Identification and characterization of novel mutations implicated in congenital fibrinogen disorders

Abstract

Introduction: Fibrinogen is a complex molecule comprised of two sets of Aα, Bβ, and γ chains. Fibrinogen deficiencies can lead to the development of bleeding or thromboembolic events. The objective of this study was to perform DNA sequence analysis of patients with clinical fibrinogen abnormalities, and to perform genotype-phenotype correlations.

Materials and methods: DNA from 31 patients was sequenced to evaluate disease-causing mutations in the three fibrinogen genes: FGA,FGB, and FGG. Clinical data were extracted from medical records or from consultation with referring hematologists. Fibrinogen antigen and functional (Clauss method) assays, as well as reptilase time (RT) and thrombin time (TT) were obtained for each patient. Molecular modeling was used to simulate the functional impact of specific missense variants on the overall protein structure.

Results: Seventeen mutations, including six novel mutations, were identified in the three fibrinogen genes. There was little correlation between genotype and phenotype. Molecular modeling predicted a substantial conformational change for a novel variant, FGG p.Ala289Asp, leading to a more rigid molecule in a region critical for polymerization and alignment of the fibrin monomers. This mutation is associated with both bleeding and clotting in the two affected individuals.

Conclusions: Robust genotype-phenotype correlations are difficult to establish for fibrinogen disorders. Molecular modeling might represent a valuable tool for understanding the function of certain missense fibrinogen mutations but those should be followed by functional studies. It is likely that genetic and environmental modifiers account for the incomplete penetrance and variable expressivity that characterize fibrinogen disorders.

Keywords: afibrinogenemia; dysfibrinogenemia; fibrinogen disorders; fibrinogen mutations; molecular modeling.

Figure 1

Location of mutations in relation to the exons of the three fibrinogen genes. FGG,FGA, and FGB are located contiguously on chromosome 4q23. FGG and FGA are transcribed in the reverse direction, opposite of FGB. FGG,FGA, and FGB consist of 10, 6, and 8 exons, respectively. Novel mutations are indicated by *, nonsense mutations are colored blue, missense mutations are black, frameshift mutations are purple, and splice site mutations are green. There are missense mutations located throughout the three genes, and the more deleterious mutations (nonsense, frameshift and splice site) are located only in FGG and FGA

Figure 2

Predicted structural and functional changes of the FGG mutant. (A) Forward facing view of the overlay of the energy minimized WT (cyan) and FGG A289D mutant (blue) human fibrinogen polymerization interface. Arrows highlight major predicted structural alterations in response to the mutation. Insets highlight the predicted local differences between the WT (right) and mutant (bottom). Dashed lines indicate predicted non‐bond interactions (green = hydrogen bonds, orange = pi‐anion, magenta = pi‐pi) (B) Predicted protein–protein interaction energy between two WT or two A289D human fibrinogen molecules. *P = .016

Figure 3

Molecular modeling simulation of WT and FGB mutants. Structural overlays of energy minimized WT (cyan) and (A) Gly272Arg (magenta) or (B) Arg455Lys human fibrinogen. Substantial conformational changes observed between the mutant and WT structures are indicated with arrows. Insets depict the regions surrounding the mutations of each structure separately. Mutated residues are highlighted in yellow and dashed lines indicate predicted non‐bond interactions (green = hydrogen bond, cyan = hydrogen‐pi, orange = electrostatic and pi‐cation)