Abstract
There are limited data on the influence of genetic polymorphisms in atrial fibrillation (AF) stroke risk. We hypothesized that a functional haemostatic polymorphism, that is, the factor VII −323 Del/Ins polymorphism, would influence the prothrombotic state associated with AF, as well as stroke risk. Other functional polymorphisms were also tested.
Methods. We performed a cross-sectional study of 119 AF patients, who were compared to 96 patients with stroke secondary to AF. In the first patient group, we analysed plasma prothrombin fragment 1+2 levels (F1+2, an index of thrombin generation) to reflect the prothrombotic state of AF.
Results. AF patients carrying the −323 Ins allele had lower plasma F1+2 levels (P=0.015). After multivariate analysis adjusted by age, sex and clinical risk factors, advanced age and 807C/T polymorphism of glycoprotein Ia (GPIa) gene were associated with higher risk of ischaemic stroke (OR: 1.06; P=0.003 and OR: 1.91; P=0.025), whilst FVII Ins −323 allele was associated with lower stroke risk (OR: 0.41; P=0.017).
Conclusion. FVII −323 Ins allele may modulate the prothrombotic state associated with AF. Despite the small sample size, we found that FVII Ins −323 allele could be associated with a lower stroke risk in AF, whereas the 807C/T polymorphism may increase the risk.
Introduction
Atrial fibrillation (AF) is associated with an increased risk of stroke and thromboembolism, but this risk is not homogeneous Citation1. AF confers a prothrombotic or hypercoagulable state, which may contribute to the risk of stroke and thromboembolism associated with this arrhythmia Citation2, Citation3.
We previously reported that elevated levels of prothrombin fragment 1 + 2 (F1 + 2, an established index of thrombin generation) were independently associated with the presence of AF Citation4. However, it is unclear if F1 + 2 has added value in predicting stroke. There are also limited data on the influence of genetic polymorphisms on the stroke and thromboembolic risk associated with AF Citation5. Indeed, a few studies have linked genetic polymorphisms to thromboembolic risk secondary to AF, with both positive and negative results Citation6–9. In the Stroke Prevention in Atrial Fibrillation III (SPAF III) study, factor V Leiden was not associated with thromboembolic risk, although there was an independent association with raised F1 + 2 levels Citation10. Of note, we recently described that a factor XIII V34L polymorphism could be associated with the prothrombotic and inflammatory states in AF, as assessed by tissue factor and interleukin-6 levels, respectively Citation11.
Key messages
The FVII Ins −323 allele, associated with reduced plasma FVII levels, may confer a lower ischaemic stroke risk in atrial fibrillation (AF).
The FVII Ins −323 allele may influence stroke risk in AF via modulation of the prothrombotic state associated with AF.
Other polymorphisms could increase the risk of suffering a cardioembolic stroke in a manner independent of the prothrombotic state associated with AF.
A common 10-bp insertion in the factor VII (FVII) promoter region, the −323 decanucleotide Del/Ins polymorphism of the FVII gene, has been associated with a reduction of FVII coagulation activity by 20% Citation12 and activated FVII, by 27% Citation13. The FVII −323 decanucleotide Del/Ins polymorphism could also influence the variability of anticoagulation and has a role in the efficacy of the anticoagulant therapy Citation14. Those alleles, which are associated with low levels of FVII, may play a protective role against myocardial infarction Citation15. Conversely, the FVII −323 decanucleotide Del/Ins polymorphism appears to be associated with intracranial haemorrhage Citation16.
We firstly hypothesized that a functional haemostatic polymorphism, the FVII −323 decanucleotide Del/Ins polymorphism (that confers reduced levels of FVII), would influence the prothrombotic or hypercoagulable state associated with AF, and secondly, be related to ischaemic stroke risk. We tested the first hypothesis in a cross-sectional study of 119 consecutive patients with non-rheumatic AF (and no previous stroke or thromboembolism). To test our second hypothesis, these patients were compared to 96 patients with ischaemic stroke secondary to AF. We also tested other functional polymorphisms affecting genes that encode proteins of the haemostatic system that also influence the prothrombotic state and thromboembolic risk in AF patients.
Patients and methods
To test our first hypothesis relating the FVII −323 decanucleotide Del/Ins polymorphism to F1 + 2 levels, we recruited 119 consecutive patients with non-rheumatic AF lasting ≥4 weeks (as documented by electrocardiograms), who were referred by general practitioners or cardiologists to our anticoagulation clinic for the initiation of anticoagulation treatment with acenocoumarol, for primary prevention of stroke. None of the patients had previously taken anticoagulant therapy, although 56 were taking aspirin. Clinical risk factors for thromboembolism, such as age, female sex, hypertension, diabetes mellitus, heart failure, and previous stroke/thromboembolism, were recorded. All patients with recent (<3 months) venous thromboembolism, myocardial infarction, or acute coronary syndrome, as well as those with infection or inflammatory disease, surgery, malignancy, and renal/liver impairment (an increase in creatinine/liver enzymes value 2-fold the normal value of our laboratory), and those being treated with hormone replacement therapy or oral anticoagulation were excluded.
Echocardiography
Transthoracic M-mode, two-dimensional, and Doppler echocardiography (Hewlett-Packard SONOS 2500, California, USA) were performed in all AF patients. The same investigator performed all echocardiographic recordings, and the coefficient of variation for our laboratory was <5%. Echocardiographic measures were performed in the long parasternal and apical four-chamber axis (left ventricular end-diastolic and end-systolic diameters, left atrial diameter, ejection fraction, shortening fraction, and left ventricular mass index), according to the guide-lines laid down by the American Society of Echocardiography. The echocardiogram study confirmed the absence of significant cardiac valve disease. Special attention was paid to exclude other potential origins of the embolus (i.e. patent foramen ovale, interatrial aneurysm).
To test our second hypothesis, we compared the previous 119 patients above with 96 additional patients with ischaemic stroke secondary to AF, who were admitted to a neurology unit from the same health area. None of them were taking anticoagulation therapy, as the AF was discovered on admission. The same exclusion criteria of the first AF group were taken into account for this group. A cranial CT scan confirmed the stroke type; patients with haemorrhagic signs were excluded. A carotid ultrasound study was performed, and patients with a significant stenosis (>50%) in one the carotid arteries were excluded.
All recruited subjects gave their informed consent to enter the study, which was approved by the local Research Committee and was performed in accordance with the Declaration of Helsinki, as amended in Edinburgh in 2000.
Laboratory
Blood samples were drawn atraumatically and without stasis into syringes preloaded with trisodium citrate (0.011 M). Platelet-poor plasma fractions were obtained by centrifugation at 4°C for 20 minutes at 2,200 g (within 5 minutes after blood collection). Aliquots were stored at −40°C to allow batch analysis. F1 + 2 levels were assayed by an enzyme-linked immunosorbent assay (ELISA) using a commercial kit (Enzygnost F1 + 2 micro, Dade Behring Marburg GmbH, Marburg, Germany) and following the manufacturer's instructions. The inter- and intra-assay coefficients of variation were <5%. We determined levels of coagulant FVII (FVIIc) in a representative group of AF patients (36 Del/Del and 18 Del/Ins), using a commercial clotting assay with FVII-deficient plasma (Instrumentation Laboratory Co., MA, USA), both in an automated coagulometer (ACL 6000, Instrumentation Laboratory Co., MA, USA).
For the genetic analysis, in all AF patients (with and without thromboembolic events) blood samples were collected into tubes containing ethylenediamine tetra-acetic acid (EDTA) (Vacutainer, Becton Dickinson, Meylon, France). Total genomic DNA was extracted from whole blood based on the Wizard® genomic DNA purification system (Promega, Innogenetics, Madrid, Spain). The −323 decanucleotide Del/Ins polymorphism of the FVII gene, factor V Leiden, G20210A prothrombin (PT) polymorphism, FXIII V34L, human platelet alloantigen (HPA) 1b (GPIIIa L33P), and 807 C/T polymorphism (F224) of the GPIa gene were genotyped by polymerase chain reaction (PCR) as described elsewhere Citation14, Citation17–21.
Statistical analysis
Continuous variables were tested for normal distribution by Kolmogorov-Smirnov's test. Where data (e.g. F1 + 2 values) were not normally distributed, they were log-transformed before statistical analysis, although these are presented in the non-logarithmic format as median (interquartile range (IQR)). Data normally distributed are presented as mean (± standard deviation (SD)). Comparisons between two groups were performed by the unpaired t test (if relevant after log transformation). Categorical data were compared using the chi-square test, and a Fisher's exact test was performed if relevant. Correlations between the measured laboratory indices (if relevant after log transformation) and clinical and demographic data were performed using the Pearson correlation coefficient. The strength of the association of polymorphism and the occurrence of embolism was estimated by calculation of the odds ratio (OR) with EpiInfo software (CDC, Atlanta, Georgia, USA) and the Cornfield method for the calculation of 95% confidence intervals (CI). A multiple logistic regression analysis (using a stepwise method) was also undertaken with the presence of ischaemic stroke as dependent variable and clinical factors (advanced age, sex, hypertension, diabetes, heart failure, ischaemic heart disease, and FVII −323 decanucleotide Del/Ins polymorphism, and other polymorphisms) as independent variables. We included as independent variables in the model those that showed a P-value <0.150 in the unvaried analysis. All analyses were carried out using SPSS version 10.0 software (SPSS Inc., Chicago, IL, USA). A value of <0.05 was considered statistically significant.
Results
The genotypes in AF patients distribution was as follows: FVII −323 Del/Del 88 (74%), −323 Del/Ins 27 (23%), and −323 Ins/Ins 4 (3%), and the allelic frequencies of the −323 Del and −323 Ins alleles were 0.869 and 0.131, respectively, which is broadly similar to those reported in other Mediterranean populations Citation13, Citation22. There were no age- or sex-dependent differences in the frequency of these alleles, and the distribution of genotypes was not significantly different from Hardy-Weinberg proportions (data not shown).
Relationship of FVII genotype with prothrombin F1 + 2 and FVIIc levels
Clinical characteristics from the 119 consecutive AF patients are summarized in . There was a statistically significant difference in the proportion of hypertensive subjects between both allelic frequencies (P = 0.019). Patients carrying the −323 Ins allele had lower plasma FVIIc levels than patients carrying the −323 Del/Del genotype (P < 0.001), as previously reported Citation12. These patients also had significantly lower F1 + 2 levels (P = 0.015) (see ). There were no differences in F1 + 2 levels among patients who were taking aspirin or not (P = 0.495). F1 + 2 and FVIIc levels correlated significantly (r=0.465; P < 0.001). There were no significant correlations between research indices and the duration of the arrhythmia or echocardiographic parameters (data not shown).
Table I. Clinical and polymorphism data in atrial fibrillation patients.
The genetic frequencies of the rest of the polymorphisms and the values of F1 + 2 according to the presence of different alleles are resumed in . There were no statistically significant differences in F1 + 2 values according to any other tested polymorphism. Again, there were no age- or sex-dependent differences in the frequency of these alleles, and the distribution of genotypes was not significantly different from Hardy-Weinberg proportions.
Table II. Genotype distribution in first atrial fibrillation patients group (without ischaemic stroke) and F1 + 2 levels according to each allele.
Relationship of FVII genotype and ischaemic stroke
The 119 patients with no ischaemic stroke were compared with 96 AF patients who had suffered from a cardioembolic ischaemic stroke (). The prevalence of subjects carrying the −323 Ins allele (15.6%) was lower in patients without previous stroke/thromboembolism than AF patients with previous stroke/thromboembolism (26.1%) (P = 0.046). Accordingly, the −323 Ins allele was associated to a 2-fold reduction of the risk to develop an ischaemic stroke in AF (OR: 0.50, 95% CI: 0.25–0.99; P = 0.046). With other polymorphisms, only the 807 C/T polymorphism (Phe 224) of the GPIa gene showed a deleterious effect (OR: 1.75; 95% CI: 1.01–3.01; P = 0.045). We were not able to find any significant association between aspirin, the duration of the arrhythmia or echocardiographic parameters, and ischaemic embolism (data not shown).
Table III. Clinical characteristics from atrial fibrillation patients with and without ischaemic stroke.
After a multivariate analysis adjusted by age, sex, and clinical risk factors, advanced age and 807 C/T polymorphism of the GPIa gene were associated with a higher risk of ischaemic stroke (OR: 1.06, 95% CI: 1.02–1.10; P = 0.003 and OR: 1.96, 95% CI: 1.07–3.42; P=0.025), whilst the presence of the FVII Ins −323 allele was associated with a lower stroke risk (OR: 0.41, 95% CI: 0.20–0.85; P = 0.017).
Discussion
FVII levels (both coagulant activity and antigen) have a wide interindividual variation in plasma, and both genetic and environmental factors are responsible for such variation Citation23. The FVII gene has five polymorphic loci that may account for up to 30% of the variance in plasmatic FVII levels, one of them being the −323 decanucleotide Del/Ins polymorphism, which diminishes promoter activity by 33% Citation24, causing a reduction of both FVII antigen and coagulation activity Citation12, Citation13. High levels of FVII have been associated to prediction of death due to coronary artery disease Citation25, whereas congenital deficiencies predispose to spontaneous bleeding after surgery Citation26. Thus, elevated levels of FVII coagulation activity might provide a mechanism for precipitating thrombotic events, and, conversely, reduced levels of FVII coagulation activity would diminish the thrombotic process Citation16.
Renewed interest has arisen in the role of FVII levels in thrombotic disease Citation27. In two cross-sectional studies with patients with ischemic stroke, FVII -C122T and FVII -G401T polymorphisms (both related to variations of circulating levels of FVII) were associated with ischemic stroke Citation28, Citation29. One also analysed the −323 Del/Ins polymorphism, but did not find any significant association Citation29. Nevertheless, both studies were not performed in cardioembolic stroke. These studies and ours support the clinical relevance of FVII (and its functional polymorphisms) in ischaemic stroke occurrence. We had hypothesized that the hypercoagulable state described in AF, as demonstrated by elevated levels of plasma F1 + 2 Citation10, could be genetically modulated by the FVII −323 decanucleotide Del/Ins polymorphism. Indeed, we show that carriers of the −323 Ins allele had significantly lower levels of this marker of thrombin generation.
The C807T single nucleotide polymorphism of the GPIa gene has been shown to correlate with the platelet GPIa/IIa density Citation21, where the 807T allele expressed the highest receptor density and the common 807C allele the lowest receptor levels, whereas heterozygous individuals expressed intermediate levels. Several studies have supported the role of this polymorphism in thrombotic diseases Citation30, but a recent meta-analysis has shown the absence of a positive association between the C807T polymorphism and stroke, indicating that changes in GPIa surface receptor does not contribute to an increased thrombotic risk Citation31. In contrast, the study of several polymorphisms of the main platelet glycoproteins in cerebral haemorrhage did not show any association Citation32. In the present study, we find that the 807T polymorphism was associated with a higher risk of cardioembolic stroke, but it did not seem to have any significant influence on the prothrombotic state observed in AF. Abnormalities of platelet function have also been described in AF, as part of the prothrombotic state described Citation2.
The pathophysiology of stroke in AF is a multifactorial and complex process, although many strokes related to AF represent embolism of thrombus formed in the left atrium, mainly at the left atrial appendage. It has been suggested that tissue factor may have a critical role as a mediator of the prothrombotic state in AF Citation33. Of note, the factor XIII V34L polymorphism can be associated with the prothrombotic and inflammatory states in AF, as assessed by tissue factor and interleukin-6 levels, respectively Citation11. Tissue factor binds and acts on FVII, and the resulting complex activates factors IX and X, initiating the clotting cascade in vivo Citation34. Thus, patients with reduced levels of both activated and coagulant FVII would have diminished the coagulation activation process, resulting in lower F1 + 2 levels. In the SPAF III study Citation10, there was an independent association between raised levels of F1 + 2 and the presence of factor V Leiden, which is associated with activated protein C resistance and a thrombophilic state Citation17. However, neither factor V Leiden nor F1 + 2 levels were associated with thromboembolic risk in those relatively ‘low-risk’ AF patients. A few studies have linked genetic polymorphisms to thromboembolic risk secondary to AF, with both positive and negative results Citation6–9. For example, none have found any association of AF thromboembolic risk with the presence of factor V Leiden Citation8. Some controversy also exists over the association of the prothrombin G20210A mutation and the occurrence of systemic embolism in AF patients Citation7, Citation8. Finally, the α-fibrinogen Thr312Ala polymorphism may increase the susceptibility to embolization of intra-atrial clots Citation9. When testing the possible role of FVII polymorphism in AF thromboembolic risk, we studied a consecutive cohort of AF patients who had been admitted to hospital with a thromboembolic ischaemic stroke secondary to AF and compared them with AF patients without stroke. We found a lower prevalence of the FVII −323 decanucleotide Del/Ins polymorphism among patients without stroke with respect to patients with stroke, even after adjusting for clinical risk factors for thromboembolism, and this was linked to low F1 + 2 levels.
Our study is limited by its cross-sectional design, which allows us to explore associations, and no causality is implied. It has also to be noted that the small number of cases limits our results. A survival bias also cannot be avoided in a disease-association study such as ours, and the likely early mortality in patients (e.g. before hospital admission) could lead to an overestimation of the FVII −323 decanucleotide Del/Ins polymorphism in the AF-stroke group. Further research is needed to assess the role of FVII −323 decanucleotide Del/Ins polymorphism in the stroke risk stratification in AF. Raised plasma markers of a prothrombotic state (such as von Willebrand factor levels, a recognized marker for endothelial damage/dysfunction) have been shown to be predictive of stroke and vascular events Citation28 and may even be additive to clinical risk stratification Citation29. In this framework, genetic markers should perhaps be taken into account Citation5, Citation7, complementing plasma indices. Indeed, the present study suggests that the hypercoagulable state and prognosis in AF could be genetically modulated by the FVII −323 decanucleotide Del/Ins polymorphism.
In summary, the FVII Ins −323 allele, associated with reduced plasma FVII levels, may confer lower ischaemic stroke risk in AF. This allele could influence the stroke risk in AF via modulation of the prothrombotic state associated with AF. We also found that the 807T collagen polymorphism of the GPIa gene could be associated to an increasing risk of suffering a cardioembolic stroke, independently of the prothrombotic state associated with AF.
Acknowledgements
This study was supported by a grant from Fundación Española del Corazón 2005, Madrid, Spain.
Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
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