A heterozygous nonsense mutation in the FGB gene (c.1299G > A) causes congenital fibrinogen disorder across four consecutive generations

Abstract

Background

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The aim of this study was to elucidate the molecular abnormalities in a four-generation Chinese family affected by congenital fibrinogen disorder (CFD).

Case presentation

The proband was a 5-year-old Chinese boy with CFD. Routine clotting tests revealed decreased plasma fibrinogen concentration in the proband and in his father and sister. Notably, the condition presented was clinically asymptomatic. Whole exome sequencing identified a heterozygous c.1299G > A mutation in exon 8 of the FGB gene, leading to p.Trp433* (TGG > TGA). Further Sanger sequencing revealed the presence of this mutation in his great-grandmother, grandfather, father, and sister as well.

Conclusion

The FGB gene variant c.1299G > A (p.Trp433*) across four consecutive generations is associated with CFD.

Background

Congenital fibrinogen disorder (CFD) is extremely unusual, and depending on whether it affects the quantity or quality of circulating fibrinogen, it is subclassified into type I and II disorders, respectively. Type I disorders (afibrinogenemia and hypofibrinogenemia) influence the quantity of circulating fibrinogen [1], whereas type II disorders (dysfibrinogenemia and hypodysfibrinogenemia) significantly affect the quality of circulating fibrinogen [2].

Fibrinogen is a pivotal element in coagulation and hemostasis; it is converted to fibrin by thrombin and factor XIII, culminating in clot formation in several steps [3, 4]. In addition, fibrinogen also has preeminent roles in inflammatory, cell migration, and angiogenesis activities [5,6,7].

Fibrinogen is a 340-kDa glycoprotein that is synthesized by hepatocytes and is present in the plasma of healthy individuals at a concentration range of 1.5–4.2 g/L [8,9,10]. Fibrinogen comprises three diverse polypeptide chains, including Aα, Bβ, and γ chains, which are coded by three genes located on chromosomes 4q31-q32, namely FGA, FGB, and FGG [11]. Mutations in any of the three fibrinogen genes cause hereditary defects of fibrinogen [12, 13]. Characterizing these mutations is crucial for the diagnosis, confirmation, and identification of potential carriers and for establishing a familial diagnosis.

The aim of this study is to report and discuss a four-generation family with CFD. Genetic analyses revealed that five members of this family carried a heterozygous variant (c.1299G > A, p.Trp433*) in the FGB gene. Our findings enhance the comprehension of the phenotype–genotype correlations related to the FGB gene.

Case presentation

The proband (IV:2) was a 5-year-old male. He was diagnosed with obstructive sleep apnea hypopnea syndrome (OSAHS) at the age of 3 years, which was found to be caused by adenoid and tonsillar hypertrophy. The results of the coagulation test showed low fibrinogen levels of 1.2 g/L (reference range: 1.93–4.19 g/L). At the age of 4 years and 7 months, the patient was re-examined for coagulation tests, revealing that the fibrinogen levels had further decreased to 0.85 g/L. The proband had normal results for complete blood count, liver function, and kidney function tests. Until April 2025, the patient had no bleeding symptoms.

In addition, we identified potential participants, including the proband and his relatives [great-grandmother (I:2), grandfather (II:2), father (III:2), proband (IV:2), and his sister (IV:1)], through a comprehensive pedigree analysis (Fig. 1). The coagulation test results of the members of the family revealed that individuals III:2, IV:1, and IV:2 had decreased fibrinogen levels of 1.12 g/L, 0.89 g/L, and 0.85 g/L, respectively. Individual II:2 had a fibrinogen level of 1.90 g/L, which approaches the lower limit of normal. Notably, coagulation testing was not performed on the great-grandmother (I:2), who did not consent to the procedure. In addition, individuals II:2, IV:1, and IV:2 showed prolonged prothrombin time (PT), whereas individuals III:2, IV:1, and IV:2 showed prolonged thrombin time (TT). Although individuals II:2, IV:1, and IV:2 did not present with spontaneous bleeding, they showed hemorrhagic tendencies. Table 1 shows detailed results of coagulation tests of this family.

Fig. 1

Congenital fibrinogen disorder family. Filled symbols represent affected individuals, and empty symbols represent unaffected individuals. The proband (IV:2) is indicated by an arrow

Table 1 Coagulation testing results of six members of the CFD family

Untargeted whole-exome sequencing (WES) was performed to investigate genetic variations in the proband (IV:2). WES was carried out on a Salus Pro™ sequencer (Salus Biomed, China) using the sequencing-by-synthesis method, according to the manufacturer’s protocols. Variants with a minor allele frequency (MAF) of < 0.05 across all populations were retained for further analysis. In silico algorithms like Sorting Intolerant from Tolerant (SIFT), Polymorphism Phenotyping v2 (PolyPhen-2), and MutationTaster were used for pathogenicity prediction of genetic variants. By integrating information from databases like ClinVar and InterVar, known pathogenic variants or disease-associated variants were identified, and benign loci were excluded from further analysis. The WES analysis of the proband identified a variant in the FGB gene (c.1299G > A). The FGB c.1299G > A variant was not identified in the Leiden Open Variation Database (LOVD), ClinVar, Genome Aggregation Database (gnomAD), Exome Aggregation Consortium (ExAC, available via gnomAD browser), 1000 Genomes Project or dbSNP databases but was detected in Mastermind Genomic Intelligence Platform (Genomenon). MutationTaster was used to predict that the production of a significantly truncated protein, which may induce nonsense-mediated decay (NMD) due to the loss of 59 amino acids (representing over 10% of the protein sequence), very likely adversely affects protein function.

The result of the proband was further confirmed by Sanger Sequencing (shown in Fig. 2. IV:2). Sanger sequencing was performed for FGB on genomic DNA to evaluate for disease-causing mutations. The PCR primers were designed as follows: The forward primer was 5′-TCATTTTACAGATGAGATTTA-3′, and the reverse primer was 5′-CCGTCATGGTTTTCCTACGACA-3′. For sequencing, the forward and reverse primers were 5′-GTCCCTAGATGGATTGGCCA-3′ and 5′-TATGAAAGAAAATTCCAATA-3′, respectively. Sanger Sequencing was also performed on the DNA samples of the proband’s great-grandmother, grandfather, father, and sister. The results revealed that all of them carried the same mutation as the proband. Conversely, his mother and aunt had the wild-type nucleotide. Unlike the unaffected individuals, all affected individuals in this family carried the mutation (c.1299G > A, p.Trp433*) in the FGB gene (Fig. 2).

Fig. 2

Sanger sequencing of affected and unaffected individuals. The great-grandmother (I:2), grandfather (II:2), father (III:2), and sister (IV:1) of the proband (IV:2) carried the mutation (c.1299G > A, p.Trp433*) in the FGB gene, whereas the aunt (III:3) of the proband was unaffected

Discussion and conclusions

From a genetic standpoint, a diverse array of mutations contributing to CFD has been identified. The complete absence or deficiency of fibrinogen results from mutations in one of three genes, namely FGA, FGB, and FGG. Currently, over 200 CFD-associated mutations in the FGB gene have been documented in the LOVD. Numerous FGB mutations are characterized as missense or late-truncating nonsense mutations occurring within the highly conserved C-terminal domain of the β-chain [14]. In the current study, WES and Sanger sequencing analyses identified a nonsense mutation in exon 8 of the FGB gene (c.1299G > A), p.Trp433* (TGG > TGA), which was observed in five members of the four-generation family. The FGB c.1299G > A (p.Trp433*) variant was not identified in the LOVD, ClinVar, gnomAD, ExAC, 1000 Genomes Project, or dbSNP databases but was detected in Genomenon. The mutation at this site is consistent with that reported by Yan et al. [15]. However, there are notable differences. (i) In the study by Yan et al., the mutation was detected only in the proband and not in other family members, whereas in the family we investigated, the mutation was present in members encompassing four consecutive generations. (ii) Yan et al. reported that the proband carried both FGB (c.1299G > A) and FGG (c.274 C > T) gene mutations, whereas the proband in our study showed only the FGB gene mutation (c.1299G > A).

The mutation c.1299G > A, which converts the tryptophan codon into a stop codon, leads to a premature termination codon mutation, particularly the p.Trp433* mutation identified in this study. Ceznerová et al. reported a novel heterozygous nonsense mutation, c.1298G > A in exon 8 of the FGB gene (p.Trp433*; TGG > TAG). The resulting protein is consistent with our findings [16]. In addition, several nonsense mutations have been detected within the final exon, exon 8 of the FGB gene, including c.1267 C > T (p.Glu423*), c.1296G > A (p.Trp432*), c.1400G > A (p.Trp467*), c.1409G > A (p.Trp470*), and c.1421G > A (p.Trp474*) [14, 17]. If a mutant β-chain is produced, the nonsense mutations may result in the synthesis of a truncated protein [14, 18, 19]. Such deleterious truncations are often not translated due to surveillance mechanisms like NMD [20]. The MutationTaster prediction in the study was consistent with the aforementioned hypothesis, suggesting that the production of a truncated protein with a loss of 59 amino acids triggers NMD and adversely affects protein function. In addition, Chen et al. [21] reported that the p.Arg17* mutation in the fibrinogen Bβ chain impairs both the transcription and translation processes of the FGB gene, potentially through the activation of the NMD pathway. However, Yan et al. [15] performed in vitro experiments to express Trp433* in CHO cells. Their results showed that the Trp433* mutation does not influence fibrinogen transcription, which contradicts the aforementioned prediction. Furthermore, their findings suggest that this nonsense mutation leads to misfolding of the D domain, consequently affecting fibrinogen secretion. Nonetheless, given that mRNA stability was not assessed, the possibility that this mutation induces CFD via the NMD pathway cannot be completely ruled out. Functional studies should be conducted in the future to enhance our understanding of specific mechanisms leading to decreased fibrinogen levels observed in patients involved in this study.

CFD can be inherited through both autosomal dominant and autosomal recessive patterns. For instance, congenital afibrinogenemia and hypofibrinogenemia are inherited in an autosomal recessive pattern, whereas congenital dysfibrinogenemia is inherited in an autosomal dominant pattern [22,23,24]. Several molecular abnormalities show a strong correlation with clinical manifestations and predict individual hemorrhagic tendencies [4]. The nature of the mutation and its clear association with the phenotype indicate that partial fibrinogen deficiency is associated with a heterozygous phenotype, whereas complete fibrinogen deficiency is characterized by a homozygous genotype [25]. Moreover, hypofibrinogenemia is associated with a reduced incidence of bleeding, whereas afibrinogenemia is considered a severe condition, wherein the diagnosed individuals may experience life-threatening hemorrhages [26]. Therefore, in this study, a heterozygous mutation in the FGB gene (c.1299G > A) was associated with reduced fibrinogen levels rather than complete absence of fibrinogen. Although the presentation was asymptomatic, this mutation was also correlated with hemorrhagic tendencies. Based on the report by Yan et al. [15], a patient carrying the heterozygous p.Trp433* mutation was diagnosed with hypofibrinogenemia, and the patient showed prolonged bleeding after trauma. Their findings are consistent with our study. In addition, the findings of coagulation tests suggest that the impact of FGB gene mutations in this family appears to progressively intensify across generations.

The c.1299G > A mutation identified in the four-generation CFD family in this study is consistent with the mutation reported by Yan et al. [15]. In addition, its adjacent sites, c.1296G > A (p.Trp432*) and c.1298G > A (p.Trp433*), have been previously documented in the literature to be associated with CFD [14, 16]. These findings collectively suggest that this mutation site can also occur in sporadic populations. In addition, in accordance with the American College of Medical Genetics and Genomics (ACMG) guidelines [27], this mutation was classified as likely pathogenic on the basis of the following criteria. (i) In vitro experiments conducted by Yan et al. [15] showed that the FGB c.1296G > A mutation may downregulate fibrinogen secretion (PS3). (ii) It is absent from databases like LOVD, ClinVar, gnomAD, ExAC, and the 1000 Genomes Project (PM2). (iii) The proband’s phenotype and family history are highly specific for CFD caused by the c.1299G > A (p.Trp433*) mutation (PP4). Therefore, further research into this mutation is of significant importance.

Taken together, we evaluated a four-generation family with CFD for disease-causing mutations using WES and Sanger sequencing and attempted to identify a correlation of the genetic variant with fibrinogen levels and clinical presentation. Across the four consecutive generations, we identified a heterozygous nonsense mutation in exon 8 of the FGB gene (c.1299G > A), which leads to CFD. Our research further substantiates the significance of this mutation and broadens the genetic spectrum associated with the FGB gene, thus enhancing genetic counseling and prenatal genetic diagnosis. In individuals with fibrinogen deficiencies, future research should focus on enhancing diagnostic tools to obtain more comprehensive information regarding hemorrhagic tendencies and accurately predict clinical phenotypes.