A Novel Missense Mutation in FGG (c.944C>A) Encodes for An Amino Acid Change (p.Ala315Asp) in the Gamma Chain of Fibrinogen Causing Hypofibrinogenemia and a Thrombotic Phenotype

Abstract 856

Fibrinogen plays a central role in maintaining hemostasis. Disruption of its normal function may lead to hemorrhagic or thrombotic events. Fibrinogen is encoded by three genes, FGA, FGB, and FGG, clustered on chromosome 4q28-q31. Hereditary defects of fibrinogen, although uncommon, can affect the quantity (hypofibrinogenemia and afibrinogenemia) or the quality (dysfibrinogenemia) of the circulating protein. We report a family with two affected individuals (father and daughter) that presented with a mild bleeding predisposition, but also exhibited thrombotic events, i.e. unprovoked deep vein thrombosis in a father and an in utero middle cerebral artery stroke in a daughter. The Clauss fibrinogen levels for the father and daughter were 105 and 98 mg/dL (normal 150–400 mg/dL) and fibrinogen antigen levels were 138 and 152 mg/dL (normal 170–400 mg/dL), respectively. Thrombin times were prolonged in both patients. All other coagulation factors were within normal range and the patients did not carry either the prothrombin G20210A or factor V Leiden mutation.

All exons and intron-exon junctions of the three fibrinogen genes were sequenced. The father and daughter were found to be heterozygous for a novel missense mutation in the gamma chain (Ala315Asp) while this mutation was not observed in an unaffected family member (mother of the daughter).

Transient transfection experiments using CHO-K1 cells showed that gamma chain with p.Ala315Asp mutation was detected in both cell lysates and supernatant, although in reduced amount as compared with cells transfected with wild-type (WT) FGG cDNA.

Structurally, there was no significant difference in fibrin fiber diameter, as measured manually by scanning electron microscopy, between the affected (father and daughter), and the daughter's unaffected mother. Similarly, there was no difference in thrombus height between the three individuals. As expected, hydraulic permeability of platelet rich plasma clots correlated with the fibrinogen levels in each person. Clotting kinetics in plasma of affected family members was consistent with hypofibrinogenemia, but did not suggest abnormal fibrin polymerization. Clot structure and fibrin deposition was further characterized with a microfluidics flow assay, an experimental technique in which platelet and fibrin accumulation is observed in real-time using fluorescent probes in whole blood under shear stress on a collagen/tissue factor micropatterned surface. Interestingly, fibrin density and platelet accumulation, as measured by platelet and fibrin surface area coverage, was highest in the father and daughter, the two individuals with hypofibrinogenemia and thrombotic phenotype.

In order to evaluate the possible changes resulting from the Ala315Asp mutation we examined predicted alterations in protein structure, energy, and protein-protein interaction between two fibrinogen molecules via in silico molecular modeling using published protein crystal structure. The molecular modeling showed that alanine for aspartic acid substitution predicted to result in substantial changes in overall conformation of the interaction interface (distal D-domain) between the two fibrinogen molecules due to introduction of a bulkier side chain. Potential energy (energy of minimization) for Ala315Asp mutant homodimer, and for WT-mutant heterodimer were lower than the WT homodimer, predicting a more stable structure from an energetic standpoint. Overall interaction energy (measurement of the energy of association between the two molecules) was lower for Ala315Asp mutant homodimer as compared with WT homodimer or WT-mutant heterodimer, suggesting more favorable interaction for Ala315Asp mutant dimers. Overall, the in silico modeling studies predicted that Ala315Asp mutation may lead to more rapid or stronger interaction between fibrinogen molecules.

These data suggest that a novel FGG mutation, c.944C>A, that predicts pAla315Asp change, if expressed and incorporated into fibrinogen molecule in vivo, may result in a more stable or more resistant to lysis clot. Characterizing the association between a specific genetic defect and its phenotypic expression will advance the current understanding of the molecular and biochemical mechanisms of the inherited disorders of fibrinogen, and may contribute to the development of safer therapeutic interventions for the patients with fibrinogen disorders.