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Technical Briefs |
1 Institute of Medical and Chemical Laboratory Diagnostics,2 University Clinic of Neurology, and3 Department of Internal Medicine, Division of Angiology, Medical University of Vienna, Vienna, Austria;4 Hospital Barmherzige Brueder, Vienna, Austria;
aaddress correspondence to this author at: Institute of Medical and Chemical Laboratory Diagnostics, Waehringer Guertel 18-20, 1090 Vienna, Austria; fax 43-1-40400-2097, e-mail christine.mannhalter{at}meduniwien.ac.at
Abstract
Background: Coagulation factor VII (FVII) plays an important role in the activation of blood coagulation and clot formation. Recent studies have provided evidence for an association between common polymorphic markers in the FVII gene and plasma FVII concentrations. The 353R>Q sequence variation, and 3 common sequence variations in the promoter of the FVII gene—the 10-bp insertion/deletion at position –323 and the –401G>T and –402G>A sequence variations—are well-known determinants of circulating FVII concentrations.
Methods: To clarify the role of these sequence variations in the pathogenesis of ischemic stroke, we performed a case–control study with 242 patients with ischemic stroke before the age of 60 years and 239 healthy controls.
Results: The –323 insertion/deletion and the 353R>Q and –401G>T sequence variations were in strong linkage disequilibrium, and the resulting haplotypes occurred with equal frequencies in patients and controls. The variant form of FVII (–402G>A) occurred only in combination with the common (wild-type) sequences at all other loci. This haplotype was more frequent in patients than in healthy controls (28% vs 22%). The difference in the prevalence of carriers of this haplotype among patients and controls was statistically significant (P = 0.03; odds ratio = 1.6; 95% confidence interval, 1.1–2.6).
Conclusion: According to our results, the FVII –402A allele seems to increase the risk of early ischemic cerebrovascular events, whereas the 353R>Q, G–401T, and –323ins/del sequence variations, which are in close linkage disequilibrium, apparently do not influence the risk of stroke.
Coagulation factor VII (FVII) is a vitamin K–dependent protease that plays a key role in the initiation of the activation of the blood coagulation cascade. The factor VIIa–tissue factor complex initiates coagulation by limited proteolysis of factors IX and X. Increased FVII coagulant activity has been associated with cardiovascular disease (1)(2)(3), carotid intima-media thickness increase (4), and cerebrovascular disease(5). Because of controversial results in other studies (6)(7), the role of increased FVII concentrations in vascular disease remains unclear.
It has been shown that FVII concentrations are influenced not only by exogenous factors, including age, sex, diet, and hormonal status (8), but also by genetic factors. Sequence variations in the FVII gene account for approximately one third of the variations in plasma FVII concentrations (9). The FVII gene is located on chromosome 13 and contains several polymorphic sites. Among them are the 353R>Q sequence variation and 3 common variants in the promoter of the FVII gene—the 10-bp insertion/deletion at position –323, the –401G>T sequence variation, and the –402G>A sequence variation—which are well-known determinants of circulating FVII concentrations. The sequence variant at nucleotide (nt) –402 has been reported to contribute to increased FVII concentrations (10), whereas the variants at nt –401 and –323 and the 353R>Q variant have been associated with decreased factor VII concentrations (9)(10)(11). The role of these sequence variations in the development of vascular disease is still controversial.
In the present study, we examined frequencies and haplotype distributions of the 353R>Q, –401G>T, and –402G>A sequence variations and the 10-bp deletion/insertion at position –323 in individuals free of vascular disease as well as in patients with ischemic stroke before the age of 60 years, both from the Austrian population (Caucasians of middle-European origin). We also investigated possible interactions of these sequence variations with established vascular risk factors.
For this study, 242 patients with an acute ischemic stroke or transient ischemic attack before the age of 60 years (105 females, 137 males; median age, 54 years; interquartile range, 51–57 years) were tested. Patient data were compared with data from 239 controls matched for age (±2 years; median age, 53 years; interquartile range, 50–56 years) and sex (137 males, 102 females). Both groups were selected from the Vienna Stroke Registry (12). Detailed characteristics and distributions of cardiovascular risk factors of patients and controls are shown in Table 1 of the Data Supplement that accompanies the online version of this Technical Brief at http://www.clinchem.org/content/vol52/issue6/. The –323 0/10bp and the 353R>Q sequence variations were genotyped by a multiplex PCR assay followed by hybridization to sequence-specific oligonucleotides immobilized on nylon membrane strips (Roche Molecular Systems), essentially as described by Cheng et al. (13). The –401G>T and –402G>A variations were analyzed by 2 separate PCR reactions. For genotyping at the –401 locus and amplification of the –402 wild-type sequence, one PCR reaction was performed. PCR products were generated in 25-µL volumes containing 0.74 U of AmpliTaq Gold (Perkin-Elmer Cetus), 1.5 mM MgCl2, 200 mM each deoxynucleotide triphosphate (Amersham Pharmacia Biotech), 7.5 pmol of –401 wild-type primer (5'-TGCGAGTCAAATCTCAGGAC-3'), 20.5 pmol of –401 variant primer (5'-ATAGCAATCAATTGAGTCAAATCTCTAGAA-3'), 10 pmol of –401 common primer (5'-TGCCCAGCTCAGGGC-3'), 8 pmol of –402 wild-type primer (5'-TACATCCACACCCAAGATGCG-3'), 8 pmol of –402 common primer (5'-ACAGGAGAAAGGTCAGGTGA-3'; all primers from TIB Molbiol), and 
15 ng of DNA. The PCR product spanned the 10-bp insertion/deletion site at nt –323, which made it possible to confirm the decanucleotide insertion (–323 0/10bp) results obtained by the hybridization method. Amplification of the –402 variant sequence was achieved by the following second PCR, in which 8 pmol of –402 variant primer (5'-TTCAAATATTATCATCCACACCCAAGAAACA-3') and 8 pmol of –402 common primer were used. Positive and negative controls were included in each PCR run.
All amplifications were performed in an Eppendorf Thermo Cycler with the following cycling conditions: 10 min of denaturation at 95 °C, followed by 33 cycles of 95 °C for 45 s, 55 °C for 45 s, and 72 °C for 45 s, with a final extension step of 7 min at 72 °C. The resulting PCR products had lengths of 105 and 114 bp for the –401 wild-type and variant alleles, respectively, and 159 and 168 bp for the –402 wild-type and variant alleles, respectively (in case of wild-type genotype at nt –323). The PCR products were separated on 10% polyacrylamide gels (Bio-Rad). Gels were stained with SYBR Green.
Continuous data are reported as the median (interquartile range), and discrete data are reported as the numbers and percentages. The McNemar test for dependent samples was used to compare groups of categorical data. Groups of continuous data were compared by the Wilcoxon test. Associations between the haplotypes and the risk of stroke were analyzed by means of binary logistic regression models with adjustment for conventional risk factors [hypertension, diabetes, hyperlipidemia, smoking, and obesity (body mass index >27 kg/m2)]. The linearity of the logit assumption was checked for continuous predictor variables, and an analysis of residuals was performed. Regression diagnostics and overall model-fitting were performed according to standard procedures. Interactions between the FVII genotype, conventional vascular risk factors, and cerebrovascular events were tested by use of multiplicative interaction terms and log likelihood ratio 
2 tests. Calculations were performed with SPSS for Windows (Ver. 10.0; SPSS Inc.), and for the haplotypes for the 4 sequence variations, we used the program PHASE 2.0 for statistical haplotype reconstruction (14).
Haplotype analysis showed the occurrence of 6 different haplotypes, of which 3 accounted for almost 98% of the alleles observed in the study population. Sequence variations at nt –401 and –323 and the 353R>Q sequence variant were in close linkage dysequilibrium. The nt –402A variant was found only in combination with the common (wild-type) sequence at all other loci, indicating complete linkage (Table 1
). Haplotypes H3, H4, H5, and H6 were combined for all further calculations. The frequencies of the combined haplotypes, coded as haplotype H3a, were compared with those of haplotypes H1 and H2.
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The frequencies of the different haplotypes in controls and patients are shown in Table 1
. The H2 haplotype (–402A/–401G/–323del/353R) was significantly more prevalent in patients than in controls (50% vs 38.5%). This difference was statistically significant after univariate analysis (odds ratio, 1.6; 95% confidence interval, 1.1–2.3; P = 0.01) and remained so after multivariate adjustment (adjusted odds ratio, 1.6; 95% confidence interval, 1.1–2.6; P = 0.03). The number of carriers of the H3a haplotype was statistically not significantly different between patients with ischemic cerebrovascular events and the control group in either univariate or multivariate analysis. The presence of traditional vascular risk factors, such as smoking, diabetes mellitus, hypertension, obesity, and hyperlipidemia, was not significantly different between carriers and noncarriers of variant alleles in patients and controls.
Over the last few years, several studies have focused on the role of FVII sequence variations in the development of vascular disease. Published data have been discordant with respect to the association of sequence variations at nt –401 and –323; the 353R>Q sequence variant; FVII concentrations; and the risk of myocardial infarction or stroke (6)(15)(16). Confirming previous studies, we could show that these 3 sequence variations are in close linkage dysequilibrium (17). We found no association of these sequence variations with the risk of ischemic cerebrovascular events, which is in agreement with the studies of Corral et al. (18) and Heywood et al.(6).
Interestingly, the FVII –402A genotype occurred only in combination with the wild-type sequences at all other tested loci, indicating complete linkage of –402A with –401G/–323del/353R. Previously, the –402A allele had been reported to be associated with both increased FVII plasma concentrations and increased transcriptional activity in vitro (10). Recently, this sequence variant has been reported to represent a risk factor for myocardial infarction (19)(20), but its role had not been tested in stroke patients. In our study we report for the first time an association of the –402A allele with the occurrence of acute ischemic cerebrovascular events before the age of 60 years by showing that individuals carrying the –402A allele have an increased risk for developing transient ischemic attack or ischemic stroke. This supports and extends recent observations that the –402G>A sequence variation is involved in the development of vascular disease. Although the impact of a single sequence variation on a complex disease such as stroke may be influenced by patient selection, ethnic background, or sample size among others, our data justify follow-up studies in patients with stroke.
Our study population represents consecutive samples of all patients with stroke at age <60 years registered in the Vienna Stroke Database. However, we could not include patients who died within 24 h. Our findings, therefore, can be applied only to survivors of an acute cerebrovascular event. We restricted our study to persons younger than 60 years because it can be assumed that in older individuals, vascular risk factors such hypertension, diabetes mellitus, or hyperlipidemia are more likely to be present and that this may overrule the relatively modest influence of a single sequence variation.
Acknowledgments
This work was supported by research grants from the Jubilaeumsfonds der Oesterreichischen Nationalbank (Project 9996). The Vienna Stroke Registry was supported by research grants from the Medizinisch-Wissenschaftlicher Fonds des Bürgermeisters der Bundeshauptstadt Wien (Projects 1540 and 1829), of the Jubiläumsfonds der Oesterreichischen Nationalbank (Projects 6866 and 8281), and the Austrian Research Fund (FWF, P13902-MED). The Vienna Stroke Registry is sponsored by an unrestricted educational grant from Sanofi-Synthelabo and Bristol-Myers Squibb and by the Wiener Krankenanstaltenverbund (L. Kaspar, MD).
References
The following articles in journals at HighWire Press have cited this article:
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  A. A. Jackson, K. R. Cronin, R. Zachariah, and J. A. Carew CCAAT/Enhancer-binding Protein-beta Participates in Insulin-responsive Expression of the Factor VII Gene J. Biol. Chem., October 26, 2007; 282(43): 31156 - 31165. [Abstract] [Full Text] [PDF]
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