Hypodysfibrinogenaemia due to production of mutant fibrinogen alpha-chains lacking fibrinopeptide A and polymerisation knob ‘A’
08 March 2010
Accepted after major revision: 14 July 2010
24 November 2017 (online)
Inherited disorders of fibrinogen are rare and affect either the quantity (hypofibrinogenaemia and afibrinogenaemia) or the quality of the circulating fibrinogen (dysfibrinogenaemia) or both (hypodysfibrinogenaemia). Extensive allelic heterogeneity has been found for all these disorders: in congenital afibrinogenaemia for example more than 40 mutations, the majority in FGA, have been identified in homozygosity or in compound heterozygosity. Numerous mutations have also been identified in patients with hypofibrinogenaemia, many of these patients are in fact heterozygous carriers of afibrinogenaemia mutations. Despite the number of genetic analyses performed, the study of additional patients still allows the identification of novel mutations. Here we describe the characterization of a novel FGA intron 2 donor splice-site mutation (Fibrinogen Montpellier II) identified in three siblings with hypodysfibrinogenaemia. Functional analysis of RNA produced by the mutant minigene in COS-7 cells revealed that the mutation led to the in-frame skipping of exon 2. Western blot analysis of COS-7 cells expressing an exon 2 deleted FGA cDNA revealed that an alpha-chain lacking exon 2, which codes in particular for fibrinopeptide A and polymerisation knob ‘A’, has the potential to be assembled into a hexamer and secreted. Analysis of precipitated fibrinogen from patient plasma showed that the defect leads to the presence in the circulation of alpha-chains lacking knob ‘A’ which is essential for the early stages of fibrin polymerisation. Fibrin made from purified patient fibrinogen clotted with thrombin displayed thinner fibers with frequent ends and large pores.
- 1 Kant JA, Fornace Jr AJ, Saxe D. et al. Evolution and organization of the fibrinogen locus on chromosome 4: gene duplication accompanied by transposition and inversion. Proc Natl Acad Sci USA 1985; 82: 2344-2348.
- 2 Redman CM, Xia H. Fibrinogen biosynthesis. Assembly, intracellular degradation, and association with lipid synthesis and secretion. Ann NY Acad Sci 2001; 936: 480-495.
- 3 Mosesson MW, Siebenlist KR, Meh DA. The structure and biological features of fibrinogen and fibrin. Ann NY Acad Sci 2001; 936: 11-30.
- 4 Blomback M, Blomback B, Mammen EF. et al. Fibrinogen Detroit--a molecular defect in the N-terminal disulphide knot of human fibrinogen?. Nature 1968; 218: 134-137.
- 5 Hanss M, Biot F. A database for human fibrinogen variants. Ann NY Acad Sci 2001; 936: 89-90.
- 6 Haverkate F, Samama M. Familial dysfibrinogenemia and thrombophilia. Report on a study of the SSC Subcommittee on Fibrinogen. Thromb Haemost 1995; 73: 151-161.
- 7 Galanakis DK. Inherited dysfibrinogenemia: emerging abnormal structure associations with pathologic and nonpathologic dysfunctions. Semin Thromb Hemost 1993; 19: 386-395.
- 8 Maghzal GJ, Brennan SO, Homer VM. et al. The molecular mechanisms of congenital hypofibrinogenaemia. Cell Mol Life Sci 2004; 61: 1427-1438.
- 9 Neerman-Arbez M, de Moerloose P. Mutations in the fibrinogen gene cluster accounting for congenital afibrinogenemia: an update and report of 10 novel mutations. Hum Mutat 2007; 28: 540-553.
- 10 Neerman-Arbez M, de Moerloose P, Bridel C. et al. Mutations in the fibrinogen aalpha gene account for the majority of cases of congenital afibrinogenemia. Blood 2000; 96: 149-152.
- 11 Asselta R, Duga S, Tenchini ML. The molecular basis of quantitative fibrinogen disorders. J Thromb Haemost 2006; 04: 2115-2129.
- 12 Ho SN, Hunt HD, Horton RM. et al. Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 1989; 77: 51-59.
- 13 Neerman-Arbez M, Germanos-Haddad M, Tzanidakis K. et al. Expression and analysis of a split premature termination codon in FGG responsible for congenital afibrinogenemia: escape from RNA surveillance mechanisms in transfected cells. Blood 2004; 104: 3618-3623.
- 14 Vu D, de Moerloose P, Batorova A. et al. Hypofibrinogenaemia caused by a novel FGG missense mutation (W253C) in the gamma chain globular domain impairing fibrinogen secretion. J Med Genet 2005; 42: e57.
- 15 Brennan SO, Hammonds B, George PM. Aberrant hepatic processing causes removal of activation peptide and primary polymerisation site from fibrinogen Canterbury (A alpha 20 Val --> Asp). J Clin Invest 1995; 96: 2854-2858.
- 16 Brennan SO, Davis RL, Chitlur M. New fibrinogen substitution (gammaSer313Arg) causes diminished gamma chain expression and hypodysfibrinogenaemia. Thromb Haemost 2010; 103: 478-479.
- 17 Weisel JW, Nagaswami C. Computer modeling of fibrin polymerization kinetics correlated with electron microscope and turbidity observations: clot structure and assembly are kinetically controlled. Biophys J 1992; 63: 111-128.
- 18 Nation JL. A new method using hexamethyldisilazane for preparation of soft insect tissues for scanning electron microscopy. Stain Technol 1983; 58: 347-351.
- 19 Rogozin IB, Milanesi L. Analysis of donor splice sites in different eukaryotic organisms. J Mol Evol 1997; 45: 50-59.
- 20 Weisel JW. Fibrinogen and fibrin. Adv Protein Chem 2005; 70: 247-299.
- 21 Emanuelsson O, Brunak S, von Heijne G, Nielsen H. Locating proteins in the cell using TargetP, SignalP and related tools. Nat Protoc 2007; 02: 953-971.
- 22 Zhang JZ, Redman CM. Assembly and secretion of fibrinogen. Involvement of amino-terminal domains in dimer formation. J Biol Chem 1996; 271: 12674-12680.
- 23 Koppert PW, Huijsmans CM, Nieuwenhuizen W. A monoclonal antibody, specific for human fibrinogen, fibrinopeptide A-containing fragments and not reacting with free fibrinopeptide A. Blood 1985; 66: 503-507.
- 24 Blomback B, Hessel B, Hogg D. et al. A two-step fibrinogen--fibrin transition in blood coagulation. Nature 1978; 275: 501-505.
- 25 Flood VH, Nagaswami C, Chernysh IN. et al. Incorporation of fibrin molecules containing fibrinopeptide A alters clot ultrastructure and decreases permeability. Br J Haematol 2007; 138: 117-124.
- 26 Litvinov RI, Gorkun OV, Galanakis DK. et al. Polymerization of fibrin: Direct observation and quantification of individual B:b knob-hole interactions. Blood 2007; 109: 130-138.
- 27 Geer CB, Tripathy A, Schoenfisch MH. et al. Role of ‘B-b’ knob-hole interactions in fibrin binding to adsorbed fibrinogen. J Thromb Haemost 2007; 05: 2344-2351.
- 28 Okumura N, Terasawa F, Haneishi A. et al. B:b interactions are essential for polymerization of variant fibrinogens with impaired holes ‘a‘. J Thromb Haemost 2007; 05: 2352-2359.
- 29 Liu CY, Koehn JA, Morgan FJ. Characterization of fibrinogen New York 1. A dys-functional fibrinogen with a deletion of B beta(9–72) corresponding exactly to exon 2 of the gene. J Biol Chem 1985; 260: 4390-4396.