Fibrinogen Yecheon: Congenital Dysfibrinogenemia with Gamma Methionine-310 to Threonine Substitution

Fibrinogen is a 340-kD glycoprotein, one of the critical proteins of the blood coagulation and platelet aggregation (1). It is composed of six polypeptide chains, (α, β, γ)₂, which are held together by disulfide bonds and organized in a symmetrical dimeric fashion (1).

Until now, a variety of mutations in each of the fibrinogen chain genes have been reported in more than 465 families all over the world, with Aα-chain mutation being the most common form, followed by γ-chain mutation (2).

Several cases of congenital dysfibrinogenemia have been reported in Korea. The first report was Aα(517-522)delins31, designated as "fibrinogen Seoul" in a 7-yr-old girl associated with amyloidosis (3). The second was γ Ala341Asp, which was designated as "fibrinogen Seoul I", in a 66-yr-old woman with peripheral artery obstructive disease (4). The third was Aα Gln328Pro, which was designated as "fibrinogen Seoul II", in a 51-yr-old man who suffered from recurrent myocardial infarction (5).

Here we describe a γ-chain fibrinogen variant (fibrinogen Yecheon), which is a point mutation from T to C in the 1,007th nucleotide of the FGG gene, resulting in a methionine (Met)-to-threonine (Thr) substitution at the 310th residue in the carboxy-terminal region of the fibrinogen γ-chain. This is the fourth report of γ Met310 to Thr substitution in the world, and the first in Korea. As for genetic dysfibrinogenemia, this is the fourth case report in Korea.

A 20-yr-old male patient with past medical history of bleeding tendency in neonate period visited the department of otorhinolaryngology for a preoperative evaluation for operation of chronic maxillary sinusitis. He reported a family history of easy bruisability of his father and conductive hearing difficulty after meningitis in childhood. No laboratory abnormalities indicating coagulation defect had been detected after birth.

In the preoperative evaluation, the patient had normal blood counts and serum chemistry test results. However, the initial coagulation screening showed prolonged prothrombin time (PT, 24.5 sec; normal 10.4-12.5 sec) and activated partial thromboplastin time (aPTT, 47.3 sec; normal, 26.0-41.0 sec). The bleeding time was normal (2 min and 30 sec; normal, 1-5 min). The PT and aPTT mixing test result indicated factor deficiency. The fibrinogen degradation product (FDP) and d-Dimer were within normal ranges. Other coagulation tests were normal (Table 1). On the other hand, plasma fibrinogen activity of the subject examined using the Clauss method was lower than the detection limit (<25 mg/dL; normal, 140-460 mg/dL), while the antigen level measured by fibrinogen ELISA was normal (437 mg/dL). After a written informed consent was obtained, a familial genetic study was started. All exons, exon-intron boundaries, and promoter regions of the fibrinogen Aα (FGA, GenBank M64982), Bβ (FGB, GenBank M64983), and γ (FGG, GenBank M10014) chain genes were amplified and sequenced. PCR and direct sequencing revealed that only the patient in his family was heterozygous for a point mutation in the FGG gene. The mutation was a mutation substituting the 310th codon ATG (Met) to ACG (Thr), (c.1007T>C) (Fig. 1A). He had no mutations in the FGA or FGB gene. We designated the patient's abnormal fibrinogen as "fibrinogen Yecheon", naming after the local city where he and his family are residing. Collectively, congenital dysfibrinogenemia was identified. For further evaluation, fibrinogen mixing test (Table 2) was run and the results showed no significant improvements in the fibrinogen level. This implies the possibility of some inhibitors of fibrinogen activity.

Fig. 1
The chromatogram shows DNA sequencing results of fibrinogen γ-chain gene (FGG). The A indicates the sample of the patient. A heterozygous FGG mutation (c.1007T>C [p.Met336Thr]) was identified. The B indicates the sample of the father and the C indicates the sample of the mother.

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Table 1
Coagulation test results

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Table 2
Fibrinogen mixing test

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The plasma fibrinogen activities of the patient's parents by Clauss method were within normal range (309 and 254 mg/dL, respectively). His two elder sisters also showed normal coagulation test results. Molecular genetic study was performed only with his parents, including his father who had a history of easy bruising, and they both were homozygous for the wild-type allele (Fig. 1B, C).

Dysfibrinogenemia was suggested in the subject according to the initial plasma fibrinogen activity and antigen levels. DNA sequence analysis confirmed congenital dysfibrinogenemia. The patient himself decided not to take operation after the diagnosis of dysfibrinogenemia, although cryoprecipitate treatment was possible. Cryoprecipitate contains factor VIII, fibrinogen, von Willebrand factor and factor XIII, so it is generally used for dysfibrinogenemia. Although he was born with bleeding tendency, the patient had no symptoms of bleeding or thrombosis after childhood. According to Cote et al.(1), an examination of the clinical symptoms associated with γ-dysfibrinogenemia shows that <5% of the individuals experienced significant bleeding, and <30% showed thrombotic tendencies. Approximately 60% of patients with γ-dysfibrinogenemias were asymptomatic at the time of diagnosis. Unfortunately, data on long-term follow-up of dysfibrinogenemic patients are rarely available. Close observation of clinical symptoms might be the best option for treatment.

γ Met310Thr was already reported by the names of Asahi (6), Frankfurt VII (7), and Hannover XXIV (2). Details of biochemical mechanisms involved in fibrin polymerization, release of fibrinopeptides A/B, factor XIIIa-mediated cross-linking profiles, and the position of glycosylation were elucidated in the study of fibrinogen Asahi (6). Asahi fibrinogen showed normal release of fibrinopeptides A/B, but fibrin polymerization and factor XIIIa-mediated γ-chain cross-linking were severely impaired regardless of calcium ions, which was caused by N-glycosylation consensus sequence Asn (308)-Gly (309)-Thr (310) conferred by the mutation (1, 6, 8). Specifically, severely impaired polymerization of Asahi fibrin monomers may account for extremely retarded cross-linking between the two Asahi γ-chains, although the amine receptor γ Gln (398) was found to function normally (1, 6, 8). Those functional abnormalities could explain fibrinogen Asahi patient's bleeding tendency as well as that of the fibrinogen Yecheon subject, which was supported by the ultrastructure of fibrinogen Caracas II (Aα Ser434 to N-glycosylated Asn) detected with the use of scanning electron microscopy. In the microscopic image, large pores bounded by local fiber networks made up of thin fibers were observed (9). Normally, fibrinogen contains approximately 3% carbohydrate consisting of NeuAc, Gal, Man, and GlcNAc, which is linked to Asn 52 on the γ chain and Asn 364 on the Bβ chain (10). The role of glycosylation in fibrinogen function has been studied and its involvement in the clotting process was proposed (10), although contradictory results have been reported (11). Steric hindrance by the extra carbohydrate itself, the disruption of D:D interactions by the new glycosylation, and strong negative charges of sialic acids in the extra carbohydrate could be raised to account for disturbed fibrin polymerization or impaired cross-linking in Niigata (Bβ Asn160Ser), Asahi, and Lima (Aα Arg141Ser) fibrins, respectively (1, 12-14).

Being a heterozygote, in fibrinogen Yecheon, as in fibrinogen Asahi, fibrin monomer containing at least one gamma chain mutated is not likely to function on making polymerization properly. Fibrinogen mixing test results (Table 2) demonstrated the presence of inhibitor. FDP level was within normal range. Those facts, therefore, suggested extra glycosylated gamma chain not only dysfunction on polymerization but also inhibit normal fibrin and fibrinogen resulting in hardly clotting with thrombin in fibrinogen activity assay by Clauss method. In addition, the subject's coagulation abnormality was thought to be caused by extra glycosylation of γ-chain and subsequent disruption of D:D interaction because other coagulation factors were within normal ranges (Table 1). The subject with fibrinogen Yecheon showed bleeding tendency. Usually, dysfibrinogenemias having extra glycosylations concerned hemorrhagic tendency unlike Tokyo V (γ Ala327-Thr) fibrinogen which was somewhat unique in that its propositus was thrombophilic partly owing to fibrinolysis-resistant fibrin clots despite the very low clottability of the fibrin. It was suggested that tissue plasminogen activator binding to Tokyo V fibrinogen might have been affected directly. The subject of fibrinogen Yecheon presented de novo mutation in FGG, while his parents did not show any coagulation abnormalities including DNA sequencing. De novo mutations are associated with advancing paternal age (15) and pollutants (16, 17).

One thing different between Yecheon and Asahi subjects was the level of FDP, which was normal (2.3 µg/mL) for the Yecheon subject while that was markedly increased to 160 µg/mL for the Asahi patient. Because d-dimer level was within normal range, the Yecheon subject was not considered in the state of hyperfibrinolysis, which was partially supported by the normal levels of plasminogen and antithrombin. As a follow-up, a further study can be conducted to understand what brought about marked increase in FDP level for Asahi subject.

Fibrinogen Yecheon is the only congenital dysfibrinogenemia in Korea associated with bleeding tendency, and not thrombosis or amyloidosis. Our subject was born with bleeding tendency, although he had no history of serious bleeding symptoms. In general, mutations within the γ chain of fibrinogen are not associated with serious bleeding disorders. Two patients, Baltimore I (Gly292Val) and Giessen IV (Asp-318Gly), who experienced mild bleeding symptoms, also suffered from thrombotic tendencies. The only γ-dysfibrinogenemia associated with a serious bleeding diathesis is Asahi I (Met310Thr). In this instance, the bleeding symptoms were probably related to the extra glycosylation resulting from the substitution (1).

In conclusion, this report describes a variant fibrinogen, hereinafter called "fibrinogen Yecheon", using the name after the town where the patient was living at the time of diagnosis (2). Fibrinogen Yecheon has a de novo heterozygous point mutation of FGG resulting in γ Met310Thr and subsequent extra N-glycosylation at γ Asn308. Extra N-glycosylated fibrinogen is considered a main inhibitor of normal fibrinogen activity.