Dung Vu

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

Summary.  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 (Aα, Bβ, and γ). 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.

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

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 γ 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 γ 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.

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

Congenital afibrinogenemia is caused by mutations in one of the three fibrinogen-encoding genes, secretion-defective fibrinogen mutants are retained in a pre-Golgi compartment in hepatocytes. This study shows that lowering the incubation temperature can restore the secretion of mutant fibrinogen molecules in transfected COS-7 cells. Background Congenital afibrinogenemia is characterized by the absence of fibrinogen, a hexamer composed of two copies of three polypeptides, Aα. Bβ and γ. 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 βC and γC domains in secretion. Here our aim was to restore the secretion of these mutants and study the properties of the rescued mutant molecules. Design and Methods 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. Results The secretion defect of two missense mutants but not that of late-truncating mutants could be partially corrected by incubating cells at 27°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°C were incorporated into fibrin clots and formed factor XIII-mediated γ-γ 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. Conclusions 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.