The natural occurrence of human fibrinogen variants disrupting inter-chain disulfide bonds (A Cys36Gly, A Cys36Arg and A Cys45Tyr) confirms the role of N-terminal A  disulfide bonds in protein assembly and secretion

Hanss, Michel; Pouymayou, Catherine; Blouch, Marie-Thérèse; Lellouche, Franck; Ffrench, Patrick; Rousson, Robert; Abgrall, Jean-François; Morange, Pierre‐Emmanuel; Quélin, Florence; Mazancourt, Philippe de

doi:10.3324/haematol.2010.029801

Cited by 11 publications

(10 citation statements)

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“…We previously showed that we can use zebrafish as an in vivo system for the identification of the causative mutations in coagulation disorders . Because zebrafish Fga deficiency closely parallels human pathology, human FGA variants, including C55G, C64Y, Y809C (Human Fibrinogen Database) and M1V , were assessed in fga − / − zebrafish. These variants have been associated with hemorrhage in human populations (Table ); however, they have not been functionally validated in vivo .…”

Section: Resultsmentioning

confidence: 99%

“…M1V is a novel mutation that has been recently identified in patients and here we confirm it in vivo as a pathological mutation. Surprisingly, two cysteine substitutions, C55G (Fibrinogen La Seyne and Fibrinogen Quimper) and C64Y (Fibrinogen Marseilles II), were able to rescue fga − / − mutants despite the fact that both positions are highly conserved and patients with these mutations are hypofibrinogenemic (activity levels down to 20–50% of the lower limit of the normal range ). The cysteine at position 55 has been clearly shown to be important for disulfide formation with the B β subunit of fibrinogen.…”

Section: Discussionmentioning

confidence: 99%

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Loss of fibrinogen in zebrafish results in an asymptomatic embryonic hemostatic defect and synthetic lethality with thrombocytopenia

Lavik

Liu

et al. 2019

Journal of Thrombosis and Haemostasis

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Loss of fibrinogen in zebrafish has been previously shown to result in adult onset hemorrhage Hemostatic defects were discovered in early fga−/− embryos but well tolerated until adulthood Afibrinogenemia and thrombocytopenia results in synthetic lethality in zebrafish. Testing human FGA variants of uncertain significance in zebrafish identified causative mutations Summary BackgroundMutations in the alpha chain of fibrinogen (FGA), such as deficiencies in other fibrinogen subunits, lead to rare inherited autosomal recessive hemostatic disorders. These range from asymptomatic to catastrophic life‐threatening bleeds and the molecular basis of inherited fibrinogen deficiencies is only partially understood. Zinc finger nucleases have been used to produce mutations in zebrafish fga, resulting in overt adult‐onset hemorrhage and reduced survival. ObjectivesTo determine the age of onset of hemostatic defects in afibrinogenemic zebrafish and model human fibrinogen deficiencies. MethodsTALEN genome editing (transcription activator‐like effector nucleases) was used to generate a zebrafish fga mutant. Hemostatic defects were assessed through survival, gross anatomical and histological observation and laser‐induced endothelial injury. Human FGA variants with unknown pathologies were engineered into the orthologous positions in zebrafish fga. ResultsLoss of Fga decreased survival and resulted in synthetic lethality when combined with thrombocytopenia. Zebrafish fga mutants exhibit a severe hemostatic defect by 3 days of life, but without visible hemorrhage. Induced thrombus formation through venous endothelial injury was completely absent in mutant embryos and larvae. This hemostatic defect was restored by microinjection of wild‐type fga cDNA plasmid or purified human fibrinogen. This system was used to determine whether unknown human variants were pathological by engineering them into fga. ConclusionsThese studies confirm that loss of fibrinogen in zebrafish results in the absence of hemostasis from the embryonic period through adulthood. When combined with thrombocytopenia, zebrafish exhibit synthetic lethality, demonstrating that thrombocytes are necessary for survival in response to hemorrhage.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Loss of fibrinogen in zebrafish results in an asymptomatic embryonic hemostatic defect and synthetic lethality with thrombocytopenia

Lavik

Liu

et al. 2019

Journal of Thrombosis and Haemostasis

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“…Platè et al, reported that AαCys64Phe mutation severely impaired the intracellular fibrinogen assembly and secretion by affecting halfmolecule dimerization in vitro [8]. Hanss et al, observed similar results in patients carrying AαCys64Tyr mutation [9]. Moreover, AαCys64 is highly conserved among several species (Figure 1(B)), indicating the essential role of AαCys64 in fibrinogen processing.…”

Section: Resultsmentioning

confidence: 73%

Congenital fibrinogen disorder caused by digenic mutations of the FGA and FGB genes

et al. 2020

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Objectives: Congenital fibrinogen disorders (CFDs) are caused by monoallelic or biallelic mutations in FGA, FGB, and FGG genes. Quantitative CFDs include afibrinogenemia and hypofibrinogenemia, while qualitative CFDs consist of dysfibrinogenemia and hypodysfibrinogenemia. Hypofibrinogenemia and dysfibrinogenemia are autosomal dominant disorders while afibrinogenemia is a recessive one. We aimed to perform genetic diagnosis of a Chinese patient with CFD. Methods: DNA was extracted from peripheral blood mononuclear cells (PBMCs) and targeted next generation sequencing (NGS) was applied using amplicon-based panel (www.ampliseq. com) targeting all exons and splice sites of FGA, FGB, and FGG genes. Identified mutations were confirmed by Sanger sequencing. Results: We have identified a novel heterozygous FGB c.560_561delTG (p.Val187GlufsTer2) frameshift deletion and a novel heterozygous FGA c.190T > G (p.Cys64Gly) missense mutations in a Chinese patient with CFD. Conclusion: This is the first report of digenic variants causing CFD, and these findings may improve understanding of the genetic architecture of CFD.

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“…Furthermore, a pair of disulfide ring at either end of each coiled-coil domains of Aα-, Bβ-, and γ-chain are also important in stabilization of fibrinogen molecule [ 13 , 14 ]. Therefore, the disulfide bond mutation, AαCys45Tyr [ 15 ] or AαCys45Phe [ 16 ], prevents half-molecule dimerization and causes an impairment in intracellular fibrinogen processing. Our aberrant transcripts have two Cys residues as candidate disulfide bond sites.…”

Section: Discussionmentioning

confidence: 99%

A Novel Mutation in the Fibrinogen Bβ Chain (c.490G>A; End of Exon 3) Causes a Splicing Abnormality and Ultimately Leads to Congenital Hypofibrinogenemia

Taira

Matsuda

Arai

et al. 2017

IJMS

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We found a novel heterozygous mutation in the fibrinogen Bβ chain (c.490G>A) of a 3-year-old girl with congenital hypofibrinogenemia. To clarify the complex genetic mechanism, we made a mini-gene including a FGB c.490G>A mutation region, transfected it into a Chinese Hamster Ovary (CHO) cell line, and analyzed reverse transcription (RT) products. The assembly process and secretion were examined using recombinant mutant fibrinogen. Direct sequencing demonstrated that the mutant RT product was 99 bp longer than the wild-type product, and an extra 99 bases were derived from intron 3. In recombinant expression, a mutant Bβ-chain was weakly detected in the transfected CHO cell line, and aberrant fibrinogen was secreted into culture media; however, an aberrant Bβ-chain was not detected in plasma. Since the aberrant Bβ-chain was catabolized faster in cells, the aberrant Bβ-chain in a small amount of secreted fibrinogen may catabolize in the bloodstream. FGB c.490G>A indicated the activation of a cryptic splice site causing the insertion of 99 bp in intron 3. This splicing abnormality led to the production of a Bβ-chain possessing 33 aberrant amino acids, including two Cys residues in the coiled-coil domain. Therefore, a splicing abnormality may cause impaired fibrinogen assembly and secretion.

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The natural occurrence of human fibrinogen variants disrupting inter-chain disulfide bonds (A Cys36Gly, A Cys36Arg and A Cys45Tyr) confirms the role of N-terminal A disulfide bonds in protein assembly and secretion

Abstract: ABSTRACTwith the PCR primers using the dye terminator sequencing kit from Applera Courtaboeuf, France. Informed consent was obtained from all patients. This work was approved by the local ethics committees (approvals DC 2008-214 and DC 2008-880).

Cited by 11 publications

References 20 publications

Loss of fibrinogen in zebrafish results in an asymptomatic embryonic hemostatic defect and synthetic lethality with thrombocytopenia

Loss of fibrinogen in zebrafish results in an asymptomatic embryonic hemostatic defect and synthetic lethality with thrombocytopenia

Congenital fibrinogen disorder caused by digenic mutations of the FGA and FGB genes

A Novel Mutation in the Fibrinogen Bβ Chain (c.490G>A; End of Exon 3) Causes a Splicing Abnormality and Ultimately Leads to Congenital Hypofibrinogenemia

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