Missense or splicing mutation? The case of a fibrinogen Bβ-chain mutation causing severe hypofibrinogenemia

Fibrinogen is a plasma glycoprotein mainly synthesized by hepatocytes that circulates as a 340-kDa hexamer consisting of pairs of Aα, Bβ, and γ chains.¹  Its 3-dimensional structure is characterized by 2 lateral nodules (each composed of 2 globular subdomains) joined to a central nodule by coiled-coil triple-helix structures.^2,3  The Aα, Bβ, and γ chains are encoded by 3 different genes (FGA, FGB, and FGG, respectively) clustered on chromosome 4q28.⁴  Fibrinogen takes part in hemostasis by forming the fibrin clot and by mediating platelet aggregation. Moreover, it is a class II acute-phase reacting protein.⁵ 

Congenital fibrinogen defects comprise type I deficiencies (afibrinogenemia and hypofibrinogenemia), with low or not measurable antigen levels, and type II deficiencies (dysfibrinogenemia and hypodysfibrinogenemia), showing normal or altered antigen levels associated with reduced coagulant activity. While dysfibrinogenemias are autosomal dominant diseases, type I deficiencies are inherited as autosomal recessive traits. Patients affected by congenital afibrinogenemia (Mendelian Inheritance in Man, [MIM] #202400) or severe hypofibrinogenemia (MIM ^*134820, ^*134830, and ^*134850) may experience bleeding manifestations varying from mild to catastrophic.⁶  The genetic basis of these rare coagulation disorders are invariantly represented by mutations within the fibrinogen genes.⁷ 

In this study, we analyzed the genetic defects underlying severe hypofibrinogenemia in a proband originally described in 1972.⁸  The patient was found to be a compound heterozygote for a previously reported nonsense mutation and a novel missense mutation. The latter was demonstrated to exert its pathogenetic effect at the messenger RNA (mRNA) processing level by activating a cryptic acceptor splice site.

The patient is a 57-year-old Italian woman with consistently moderate hemorrhagic symptoms. Plasma fibrinogen concentration was re-evaluated by a functional assay based on fibrin polymerization time⁹  and by an enzyme-linked immunosorbent assay (ELISA).¹⁰  Resulting levels (0.2941 μM [10 mg/dL] and 0.414681 μM [14.1 mg/dL] for functional and immunoreactive fibrinogen, respectively; normal range for both tests, 4.7056-11.764 μM [160-400 mg/dL]) were concordant with previous data,⁸  confirming the diagnosis of severe hypofibrinogenemia.

The patient signed an informed consent according to the Declaration of Helsinki. The study was approved by the institutional review board of the University of Milan. All procedures were performed as described.¹¹ 

Expression of wild-type and mutant fibrinogen was achieved by transiently cotransfecting COS-1 cells with plasmids containing the cDNAs for the 3 fibrinogen chains as described.¹²  Pulse-chase experiments, immunoprecipitations, sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), and evaluation of fibrinogen antigen levels in conditioned media and cell lysates by ELISA were performed as described.¹² 

A 2125-bp fragment of FGB (introns 2 to 5), PCR-amplified from the proband's genomic DNA using the primer couple FGB-3F 5′-GTCCATGACCCAAATCCTTC-3′ and FGB-5R 5′-TGTTTCCTGGCATTTTACGG-3′, was ligated into the mammalian expression vector ptarget (Promega, Milan, Italy) as described.¹¹  Insert orientation and distinction between wild-type (pT-Bβ-In2/In5-wt) and mutant (pT-Bβ-In2/In5-5157T>A) constructs were accomplished by sequencing. Plasmids were independently transfected in HeLa cells (not expressing fibrinogen) by the calcium-phosphate technique.¹¹  One microgram of total RNA extracted 48 hours after transfection (RNAWIZ reagent; Ambion, Austin, TX) was reverse transcribed (Enhanced Avian RT-PCR Kit; Sigma, St Louis, MO), and one tenth of the reverse-transcriptase (RT) reaction was used as template to amplify wild-type or mutant transcripts with the exonic primers FGB-Ex3F 5′-CCTACAGGATGTCAGTTGC-3′ and FGB-Ex5R 5′-AACTGTCAGGTTGAATGAGAT-3′. Quantitative evaluation of transcripts by the fluorescent hot-stop PCR technique was performed as described.¹¹ 

DNA sequencing of the fibrinogen cluster revealed the patient to be a compound heterozygote for 2 point mutations in FGB. One mutation was a novel T>A transversion in exon 4 at position 5157 (numbering according to GenBank M64983), predicted to cause the Leu172Gln substitution (numbering omits the signal peptide). The other mutation was a C>T transition at position 3282 in exon 2, causing a nonsense mutation at residue 17. This mutation (Arg17Stop) was previously reported in the homozygous state in an Iranian afibrinogenemic patient.¹³ 

While the causal role of Arg17Stop was self-evident, the pathogenetic role of Leu172Gln required further characterization. First, its absence was checked in 200 haploid genomes from unrelated Italian control individuals. Second, given the unavailability of other family members for segregation analysis, the phase of association of Leu172Gln and Arg17Stop was determined by cloning a 2139-bp region of FGB (nucleotides 3185-5323) from the proband and sequencing 10 recombinant clones. All clones contained either Leu172Gln or Arg17Stop, indicating that the 2 mutations are located on different alleles. Leu172 is located in the second half of the coiled-coil region, whose conserved hydrophobic residues are predicted to play a role in fibrinogen assembly.¹⁴  Although the Leu172Gln substitution is nonconservative (hydrophobic to polar), its impact on fibrinogen assembly and folding is uncertain, since the side chain of Gln172 is comparable with that of the wild-type Leu172 and therefore could fit into the hydrophobic core of the coiled-coil region of fibrinogen.

To confirm the causal role of Leu172Gln, in vitro expression experiments were performed by transiently coexpressing the mutant Bβ protein with the wild-type Aα and γ chains in COS-1 cells, which do not express fibrinogen (Figure 1A-B). The 340-kDa fibrinogen band, corresponding to the hexameric molecule, was detectable in both wild-type and mutant samples as well as in control fibrinogen-expressing HepG2 cells, whereas it was absent in mock-transfected cells. These data (confirmed by densitometric quantitation of fibrinogen bands; Figure 1A-B right) demonstrated a normal intracellular assembly and secretion of Bβ-Leu172Gln fibrinogen. Fibrinogen levels in both conditioned media and lysates of cells transfected with the mutant plasmid were also evaluated by ELISA and they were even slightly higher than those measured in wild-type samples (Figure 1C), thus convincingly confirming that Leu172Gln does not interfere with fibrinogen synthesis, intracellular processing, and secretion.

Even though Leu172Gln had no apparent effect at the protein level, it still remained a good candidate as causative mutation. Actually, no other putative pathogenetic mutation was found in the sequenced regions of the fibrinogen cluster and the sole presence of Arg17Stop in the proband was not sufficient to account for severe hypofibrinogenemia. In fact, this same mutation was previously reported to be associated with mildly reduced fibrinogen levels in both heterozygous parents of an Iranian afibrinogenemic patient.¹³ 

A possible effect of Leu172Gln at the RNA level was therefore postulated. A closer inspection of the nucleotide sequence surrounding the mutation revealed that it determines the formation of a sequence (CAGG) that might act as a cryptic acceptor splice site (Figure 2B), causing the skipping of the first 116 nucleotides of exon 4 from the mature transcript.

To test whether Leu172Gln (hereafter referred to as 5157T>A) was itself a splicing mutation, part of the Bβ transcript (either wild type or containing 5157T>A) was produced in HeLa cells by transient transfection with 2 minigene constructs (pT-Bβ-In2/In5-5157T>A and pT-Bβ-In2/In5-wt; see “Study design” and Figure 2A). RT-PCR on total RNA from cells transfected with pT-Bβ-In2/In5-5157T>A gave rise to a major product (shorter than expected; Figure 2B) and a minor 466-bp product corresponding to the wild-type one, suggesting low levels of residual wild-type splicing. Sequencing revealed that the shorter fragment (350 bp) resulted from the use of a cryptic acceptor splice site generated by the 5157T>A mutation (Figure 2B). This would result in a frameshift and a premature stop at residue 146 of the mutant protein, preceded by a scrambled sequence of 12 residues.

Therefore 5157T>A represents the first exonic splicing mutation identified so far in the fibrinogen genes. A residual wild-type splicing of approximately 10% was calculated by the fluorescent hot-stop PCR technique¹¹  (Figure 2C). Assuming that this result does mimic the in vivo situation, it might explain the low but measurable level of immunoreactive fibrinogen in the proband's plasma.

These findings strengthen the concept that no safe prediction of phenotypes of mutations is feasible until an accurate functional analysis at the protein and/or RNA level has been performed.