EARLY-ONSET ISCHAEMIC STROKE IN PATIENT WITH NOVEL F2 C.1824C>T GENE VARIANT AND PAI-1 4G/4G, MTHFR 677TT GENOTYPE

Background/Aim. Ischemic stroke is a heterogeneous disorder caused by several genetic and environmental risk factors. It was suggested that coagulation disorders cause 1-4% of cases with ischemic stroke, especially in patients with early-onset of ischemic stroke. Case report. Here, we describe a case of patient who developed an unprovoked ishemic stroke in young adult. Biochemical, immunological and thrombophilia screening, as well DNA sequencing were performed in order to reveal molecular pathology underlying stroke of patient. Thrombophilia testing showed that patient was homozygous carrier for PAI-1 4G/5G and MTHFR C677T mutations. Additional genetic analysis revealed the presence of recently reported FII c.1824C>T gene variant, which is located in the last exon of prothrombin gene and previously shown to cause hyperprothrombinemia, hypofibrinolysis and altered fibrin clot phenotype. Conclusion. Our results suggest that newly reported FII c.1824C>T gene variant might have synergistic effect with PAI 4G/4G and MTHFR 677TT genotype in formation of altered fibrin clot phenotype characterized by thin, densely packed fibrin fibers, which makes clot less susceptible to fibrinolysis and greatly increases the risk for early ischemic stroke onset.


Introduction
Ischemic stroke (IS) is a heterogeneous disorder that counts for 3/4 of all stroke cases and could be provoked by multiple risk factors, both genetic as well as environmental (1, 2). A great number of genetic mutations has been shown to have a role in the etiology of several subtypes of stroke (2), but it is still challenging to identify specific causative mutations (3). Studies performed on twins and siblings, shown that IS onset is greatly affected by inherited risk factors (4). Majority of genetic factors for IS have rather polygenic than monogenic influence. Additionally, IS could have a wide range of phenotypes, which could differ in their genetic background. Almost all human studies to date have employed a candidate gene approach (4).
Previous studies have suggested that coagulation disorders are the major cause of only a 1% to 4% of all IS, but may be of relevance for the pathogenesis of subgroups of stroke patients such as strokes in young ones (5,6). The perturbation of the coagulation cascade, due to the gene variants found in several genes involved in hemostasis (fibrinogen, prothrombin, FV, FVII, FXIII, thrombomodulin, PAI-1, TAFI) are associated with increased coagulability and thrombotic risk (4). The most frequently studied genetic variants in the pathogenesis of IS are FV G1691A (FV Leiden), FII G20210A and the MTHFR C677T (7).
One of the potential candidate genes involved in the pathogenesis of IS is prothrombin (FII) gene. 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).
We present a case of the patient who suffered from early-onset unprovoked IS. Since the cause of this IS remained unknown after routine testing, we performed additional genetic analyses which revealed that patient is heterozygous carrier of the recently described FII c.1824C>T gene variant.

Case report
The patient, 42-year-old, right-handed male, nonsmoker, was initially presented to emergency department of the local hospital due to the sudden onset of left-sided weakness.
Upon the arrival, he was alert and cooperative, while left facial palsy and severe left-sided hemi paresis were noted. Findings of the other neurological examination was normal.
Computed Tomography (CT), performed on admission, revealed no evidence of acute cerebral infarction or any other brain pathology (Figure 1 stroke was unknown we performed routine PCR tests which included genotyping for FV Leiden, FII G20210A, MTHFR C677T and PAI-1 4G/5G mutations. PCR tests showed that patient is homozygous carrier of PAI-1 4G/5G and MTHFR C677T mutations, non-carrier for FV Leiden and FII G20210A mutations. As it was previously shown that certain gain of function variants within 3` end of the FII gene could be associated with early onset of IS (10), we performed additional analysis: DNA sequencing of this region in FII gene and screening for variants, as well as determination of patients plasma prothrombin level by Western blot analysis. DNA sequencing analysis, performed as described previously (11)

Discussion
Here we present a patient with early-onset ischemic stroke of unknown etiology.
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. Impaired fibrinolysis and decreased fibrin network permeability are shown to represent substantial prothrombotic mechanisms contributing to the IS onset (11). The PAI-1 (plasminogen activator inhibitor 1) has a crucial role in the inhibition of two types of plasminogen activatiors, tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (u-PA), thus representing one of the most potent inhibitors of plasma fibrinolytic activity (12). The common PAI-1 4G/5G polymorphism within the promoter region of PAI-1 gene influences transcription of the gene and expression level of PAI-1 protein. The additional guanine in the DNA strand of the promoter region (5G allele) creates a repressor protein binding site that is absent in 4G allele. This reflects on PAI-1 expression and its plasma concentrations with 4G homozygotes having the highest and the 5G homozygotes the lowest PAI-1 concentrations (13). High levels of PAI-1 in 4G/4G carriers lead to suppressed fibrinolysis and consequent pathological fibrin deposition and tissue damage (14). The results on the association of PAI-1 4G/5G polymorphism with IS are conflicting, however the meta-analysis which included population-based association studies from 1966 up to 2006 showed that PAI-1 4G/4G genotype is likely to be associated with IS (15).
Elevated plasma homocysteine (hiperhomocysteinemia) and homozygosity for MTHFR C677T variant has been associated with the increased risk for IS, however the exact mechanism is not fully determined (16,17). MTHFR (Methylenetetrahydrofolate Reductase) is a key enzyme in the folate pathway and homocysteine conversion to methionine. The presence of C677T variant leads to reduced enzyme activity and remethylation of homocysteine to methionine, resulting in elevated homocysteine plasma levels (17). Homocysteine or its metabolites interact with plasma coagulation proteins and affect their function in vivo (16). It has been shown that elevated homocysteine level alters coagulation factor V in vitro and inhibits its cleavage by activated protein C (18).
Hyperhomocysteinemia might also promote fibrinogen modification thereby impairing activity of fibrinolytic enzymes and fibrin polymerization. This results in altered fibrin clot structure, composed of thinner and tightly packed fibers resistant to fibrinolysis (16), which correlates with the fibrin clot phenotype observed in IS patients (17).
The third most common genetic risk factor associated with IS are gain of function variants in FII gene, which cause elevated prothrombin level, hypercoagulability and tendency towards hyper production of fibrin clot (19,20). One of the most commonly tested prothrombotic variants in IS patients is FII G20210A mutation (19,21). 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. which leads to development of a prothrombotic phenotype (10). In this case study, we did not investigated the fibin clot structure and thickness of fibrin fibers in our IS patient as did the Undas et al (22). However, electron microscopy scanning of fibrin clot structure from FII c.1824C>T carriers, FII G20210A and healthy noncarriers by Pruner et al, revealed that fibrin fibers in plasma are denser and thinner in case of FII c.1824C>T compared to clots in FII G20210A and healthy noncarriers (10).
Fibrin clot phenotype is dictated by number of variables, involving pH, ionic strength, and concentrations of calcium, fibrinogen (23). However, thrombin concentration during clot formation has a crucial role in the density and stability of fibrin clot. Higher thrombin concentrations produce thin fibrin fibers that are densely packed, less susceptible to fibrinolysis and associated with thrombosis, whereas lower concentrations lead to production of thick, loosely-woven, permeable clots and bleeding disorders (23). Fibrin clot structure differs in terms of stability depending on the type of stroke, characterized as clots more prone to lysis in acute intracerebral hemorrhage, as opposed to more stable clots in the case of IS (24). Taking into account that our IS patient had elevated prothrombin level and previous results that FII c.1824C>T variant leads to the production of denser clots (10), we could hypothesize that this mechanism of clot formation is involved in the pathogenesis of early onset IS, but further studies concerning the association of fibrin clot phenotype and early onset IS are necessary.
Genetic predisposition for cerebral ischemia may result from an additive effect of several genes or from synergistic co-effects. Several studies have shown that PAI-1 4G/4G and MTHFR 677TT genotype could affect the fibrin clot microstructure. High levels of PAI-1 lead to suppressed fibrinolytic activity, while high homocysteine levels modify fibrinogen to be more resistant to cleavage by fibrinolytic enzymes (14,16,25,26).
Altogether, the conjunct effect of PAI-1 4G/4G and MTHFR 677TT genotypes observed in our patient might influence the susceptibility of fibrin clot to lysis and cause reduced clot permeability.

Conclusion
Based on our findings, we hypothesize that PAI-1 4G/5G and MTHFR C677T variants in synergy with hyperprothrombinemic and, hypofibrinolytic FII c.1824C>T variant, could lead to formation of altered fibrin clot phenotype characterized by densely packed, fibrinolysis resistant fibrin fibers, which could contribute to the early-onset ischemic stroke in our patient.