Congenital hypofibrinogenemia associated with γK232T

Summary

We have previously reported a case of congenital hypofibrinogenemia caused by a novel heterozygous A→C transition at nucleotide 5864 of FGG, leading to the K232T substitution in the fibrinogen γ-chain. However, the pathogenic mechanism is still unclear. To further reveal its molecular basis, we examined the effects of γK232T substitution on fibrinogen synthesis, stability, and secretion through in vitro expression of mutant γ232T in Chinese hamster ovary (CHO) cells. Quantitative RT-PCR of the variant γ-chain mRNA showed that the γ232T transcribed the variant cDNA. Enzyme-linked immunosorbent assay and Western blot analysis of the cell lysates and culture media showed that the CHO cells transfected with successfully synthesized the variant fibrinogen, but failed to secrete it into the culture medium. Furthermore, fibrinogen purified from the plasma of patient showed a normal thrombin-catalyzed fibrin polymerization, also indicating the impeding secretion of variant γ232T fibrinogen. In conclusion, our data reveal that the γK232T is responsible for the congenital hypofibrinogenaemia through interfering with the correct secretion of fibrinogen.

Zusammenfassung

Wir haben bereits über einen Fall von kongenitaler Hypofibrinogenämie berichtet, die durch eine neue heterozygote A→C Transition im Nukleotid 5864 des FGG, welche zur Substitution von K232T in der Fibrinogen-γ-Kette führt, verursacht wird. Der pathogene Mechanismus ist jedoch immer noch unklar. Urn die molekularen Grundlagen weiter aufzuklären, untersuchten wir mittels in vitro-Expression von mutiertem γ232T in chinesischen Hamster-Ovar (CHO)-Zellen die Wirkungen einer γK232T-Substitution auf Synthese, Stabilität und Sekretion von Fibrinogen. Die quantitative RT-PCR der varianten γ-Ketten-mRNA ergab, dass das γ232T die variante cDNA transkribierte. Die Analyse der Zelllysate und der Kulturmedien mittels ELISA und Western Blot ergab, dass die mit transfizierten CHO-Zellen erfolgreich die Fibrinogen-Variante synthetisierten, diese aber nicht in das Kulturmedium abgeben konnten. Des Weiteren zeigte das aus dem Plasma des Patienten aufgereinigte Fibrinogen eine normale, durch Thrombin katalysierte Fibrin-Polymerisation, was ebenfalls fur eine beein-trächtigte Sekretion des varianten γ232T-Fibrinogens spricht.Wir schließen aus unseren Daten, dass das γK232T über eine Störung der adäquaten Sekretion von Fibrinogen für die kongenitale Hypofibrinogenämie verantwortlich ist.

Keywords

Congenital hypofibrinogenemia - fibrinogen γ-chain mutation - in vitro expression - fibrinogen secretion

Schlüsselwörter

Kongenitale Hypofibrinogenämie - Fibrinogen γ-Ketten-Mutation - in vitro-Expression - Fibri-nogensekretion

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