Fibrin clot properties to assess the bleeding phenotype in unrelated patients with hypodysfibrinogenemia due to novel fibrinogen mutations

Abstract

Congenital hypodysfibrinogenemia is a rare fibrinogen disorder, defined by decreased levels of a dysfunctional fibrinogen. We present the functional and structural characterization of two new fibrinogen variants. A duplication of 32 bases in FGA exon 5, p.Ser382GlyfsTer50 was identified in a patient (P1) with history of hemoptysis and traumatic cerebral bleeding. A missense mutation in FGG exon 8, p.Ala353Ser was identified in two siblings (P2 and P3) with tendency to bruising and menorrhagia. Fibrin polymerization was studied in plasma and in purified fibrinogen by turbidimetry. Fibrin structure was studied by a permeability assay, laser scanning confocal microscopy (LSCM) and scanning electron microscopy (SEM). In both plasma and purified fibrinogen samples, all patients had an abnormal polymerization characterized by a decreased maximal absorption compared to controls. The permeation constant (Ks) was markedly increased in all patients: 31 ± 9 × 10 − 9 cm 2 in P1, and 20 ± 0.1 × 10 − 9 cm 2 in P2 and P3, compared to 6 ± 2 × 10 − 9 cm 2 in the control (p < 0.05). The presence of very large pores that accounts for the increased Ks was confirmed by LSCM and SEM patients' clots images. By SEM, the patients' fibrin fibers diameters were thicker: 90 ± 25 nm in P1, 162 ± 64 nm in P2 and 132 ± 46 nm in P3 compared to 74 ± 25 nm in control (p < 0.0001). In conclusion, both new causative fibrinogen mutations altered clot structure by forming thick fibers, diminishing fiber branching, and increasing pore filling space. These structural changes to clots explain the patients' bleeding phenotypes.

Congenital fibrinogen disorders: Strengthening genotype–phenotype correlations through novel genetic diagnostic tools

“…fibrinopeptide release and turbidimetry), viscoelastic assays and structural studies (e.g. electron microscopy) 36,38,39,41,42,45,46,48 . In silico molecular modelling utilises computational algorithms to predict what effect genetic variation at a specific site will have at a nucleotide and amino acid level.…”

“…Given the heterogeneity of genetic lesions that occur in CFDs, the modality for molecular testing of these patients Molecular typing for fibrinogen disorders, in most case series, has historically been performed by Sanger sequencing each of the three fibrinogen genes including all exons, exonintron boundaries and promoter regions. [34][35][36][37][38][39][40][41][42] In patients with dysfibrinogenaemia, a step-wise approach was frequently used by initially sequencing exon 2 of FGA and exon 8 of FGG with subsequent sequencing of the entirety of the remaining exons of these genes if mutations were not identified. 43,44 Termed 'hotspots', these regions are enriched for mutations in these genes.…”

“…electron microscopy). 36,38,39,41,42,45,46,48 In silico molecular modelling utilises computational algorithms to predict what effect genetic variation at a specific site will have at a nucleotide and amino acid level. Degree of conservation at a particular genomic location, physiochemical differences between the original and altered amino acid, and predicted effect on splicing are some of the properties these scores incorporate in their pathogenicity estimation.…”

Addressing some challenges of congenital fibrinogen disorders in 2023 and beyond

“…47 On one side, patients with a bleeding phenotype have abnormal polymerization profiles, increased lysis, and thick fibrin fiber with large pores. 48 On the other side, patients with a thrombotic phenotype have fibrin networks composed of thin fiber strands that have small pores and are more rigid and less permeable. 49 If these methods are often investigated to explain the clinical presentation of single cases, data on large series are limited.…”

Two Novel Heterozygous Mutations (p.γPhe230Val and p.AαAsn839Thr) Cause Hereditary Hypodysfibrinogenemia in Two Chinese Independent Families

“…According to the degree of Fg:C and Fg:Ag reduction, inherited fibrinogen disorder can be divided into two types, type I and type II. Type I defects include hereditary afibrinogenemia and hereditary hypofibrinogenemia; type II defects include dysfibrinogenemia or hypodysfibrinogenemia [2]. There are few reports of hypodysfibrinogenemia, which is characterized by varying degrees of reduced fibrinogen levels and abnormal function, and the pathogenic mechanism may be caused by abnormal assembly, secretion, or increased degradation of fibrinogen chain [3].…”