Congenital hypofibrinogenemia associated with γK232T

Zhao Misheng; Wang Mingshan; Lou Zhefeng; Chen Xiaoli; Yu Dandan; Li Xiaolong; Xia Wenli; Wang Han; Gao Shenmeng; Zhu Liqing

doi:10.5482/HAMO-17-03-0014

Hämostaseologie, Table of Contents

Hamostaseologie 2018; 38(01): 43-48
DOI: 10.5482/HAMO-17-03-0014

Original Article

Schattauer GmbH

Congenital hypofibrinogenemia associated with γK232T

In vitro expression demonstrates defective secretion of the variant fibrinogenKongenitale Hypofibrinogenämie in Verbindung mit γK232T In vitro-Expression zeigt mangelhafte Sekretion der Fibrinogen-Variante

Authors

Zhao Misheng

¹Department of Clinical Laboratory, The First Hospital of Wenzhou Medical University, Nanbaixiang, Ouhai District, Wenzhou, 325000, China

²Department of Clinical Laboratory, Wenzhou People’s Hospital, Lucheng District, Wenzhou, 325000, China
Wang Mingshan

¹Department of Clinical Laboratory, The First Hospital of Wenzhou Medical University, Nanbaixiang, Ouhai District, Wenzhou, 325000, China
Lou Zhefeng

³School of Laboratory Medicine and Life Science, Wenzhou Medical University
Chen Xiaoli

¹Department of Clinical Laboratory, The First Hospital of Wenzhou Medical University, Nanbaixiang, Ouhai District, Wenzhou, 325000, China
Yu Dandan

¹Department of Clinical Laboratory, The First Hospital of Wenzhou Medical University, Nanbaixiang, Ouhai District, Wenzhou, 325000, China
Li Xiaolong

¹Department of Clinical Laboratory, The First Hospital of Wenzhou Medical University, Nanbaixiang, Ouhai District, Wenzhou, 325000, China
Xia Wenli

²Department of Clinical Laboratory, Wenzhou People’s Hospital, Lucheng District, Wenzhou, 325000, China
Wang Han

²Department of Clinical Laboratory, Wenzhou People’s Hospital, Lucheng District, Wenzhou, 325000, China
Gao Shenmeng

⁴Department of Internal Medicine, The First Hospital of Wenzhou Medical University, Nanbaixiang, Ouhai District, Wenzhou, 325000, China
Zhu Liqing

¹Department of Clinical Laboratory, The First Hospital of Wenzhou Medical University, Nanbaixiang, Ouhai District, Wenzhou, 325000, China

Abstract

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

Full Text

References

References
1 Kani S, Terasawa F, Lord ST. et al. In vitro expression demonstrates impaired secretion of the gammaAsn319, Asp320 deletion variant fibrinogen. Thromb Haemost 2005; 94: 53-59.
2 Mosesson MW. Fibrinogen and fibrin structure and functions. J Thromb Haemost 2005; 03: 1894-1904.
3 Weisel JW, Litvinov RI. Mechanisms of fibrin polymerization and clinical implications. Blood 2013; 121: 1712-1719.
4 Podolnikova NP, Yakovlev S, Yakubenko VP, Wang X, Gorkun OV, Ugarova TP. The interaction of integrin alphaIIbbeta3 with fibrin occurs through multiple binding sites in the alphaIIb beta-propeller domain. J Biol Chem 2014; 289: 2371-2383.
5 Doolittle RF. Fibrinogen and fibrin. Annu Rev Biochem 1984; 53: 195-229.
6 Mosesson MW, Siebenlist KR, Meh DA. The structure and biological features of fibrinogen and fibrin. Ann N Y Acad Sci 2001; 936: 11-30.
7 Redman CM, Xia H. Fibrinogen biosynthesis. Assembly, intracellular degradation, and association with lipid synthesis and secretion. Ann N Y Acad Sci 2001; 936: 480-495.
8 de Moerloose P, Casini A, Neerman-Arbez M. Congenital fibrinogen disorders: an update. Semin Thromb Hemost 2013; 39: 585-595.
9 Zhu L, Wang M, Xie H, Jin Y, Yang L, Xu P. A novel fibrinogen mutation (gamma Thr277Arg) causes hereditary hypofibrinogenemia in a Chinese family. Blood Coagul Fibrinolysis 2013; 24: 642-644.
10 Zhu L, Wang Y, Zhao M. et al. Novel mutations (gammaTrp208Leu and gammaLys232Thr) leading to congenital hypofibrinogenemia in two unrelated Chinese families. Blood Coagul Fibrinolysis 2014; 25: 894-897.
11 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.
12 Kotlin R, Pastva O, Stikarova J. et al. Two novel mutations in the fibrinogen gamma nodule. Thromb Res 2014; 134: 901-908.
13 Zhang J, Zhao X, Wang Z. et al. A novel fibrinogen B beta chain frameshift mutation causes congenital afibrinogenaemia. Thromb Haemost 2013; 110: 76-82.
14 Spraggon G, Everse SJ, Doolittle RF. Crystal structures of fragment D from human fibrinogen and its crosslinked counterpart from fibrin. Nature 1997; 389: 455-462.
15 Brennan SO, Sheen CR, George PM. Novel gamma230 Asn-->Asp substitution in fibrinogen Middlemore associated hypofibrinogenaemia. Thromb Haemost 2005; 93: 1196-1197.
16 Hanss M, Ffrench P, Vinciguerra C, Bertrand MA, Mazancourt P. Four cases of hypofibrinogenemia associated with four novel mutations. J Thromb Haemost 2005; 03: 2347-2349.
17 Asselta R, Robusto M, Plate M, Santoro C, Peyvandi F, Duga S. Molecular characterization of 7 patients affected by dysor hypo-dysfibrinogenemia: Identification of a novel mutation in the fibrinogen Bbeta chain causing a gain of glycosylation. Thromb Res 2015; 136: 168-174.
18 Iida H, Ishii E, Nakahara M. et al. A case of congenital afibrinogenemia: fibrinogen Hakata, a novel nonsense mutation of the fibrinogen gamma-chain gene. Thromb Haemost 2000; 84: 49-53.
19 Terasawa F, Fujita K, Okumura N. Residue gamma153Cys is essential for the formation of the complexes Aalphagamma and Bbetagamma, assembly intermediates for the AalphaBbetagamma complex and intact fibrinogen. Clin Chim Acta 2005; 353: 157-164.
20 Okumura N, Terasawa F, Tanaka H. et al. Analysis of fibrinogen gamma-chain truncations shows the C-terminus, particularly gammaIle387, is essential for assembly and secretion of this multichain protein. Blood 2002; 99: 3654-3660.
21 Dear A, Dempfle CE, Brennan SO, Kirschstein W, George PM. Fibrinogen Mannheim II: a novel gamma307 His-->Tyr substitution in the gammaD domain causes hypofibrinogenemia. J Thromb Haemost 2004; 02: 2194-2199.
22 Suzuki K, Tsunekawa Y, Hernandez-Benitez R. et al. In vivo genome editing via CRISPR/Cas9 mediated homology-independent targeted integration. Nature 2016; 540: 144-149.