Quality control of fibrinogen secretion in the molecular pathogenesis of congenital afibrinogenemia

Congenital afibrinogenemia is a rare bleeding disorder characterized by the absence in circulation of fibrinogen, 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 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 Bβ 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 βC core. Immunofluorescence and immuno-electron microscopy studies indicate that secretion-impaired mutants are retained in a pre-Golgi compartment. In addition, expression of Bβ, γ and angiopoietin-2 chimeric molecules demonstrated that the βC domain prevents the secretion of single chains and complexes, whereas the γC 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 afibrinogenemi

Targeted mutation of zebrafish fga models human congenital afibrinogenemia

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 3 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 with 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

Quality control of fibrinogen secretion in the molecular pathogenesis of congenital afibrinogenemia

Congenital afibrinogenemia is a rare bleeding disorder characterized by the absence in circulation of fibrinogen, a hexamer composed of two sets of three polypeptides (Aalpha, Bbeta and gamma). 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 Bbeta 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 betaC core. Immunofluorescence and immuno-electron microscopy studies indicate that secretion-impaired mutants are retained in a pre-Golgi compartment. In addition, expression of Bbeta, gamma and angiopoietin-2 chimeric molecules demonstrated that the betaC domain prevents the secretion of single chains and complexes, whereas the gammaC 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

A homozygous duplication of the FGG exon 8-intron 8 junction causes congenital afibrinogenemia. Lessons learned from the study of a large consanguineous Turkish family

Congenital afibrinogenemia is the most severe congenital fibrinogen disorder, characterised by undetectable fibrinogen in circulation. Causative mutations can be divided into two main classes: null mutations with no protein production at all and missense mutations producing abnormal protein chains that are retained inside the cell. The vast majority of cases are due to single base pair mutations or small insertions or deletions in the coding regions or intron-exon junctions of FGB, FGA and FGG. Only a few large rearrangements have been described, all deletions involving FGA. Here we report the characterization of a 403 bp duplication of the FGG exon 8-intron 8 junction accounting for congenital afibrinogenemia in a large consanguineous family from Turkey. This mutation, which had escaped detection by Sanger sequencing of short PCR amplicons of coding sequences and splice sites, was identified by studying multiple alignments of reads obtained from Whole Exome Sequencing of a heterozygous individual followed by PCR amplification and sequencing of a larger portion of FGG. Because the mutation duplicates the donor splice site of intron 8, we predicted that the impact of the mutation would be on FGG transcript splicing. Analysis of mRNAs produced by cells transiently transfected with normal or mutant minigene constructs showed that the duplication causes production of several aberrant FGG transcripts generating premature truncating codons