Homozygosity of the T Allele of the 46 C→T Polymorphism in the F12 Gene Is a Risk Factor for Ischemic Stroke in the Spanish Population
Background and Purpose— Ischemic stroke (IS) is a complex disease that involves genetic and environmental factors. In a family-based study (the Genetic Analysis of Idiopathic Thrombophilia [GAIT] Project) that included a genome-wide scan, we demonstrated that a common polymorphism (46 C→T) in the exon 1 of the F12 gene jointly influences variability of plasma Factor XII levels and susceptibility to thrombotic disease. We have investigated the risk of IS related to this polymorphism in a case–control study.
Methods— We studied 436 individuals: 205 diagnosed with IS and 231 age–gender–ethnic control subjects. We measured Factor VIIIc, fibrinogen, and Factor XIIc levels, and we genotyped the 46 C→T polymorphism in the F12 gene.
Results— There were 91 women and 114 men in the IS group and 109 women and 122 men in the control group. We confirmed our previous observation that individuals with different genotypes for the 46 C→T polymorphism showed significant differences in Factor XIIc levels. Most importantly, the mutated T allele in the homozygous state (genotype T/T) was associated with an increased risk of IS with an adjusted odds ratio of 4.1 (95% CI, 1.1 to 15.9).
Conclusions— This study suggests that the 46 C→T polymorphism is a genetic risk factor for IS in the Spanish population. In addition, our results confirm that the use of genetic linkage studies along with a case–control association study is an extremely valuable approach for identifying DNA variants that affect complex diseases.
As a result of the success of the Human Genome Project to identify genes that influence the pathogenesis of diseases, there has been a renewed interest in the knowledge of new, putative “stroke genetic risk factors.”1 Current research on complex diseases, such as ischemic stroke (IS), now focuses on identifying genetic variants that increase the susceptibility to thrombotic disorders.2–4
Ischemic stroke is a complex disease resulting from the interaction between environmental and genetic factors.5–7 Although results are controversial, some hemostatic factors8 including Factor XII have been associated with the risk of arterial thrombotic disease, including IS.9–11
Factor XII is a plasma protein and a member of the serin-protease family.12–14 Once activated, it initiates the contact activation system; it also has fibrinolytic properties.15 Severe congenital Factor XII deficiencies have been described, but its association with thromboembolic complications is controversial.10 Some studies have reported that variability of plasma Factor XII levels are associated with the predisposition to arterial thrombosis.13,14 A common genetic polymorphism (46 C to T substitution) in the 5′-untranslated region of the coagulation Factor XII gene has been associated with low translation efficiency and variability of Factor XII levels in the general population.12
Association studies have shown the relationship of Factor XII levels and the 46 C→T polymorphism in the development of arterial disease, but the results are controversial.13,14,16–18 To our knowledge, only 1 study has explored the association between IS and this polymorphism.8
Association studies to determine the risk of thrombosis related to a polymorphism must be done with great caution.19,20 New guidelines have been provided in 2 editorials.21,22 They contain recommendations on the desirable features of a genetic association study. If these guidelines are followed, association studies can be powerful tools to provide information about the prevalence of genetic risk factors and their contribution to the development of IS or other thombotic diseases.
We followed these guidelines in our study. We took into account the results from our GAIT (Genetic Analysis of Idiopathic Thrombophilia) Project, a family-based study.23–25 The GAIT Project, with the benefit of the results of a genome-wide scan, demonstrated a substantial overlap in the genetic contribution to venous and arterial thrombosis.23 It is safe to conclude that there are common genes involved in the pathogenesis of both venous and arterial thromboses. Also, within the framework of the GAIT Project, we demonstrated that a polymorphism in exon 1 of the F12 gene (a C→T transition at nucleotide 46) jointly influences Factor XII levels and the risk of thrombosis.25 To strengthen these results, we constructed an age–gender–ethnic-matched case–control study of an independent sample of Spanish individuals with IS.
Materials and Methods
Our population-based case–control study consisted of 205 patients diagnosed with IS and 231 age–gender–ethnic-matched control subjects. Patients with IS were admitted to the Stroke Unit of our hospital between 1998 and 2003. The patients were aged 20 to 80 years and each had at least 1 episode of IS. The stroke patients were enrolled in our study at least 1 month after the acute event.
The diagnosis and classification of the different types of IS were based on an extensive diagnostic evaluation, including findings of medical history, general clinical and neurological examination, laboratory investigation, CT or MR imaging of the brain, cerebrovascular imaging (extracranial carotid and vertebral ultrasonography, carotid transcranial Doppler ultrasound, MR angiography, or conventional cerebral angiography), and also transesophageal or transthoracic echocardiography. Patients were classified into different stroke categories based on criteria in the Trial of Org 10172 in Acute Treatment (TOAST) study.26 Cardioembolism, small vessel disease, large vessel atherosclerosis, and cryptogenic stroke were included; strokes of unusual causes were excluded.
Control subjects were friends and spouses of patients who were recruited at the same time as the patients; they were included only if they had no personal history of thromboembolic disease, including venous and arterial thrombosis, cirrhosis, nephrotic syndrome, or active cancer. In addition, they were excluded if they were using oral anticoagulants. The control group was matched for age and sex with the patient group. To avoid genetic stratification, both groups were collected from the same geographical area (all with both family names of Spanish origin).
Patients and controls gave informed consent. The interview included questions on personal and family history of cardiovascular diseases; conventional cardiovascular risk factors such as hypertension, diabetes, and hypercholesterolemia; if they had ever been diagnosed by a physician as such; or were currently taking prescription drugs for these conditions. Also, we recorded other cardiovascular risk factors such as smoking, alcohol intake, and morbid obesity. Average alcohol intake >300 g per week was considered significant and was recorded as such; alcohol intake <300 g per week was recorded as nonsignificant intake. Morbid obesity was recorded if the body mass index (calculated as body weight (kg) divided by height squared (m2) was ≥30 kg/m2. A subject was considered morbidly obese when body mass index was >30 kg/m2 and not morbidly obese when body mass index was <30 kg/m2. Smokers were defined as persons who were smoking at least 5 cigarettes per day during the 5 years before the study; a subject was considered a nonsmoker if <5 cigarettes per day were smoked. The family history was positive if at least a first- or second-degree family member had venous or arterial thrombosis including IS or acute coronary artery disease.
Blood samples were obtained from the antecubital vein at least 1 month after the acute episode. Samples for hemostatic tests were collected in 1/10 volume of 0.129 mol/L sodium citrate. Fibrinogen was measured by Clauss method as described elsewhere. Assays for Factor VIII were performed immediately on fresh plasma samples; the remaining plasma samples were stored at −80°C until used. Factor VIIIc and Factor XIIc were assayed using deficient plasma from Diagnostica Stago.
DNA was isolated from peripheral blood leukocytes by a standard protocol.27 The 46 C→T variant was screened using the primers described previously,12 with minor modification in the reaction conditions.25
Statistical analyses were performed using SPSS software. The values are expressed as mean±SD. Student t test was used to calculate the mean differences between groups. Conventional cardiovascular risk factors were considered as dichotomous covariables. We performed the χ2 test for group comparison of the frequencies. P<0.05 were considered significant. According to receiver operating characteristic (ROC) curves, we considered elevated Factor VIII levels if >176% and elevated fibrinogen levels if >3.5 g/L. To determine the adjusted odds ratio (OR) of IS, we converted the Factor VIII and the fibrinogen levels in binary variables using the above mentioned values as cutoffs. We analyzed the Factor XII as a dichotomous variable. This problem occurs frequently in the hemostatic factors because of its complex and paradoxical hemostatic effects. That is why we often use dichotomized variables to establish the risk associated with thrombosis. For Factor XIIc, we used levels lower than the 10th percentile (<74%) as a cutoff.
The plasma levels of Factor XIIc were compared among different genotypes of 46 C→T by 1-way ANOVA. ORs were calculated as relative risk for IS adjusted for age, sex, and other covariables by logistic regression. For analysis we considered 2 combinations, one considering the genotypes C/C and C/T as the reference group and another considering the genotype C/T and T/T as the reference group.
The basic characteristics of the sample population are given in Table 1. Mean age at the time of the first episode of IS was 56 years (range, 23 to 80). The mean age of the controls was 54 years (range, 21 to 80). Ninety-one women and 114 men made up the IS group; 109 women and 122 men, the control group. All of the major cardiovascular conventional risk factors, such as hypercholesterolemia, hypertension, or diabetes mellitus, were associated with a significant increased risk of IS. Only smoking and morbid obesity were not associated with an increased risk of IS. Patients with positive family histories of arterial thrombosis showed an increase risk of IS (4.5; 95% CI, 2.9 to 6.9). Factor VIIIc (mean 187.9%; range, 62 to 516) and fibrinogen levels (3.7 g/L; range, 1.9 to 8.4) were increased significantly in patients compared with the controls (155.5%; range, 48 to 360; and 3.5 g/L; range, 2.1 to 7.8). The levels of Factor VIIIc >176% and fibrinogen levels >3.5g/L were associated with a higher risk of IS (2.2; 95% CI, 1.5 to 3.2; P<0.001; and 1.9; 95% CI, 1.2 to 2.9; P<0.01, respectively). Cryptogenic stroke was diagnosed in 107 patients. Cardioembolism was diagnosed in 26 patients and small vessel disease in 39 patients. Large vessel atherosclerosis was diagnosed in 33 patients. Genotype T/T was found in 6 patients with cryptogenic stroke, 1 patient with cardioembolism, 2 patients with small vessel disease, and 3 patients with large vessel atherosclerosis.
The distribution of the 46 C→T polymorphism of the F12 gene are shown in Table 2. The prevalence of genotype C/C was ≈62% in patients and 61% in controls. The prevalence of genotype C/T was ≈33% in patients and 38% in controls. The differences of the prevalences were not statistically significant. In contrast, the prevalence of genotype T/T was almost 6% in patients and <2% in controls. Here the difference was statistically significant (P<0.001). The frequency of the 46 C/T polymorphism was in the Hardy–Weinberg equilibrium. This frequency is included in the confidence interval of our controls in the Spanish population (2%; 95% CI, 0.7 to 4.6).24 There were no statistically significant differences in mean values of plasma Factor XIIc levels between patients (112.3±35.1) and controls (114.6±25.9) (data not shown). The distribution of plasma Factor XIIc levels according to the genotype is shown in the Figure. The Factor XIIc levels, analyzed as a function of the 46 C→T polymorphism, showed statistically significant differences between the different genotypes (P<0.0001). Genotype T/T showed the lowest levels of Factor XIIc (57.9%; range, 43 to 94) compared with the levels of the other genotypes (C/C genotype: 127.6%; range, 62 to 250 and C/T genotype: 95.2; range, 46 to 175).
The risk of IS associated with plasma Factor XIIc plasma levels and with C/C, C/T, and T/T genotypes are shown in Table 3. The crude OR of IS associated with low levels of Factor XIIc (<74%) in patients compared with controls was 2.7 (95% CI, 1.4 to 5.2). When we adjusted for sex, age, and other covariables, such as Factor VIIIc, hypercholesterolemia, hypertension, diabetes, and fibrinogen, the OR was 2.9 (95% CI, 1.3 to 6.2) for IS patients compared with the controls.
We studied the risk of IS associated with the genotype C/C. The crude OR of IS associated with the 46 C→T polymorphism for the genotype C/C was 1.0 (95% CI, 0.7 to 1.5). When we analyzed the risk adjusted for sex, age, and other covariables, such as Factor VIIIc, smoking, hypercholesterolemia, hypertension, diabetes, and fibrinogen, the OR was 1.0 (95% CI, 0.7 to 1.5) for IS patients compared with the controls. We also studied the risk of IS associated with the genotype C/T. The crude OR of IS associated with the 46 C→T polymorphism for the genotype C/T was 1.3 (95% CI, 0.9 to 1.9). When we analyzed the risk adjusted for sex, age, and other covariables, the OR was 1.2 (95% CI, 0.8 to 1.9) for IS patients compared with the controls. The crude OR of IS associated with the 46 C→T polymorphism for the T/T genotype was 4.7 (95% CI, 1.3 to 16.9). When we analyzed the risk adjusted for sex, age, and other covariables, such as Factor VIIIc, smoking, hypercholesterolemia, hypertension, diabetes, and fibrinogen, the OR was 4.1 (95% CI, 1.1 to 15.9) for IS patients compared with the controls. This suggests that genotype T/T is an independent risk factor for IS.
Our case–control study showed an adjusted OR of 4.1 (95% CI, 1.1 to 15.9) for IS associated with the genotype T/T of the 46 C→T polymorphism, and confirms and extends the previous observation that individuals with different genotypes for the 46 C→T polymorphism show statistically significant differences in their plasma Factor XIIc levels.24 Also, in our study, conventional cardiovascular risk factors such as hypertension and diabetes, were associated with the risk of IS. Hemostatic risk factors, such as fibrinogen and Factor VIIIc levels, also increase the risk of IS. These results agree with those of other studies.7,8
Several studies have reported an association between the 46 C→T polymorphism and cardiovascular disease, ie myocardial infarction. However, this association was not confirmed in subsequent studies.16,17 Only 1 study in a Japanese population has explored this association with IS, and there was no statistically significant association of this polymorphism with the risk of IS.9
Our study is unique because our strategy was designed to avoid the well-known biases of association studies with regard to establishing a polymorphism as a risk factor for the development of thrombotic disease.20 From the GAIT Project (a family-based study), we knew that there was a linkage between the 46 C→T polymorphism in the F12 gene and the thromboembolic disease, including cardiovascular disease.24 We demonstrated a high heritability (h2: 67%) of Factor XII and we reported also that the structural F12 gene influenced both susceptibility to thrombosis and Factor XII plasma levels.24,25 Following these results, we were able to design an age–gender–ethnic-matched case–control study in an independent population that replicated the GAIT Project results.
The small sample, which is constricted by the fact that only survivors of IS are included in this case–control study, is one of the limitations and a major problem in many cardiovascular case–control studies, but only prospective studies shed some light on this inherent design problem. Despite these provocative results, the role of Factor XII levels in the development of IS is still not well understood. It has been suggested that activated Factor XII has a pivotal role in several pathways concerned with tissue defense and repair, including the initiation of the intrinsic pathways in blood coagulation. It acts as a potent activator of plasminogen14 and contributes to fibrinolysis.15 It has been hypothesized that Factor XII is implicated in the pathogenesis of atherosclerosis. It appears that human endothelial cells possess receptors for Factor XII, and Factor XII is activated on contact with biological surfaces.11 It may be that low levels of Factor XII reflect vascular lesions and a lower fibrinolysis activity. Both mechanisms of action might increase the risk of IS.14,15 In our study, we found that low levels of Factor XIIc (<74%) increased the risk of IS. These data are in agreement with the observed risk of thromboembolic complications in some patients with Factor XII deficiency but not in other patients.10 It is well-known that the development of IS as a complex disease depends on the interaction of multiple environmental and genetic factors. This complexity may explain the liability to develop IS in some patients with low levels (including severe deficiencies) of Factor XII levels. It is clear that further studies are needed to elucidate the role of Factor XII levels in IS.
In conclusion, we found a 4-fold higher risk of IS associated with the homozygosity of the T allele of the F12, 46 C→T polymorphism in the Spanish population. We hope that this study along with others will help assess the feasibility of developing new strategies to prevent IS and to develop therapeutical approaches.
We are indebted to Professor W.H. Stone for his help in discussing and revising the manuscript.
- Received October 1, 2004.
- Revision received April 13, 2004.
- Accepted April 29, 2004.
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