hypofibrinogenaemia and afibrinogenaemia) or the quality of the circulating fibrinogen (dysfibrinogenaemia). Extensive allelic heterogeneity has been found for all three disorders: in congenital afibrinogenaemia .30 mutations, the majority in FGA, have been identified in homozygosity or in compound heterozygosity. Several mutations have also been identified in patients with hypofibrinogenaemia; many of these are heterozygous carriers of afibrinogenaemia null mutations. Objective: To report the case of a patient from Slovakia diagnosed with hypofibrinogenaemia characterised by fibrinogen concentrations of around 0.7 g/l. Results: The patient was found to be heterozygous for a novel missense mutation W253C (W227C in the mature protein) in the C-terminal globular domain of the fibrinogen c chain. Co-expression of the W253C FGG mutant cDNA (fibrinogen Bratislava) in combination with wild-type FGA and FGB cDNAs showed that fibrinogen molecules containing the mutant c chain can assemble intracellularly but are not secreted into the media, confirming the causative nature of the identified mutation. Conclusions: Current analysis of fibrinogen Bratislava indicates that the domains important for the processes of hexamer assembly and hexamer secretion should not be considered as strictly restricted to one or other fibrinogen chain.

Recombinant variant fibrinogens substituted at residues γ326Cys and γ339Cys demonstrated markedly impaired secretion of assembled fibrinogen

“…Although there is a possibility that characteristics of secretion of fibrinogen might be somewhat different between COS-1 and CHO cells, variant fibrinogens substituted at residues γ326 or γ339 were essentially not secreted or only secreted at very low levels into the culture media. In the same fashion, Vu et al demonstrated that truncated γ-chain variant, γ387X, was assembled into fibrinogen inside the COS-7 cells, yet this was not secreted into medium [17]. This result appears to contradict data from our study showing no fibrinogen is detected when this truncated γ-chain is transfected in CHO cells [15].…”

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

“…40 This discrepancy may be attributable to a greater permissiveness of COS-7 cells in comparison with CHO cells regarding secretion of mutant fibrinogen molecules, as we had previously found for assembly of truncated γ chains into hexamers. 12 In this regard, half-molecules, [Aαγ] complexes and free Aα or γ chains in the culture medium of COS-7 cells (Figures 1, 2) were also observed in that of hepatoma cell lines such as Hep3B and HepG2 cells, transfected BHK, CHO and COS-1 cells 14,21,41,42 and even in the plasma of afibrinogenemic patients. 43,44 It is difficult to determine which cell model reflects the in vivo situation best, since these differences are also found in patients, possibly reflecting polymorphic differences in modifier genes, which remain to be identified.…”

“…To investigate whether the secretion of the fibrinogen FGB p.G444S (p.G414S), 7 FGB p.W467X (p.W437X) 9 and FGG p.W253C (p.W227C) 12 mutants can be rescued, we co-transfected COS-7 cells with empty vectors, the three normal fibrinogen cDNA constructs, or the mutant FGB G444S, FGB W467X, FGG W253C cDNA (residues numbered from the initiator codon) combined with the other two corresponding normal cDNA. Cells were exposed to one of the three common chemical chaperones with concentrations found to be successful in other studies (10 mM 4-phenylbutyrate (4-PBA), 2% dimethylsulfoxide (DMSO), and 100 mM trimethylamine N-oxide (TMAO).…”

Mutations in the fibrinogen gene cluster accounting for congenital afibrinogenemia: an update and report of 10 novel mutations

“…For the majority of these mutations analysis of patient plasma fibrinogen by mass spectrometry has confirmed absence of the mutant gamma-chain in the circulation. Others have been studied at the functional level in transfected cells: fibrinogen Matsumoto IV p.Cys179Arg [Terasawa et al, 1999] was found to impair intracellular hexamer assembly, while fibrinogen Bratislava p.Trp253Cys [Vu et al, 2005a] was found to impair fibrinogen secretion. Again, in all likelihood homozygosity for these mutations would cause afibrinogenemia.…”