The Silence Speaks, but We Do Not Listen: Synonymous c.1824C>T Gene Variant in the Last Exon of the Prothrombin Gene as a New Prothrombotic Risk Factor

Abstract

Background Thrombosis is a major global disease burden with almost 60% of cases related to underlying heredity and most cases still idiopathic. Synonymous single nucleotide polymorphisms (sSNPs) are considered silent and phenotypically neutral. Our previous study revealed a novel synonymous FII c.1824C>T variant as a potential risk factor for pregnancy loss, but it has not yet been associated with thrombotic diseases. Methods To determine the frequency of the FII c.1824C>T variant we have sequenced patients’ DNA. Prothrombin RNA expression was measured by quantitative PCR. Functional analyses included routine hemostasis tests, western blotting and ELISA to determine prothrombin levels in plasma, and global hemostasis assays for thrombin and fibrin generation in carriers of the FII c.1824C>T variant. Scanning electron microscopy was used to examine the structure of fibrin clots. Results Frequency of the FII c.1824C>T variant was significantly increased in patients with venous thromboembolism and cerebrovascular insult. Examination in vitro demonstrated increased expression of prothrombin mRNA in FII c.1824T transfected cells. Our ex vivo study of FII c.1824C>T carriers showed that the presence of this variant was associated with hyperprothrombinemia, hypofibrinolysis, and formation of densely packed fibrin clots resistant to fibrinolysis. Conclusion Our data indicate that FII c.1824C>T, although a synonymous variant, leads to the development of a prothrombotic phenotype and could represent a new prothrombotic risk factor. As a silent variant, FII c.1824C>T would probably be overlooked during genetic screening, and our results show that it could not be detected in routine laboratory tests.

Early-onset ischaemic stroke in a patient with the novel F2 c.1824C>T gene variant and PAI-1 4G/4G, MTHFR 677TT genotype

“…Thrombophilia testing showed that patient was homozygous carrier for PAI-1 4G/5G and MTHFR C677T mutations. Additional analysis revealed the presence of recently reported FII c.1824C>T gene variant in heterozygous state (10) and elevated plasma prothrombin level.…”

“…Taking into account that thrombophilia testing showed our patient is not a carrier of this variant, we decided to look into novel potential variants in FII gene associated with early IS onset and did sequencing of 715 bp within its 3` end, where a recently described FII c.1824C>T gene variant was detected. The FII c.1824C>T represents a synonymous FII variant leading to the CGC to CGT codon replacement, on the Arg608 position in the protein (10). The study by Pruner et al showed increased frequency of c.1824C>T gene variant in patients who suffered IS compared to healthy controls (4.8% vs. 0.9%), and 5.4-fold greater risk for IS occurrence in variant carriers (10), in vitro examination demonstrated that FII c.1824T allele is associated with elevated expression of prothrombin mRNA (10).…”

“…The FII c.1824C>T represents a synonymous FII variant leading to the CGC to CGT codon replacement, on the Arg608 position in the protein (10). The study by Pruner et al showed increased frequency of c.1824C>T gene variant in patients who suffered IS compared to healthy controls (4.8% vs. 0.9%), and 5.4-fold greater risk for IS occurrence in variant carriers (10), in vitro examination demonstrated that FII c.1824T allele is associated with elevated expression of prothrombin mRNA (10). In addition, ex vivo study indicated that FII c.1824C>T variant leads to hyperprothrombinemia, hypofibrinolysis and formation of denser and thinner fibrin fibers within the clot, which makes it resistant to fibrinolysis (10).…”

“…As additional control, plasma of symptomatic heterozygous carrier of FII G20210A mutation, suffered from deep venous thrombosis and plasma of healthy volunteer, who was non-carrier for all known mutations in the 3`end of the prothrombin gene (confirmed by direct sequencing), was used. The plasma dilution, protein transfer, membrane blocking, antibodies used for protein detection and protein quantification were performed as described previously (10). Results of Western blot analysis showed that examined stroke patient had elevated prothrombin level…”

“…Its unusual non canonical architecture makes the 3` end of prothrombin gene sensitive to gain-of-function mutations, which are associated with increased prothrombin expression (8). Recent studies reported two novel gain-of-function variants in this region of prothrombin gene, FII c.1787G>A (prothrombin Belgrade) and FII c.1824C>T, which are recognized as significant risk factors for the IS occurrence (9,10).…”