Differences in the function and secretion of congenital aberrant fibrinogenemia between heterozygous γD320G (Okayama II) and γΔN319-ΔD320 (Otsu I)

Abstract

Background:We encountered two patients with hypodysfibrinogenemia and designated them as Okayama II and Otsu I. Although the affected residue(s) in Okayama II and Otsu I overlapped, functionally determined fibrinogen levels and the ratio of functionally to immunologically determined plasma fibrinogen levels were markedly different. Methods: DNA sequence and functional analyses were performed for purified plasma fibrinogen. A recombinant protein was synthesized in Chinese hamster ovary (CHO) cells to determine the secretion of variant fibrinogens. Results: A heterozygous ANG in FGG, resulting in γ320AspNGly for Okayama II, and a heterozygous deletion of AATGAT in FGG, resulting in the deletion of γAsn319 and γAsp320 (γΔN319-ΔD320) for Otsu I, were obtained. SDS-PAGE and Coomassie staining revealed that the variant γ-chain was not clear in Okayama II, but was clearly present in Otsu I. The lag period for the fibrin polymerization of Okayama II was slightly slower than that of the normal control, whereas Otsu I fibrinogen indicated no polymerization within 30 min. Both variant γ-chains were synthesized in CHO cells and assembled into fibrinogen; however, the fibrinogen concentration ratio of the medium/cell lysate of γ320Gly was six-fold lower than that of γΔN319-ΔD320. Conclusions: We concluded that the plasma fibrinogen of Okayama II, constituted by a lower ratio of the variant γ-chain, led to the almost normal functioning of fibrin polymerization. However, the plasma fibrinogen of Otsu I, with a higher ratio of the variant γ-chain, led to marked reductions in fibrin polymerization.

Genetics, diagnosis and clinical features of congenital hypodysfibrinogenemia: a systematic literature review and report of a novel mutation

“…Pulse‐chase analysis of recombinant Suhl (Frostburg) and Cordoba Fibrinogen in CHO or COS‐1 cells showed that the rate of both assembly and secretion were lower than that of normal fibrinogen . On the other hand, Fibrinogen Otsu I (Vlissingen, Frankfurt IV) and Okayama II were normally synthetized and assembled but only partially secreted . Expression studies of recombinant protein are important to determine the reason of the ‘hypofibrinogenemic’ phenotype.…”

“…Seven additional hypodysfibrinogenemia cases were a result of compound heterozygosity for mutations on the same or different fibrinogen genes [4,8,[14][15][16][17][18]. Expression of recombinant fibrinogens was performed for seven fibrinogen variants [5][6][7][18][19][20][21].…”

“…On the other hand, Fibrinogen Otsu I (Vlissingen, Frankfurt IV) and Okayama II were normally synthetized and assembled but only partially secreted [6]. Expression studies of recombinant protein are important to determine the reason of the 'hypofibrinogenemic' phenotype.…”

“…The dysfibrinogenemic characteristics were confirmed by functional analyses in purified or plasmatic systems, showing for most variants defective polymerization, defective calcium binding and/or hypofibrinolysis [6,25,[44][45][46]. Other mutations have a different pathophysiology: the FGA Val39Asp mutation (Fibrinogen Canterbury) introduces an inappropriate protein cleavage sequence of the Aa chain, leading to removal of fibrinopeptide A before the fibrinogen molecule enters the circulation [47].…”

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

“…Recombinant mutant fibrinogens were prepared as described previously [ 21 , 22 ]. Briefly, the mutant Bβ-fibrinogen expression vector was prepared by the insertion of the 99-bp nucleotides of intron 3 into the pMLP-Bβ plasmid (kindly provided by Lord ST, University of North Carolina, Chapel Hill, NC, USA), which contained wild-type Bβ-chain cDNA, with restriction enzymes (Eco81I in exon 2 and Bst1107I in exon 5).…”