A novel fibrinogen gamma-chain mutation, p.Cys165Arg, causes disruption of the γ165Cys–Bβ227Cys disulfide bond and ultimately leads to hypofibrinogenemia

Highlights

• This study describes a case of congenital hypofibrinogenemia in China, revealing that a γCys165Arg mutation in fibrinogen induces damage to the interchain disulfide bond (γ165Cys Bβ227Cys) and results in dysfunctional fibrinogen secretion.

• Absence of the mutated chain in patient plasma confirmed the diagnosis of hypofibrinogenemia rather than hypodysfibrinogenemia, which was confirmed by MALDI-TOF MS.

• Reduced fibrinogen levels increase the risk of bleeding during the early and postpartum periods of pregnancy.

• Maintaining fibrinogen levels is crucial for minimizing hemorrhagic risk in pregnant patients with congenital hypofibrinogenemia.

Abstract

Background

Congenital hypofibrinogenemia is a type of hereditary disease characterized by impaired fibrinogen synthesis and/or secretion induced by mutations in the fibrinogen gene.

Objectives

We investigated the phenotypes, genotypes, and pathogenesis of congenital hypofibrinogenemia in an affected family.

Patients/methods

The proband had a risk of bleeding; therefore, conventional coagulation screening was performed for the proband and her family members. Mutation sites in all exons and flanking sequences of FGA, FGB, and FGG were identified, with matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) performed to indicate the expression of abnormal chains. The effect of the mutation sites on fibrinogen structure and function was predicted by molecular modeling, and purified plasma fibrinogen from the proband was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and scanning electron microscopy. Thromboelastography was applied to assess the risk of bleeding and clotting in the proband.

Results

Fibrinogen levels in the proband were 1.21 g/L, 1.31 g/L, and 1.38 g/L according to Clauss assay, the prothrombin time method, and enzyme-linked immunosorbent assay, respectively. A novel heterozygous mutation (γCys165Arg), a heterozygous mutation (AαIle6Val), and two genetic polymorphisms (AαThr331Ala and BβArg478Lys) in fibrinogen were found in the proband, and MALDI-TOF MS indicated absence of the mutated chain in patient plasma. Additionally, the heterozygous mutation (γCys165Arg) displayed substitution of a nonpolar γ165Cys (low mass) with a positively charged Arg (high mass) along with a small fiber diameter and loose network structure.

Conclusions

Fibrinogen γCys165Arg mutations cause damage to the interchain disulfide bonds of fibrinogen and hinder fibrinogen secretion, possibly explaining the pathological mechanism associated with congenital hypofibrinogenemia.

Introduction

Fibrinogen is a symmetric dimeric molecule comprising two homologous subunits, each consisting of three peptide chains: Aα, Bβ, and γ. Fibrinogen synthesis is a highly ordered process that occurs in hepatocytes. Molecular chaperones such as GRP78 and calnexin facilitate accurate processing and folding of the new fibrinogen polypeptide chain in the endoplasmic reticulum [1]. Under hydrophobic conditions, fibrinogen assembles to form Aαγ and Bβγ intermediates [2], which bind to the Bβ or Aα chain, respectively, to form the AαBβγ-subunit structure [3]. Two homologous subunits dimerize through disulfide bonds, with glycosylation, sulfation, and specific side-chain phosphorylation subsequently performed in the Golgi complex and resulting in extracellular secretion following modification. During coagulation, fibrinogen molecules are cleaved by thrombin and converted to fibrin, which serves as a major structural protein of blood clots in order to exert hemostatic functions.

Congenital hypofibrinogenemia is a type of hereditary disease characterized by impaired fibrinogen synthesis and/or secretion induced by mutations in the fibrinogen gene [4]. Heterozygous mutations occur most commonly in congenital hypofibrinogenemia. The clinical phenotypic characteristics include low fibrinogen levels (<1.5 g/L). Additionally, coagulation tests depending on the formation of fibrin are variably prolonged, the most sensitive assay being the thrombin time (TT) [5,6]. Some patients experience symptoms such as spontaneous bleeding, thrombosis, and in women, spontaneous abortion [7,8], the rate of spontaneous bleeding usually approaching 20% and potentially reaching 80% following traumatic injury [9].

The present study investigated the phenotypes, genotypes, and related functional structures of fibrinogen in a family presenting with congenital hypofibrinogenemia, and elucidated the possible patho-mechanism through DNA sequencing, thromboelastography, matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE), and scanning electron microscopy (SEM).