Contribution of Factor VII Genotype to Activated FVII Levels
Differences in Genotype Frequencies Between Northern and Southern European Populations
Abstract The relationship between coagulation factor VII (FVII) levels in plasma and FVII genotypes, determined by three polymorphisms (5′F7, IVS7, and 353R/Q), were studied in 500 control subjects enrolled in a European multicenter study. The selection of particular FVII genotypes and the analysis of variance clearly indicated the independent contribution of a single 5′F7 insertion (A2) or 353Q (M2) allele to lowering plasma levels of activated FVII (FVIIa) (by a mean 25%). The M2 allele alone was found to make a major contribution to the genetically determined component of the FVIIa levels. Genotypes associated with low FVII levels were significantly rarer in the northern part of Europe (Oslo) than in the southern part (Rome, Murcia). The contribution made by the FVII genotype to the total variance of FVIIa levels was higher (30%) than that made to either FVII activity (25%) or FVII antigen (12%). Subjects with different FVII genotypes showed up to fivefold differences in mean FVIIa values, thus allowing attribution of a substantial part of the considerable interindividual variation to genetic variation, which may be of assistance in the interpretation of FVIIa levels on an individual basis. When FVII levels were adjusted by age and by triglyceride levels, the contribution of FVII genotypes to the FVII phenotypic variance was virtually unchanged. Taken together, these data indicate that in healthy control subjects the FVII genotype is a major predictor of plasma FVIIa levels and would support further study on the role of FVII genetic components in the development of cardiovascular disease.
- Received January 3, 1997.
- Accepted June 25, 1997.
Prospective studies have identified elevated levels of FVIIc as a risk factor, especially for fatal events, in ischemic heart disease among middle-aged men.1 Moreover, increased activity in the extrinsic pathway is currently believed to be associated with a hypercoagulable state.2,3 Differences found4–6 in the degree of association between FVII levels and ischemic heart disease can, however, be due to differences in the techniques used for measuring FVIIc levels7 and to the lack, at that time, of assays for the enzymatically active form of the protein, FVIIa.8–10 Although most FVII circulates in the zymogen form, a priming role in triggering the coagulation cascade has been assigned to FVIIa, which is not neutralized by plasma inhibitors and has a much longer half-life than other vitamin K-dependent proteases. Measuring FVIIa levels may be the most reliable way to study its relationship with cardiovascular disease. Because FVIIa is the first active protease in the coagulation pathway and could contribute to extensive initiation of the clotting cascade, its levels may be important for the outcome of a thrombotic event, for instance, in determining the size of an occlusive thrombus after the rupture of atherosclerotic plaques.
Previous studies11–13 have shown that FVII gene polymorphisms are major determinants of plasma FVIIc and FVIIAg levels, whereas few data are available14,15 on genetic components that may determine plasma levels of FVIIa. The availability of a specific assay for the determination of FVIIa16–18 in human plasma makes it possible to investigate the role of variations in the FVII locus in determining FVIIa levels.
Because differences in FVII activity levels and in genotype frequencies have been reported in different ethnic groups,19,20 and because the risk of acute myocardial infarction is lower in southern European countries than in northern European countries, we designed a multicenter study to enroll volunteer subjects from several European countries with considerable geographic and ethnic differences and different lifestyles. This population study, a reference for subsequent studies of patients with heart disease, illustrates FVII genotypic and phenotypic variations and their relationships.
Apparently healthy volunteers (aged 19–75 years) from five different European countries (France, Italy, The Netherlands, Norway, and Spain) were, after giving informed consent, enrolled in the European Community Concerted Action Trial (CLOTART). Five hundred blood samples were collected for phenotypic and genotypic analyses. The genotypic analysis was extended to an additional 237 subjects to further refine the estimate of the differences in genotype frequencies among centers. The number of subjects investigated in each center is reported in Table 1⇓.
All blood collections were carried out irrespective of the use of oral contraceptives, time of day, or fasting status of the subject, except in Oslo, where only fasting subjects were enrolled. All participants declared themselves free of cardiovascular disease, diabetes, and cancer. Blood for coagulation studies was drawn into 5-mL Vacutainers containing 0.1 volume of 0.129 mol/L buffered sodium citrate. All samples were centrifuged within 30 min at 2000×g for 15 min. Plasmas were harvested and aliquoted in plastic tubes with colored caps, the cap color indicating the analyzing laboratory. Samples were frozen to − 80°C in cryotubes and cryoboxes and subsequently sent in dry ice to the central repository in the coordinating institution (Thrombosis Center, University of Rome) for redistribution.
For the DNA-based genetic evaluations, pellets from the citrated blood samples were harvested in plastic tubes and frozen (−10°C). Each laboratory carried out phenotypic or genotypic evaluations on the whole population using standard methods and equipment.
FVIIc was assayed by a one-stage method, and FVIIAg was assayed using an enzyme immunoassay as previously described.12 Pooled plasma (20 fasting males and 20 fasting females, from Rome) was used as a standard. aPTT and prothrombin time were performed on each sample. Samples, which were all within the normal range, were then pooled.
FVIIa was assayed with a kit (Staclot VII-arTF, Diagnostica Stago, Asniere, France); values were expressed in milliunits per milliliter, 30 such units being equivalent to 1 nanogram of FVIIa. For the FVIIa assay, the standard was a recombinant FVIIa (Novo-Nordisk, Bagsvd, Denmark) supplied with the kit. All assays of one type were carried out in a single laboratory.
Genomic DNA was extracted in Oslo from deep frozen cell pellets and purified as described by the manufacturer, using an Applied Biosystems 341 Genepure instrument. The detection of FVII markers11,21,22 was improved by the use of multiplex PCR of the 5′F7 and IVS7 polymorphisms, followed by agarose-gel electrophoresis of crude PCR samples. The exon 8 polymorphism (353R/Q) was detected as previously described.12
Primers for PCR amplification, derived from the sequence of O’Hara et al,23 were as follows: 5′F7 polymorphism (5′-AG GCTCTCTTCAAATAATTACATC-3′, nt −439 to −416, and 5′-AGAGCGGACGGTTTGTT-3′, nt −237 to −254); IVS7 polymorphism (5′-AATGTGACTTCCACACCTCC-3′, nt 9568 to 9587, and 5′-GATGTCTGTCTGTCTGTGGA-3′, nt 10 009 to 9990). Multiplex PCR (Perkin-Elmer 9600 Thermocycler) of the 5′F7 and IVS7 polymorphisms were run for 30 cycles as follows: 20 s of denaturation at 93°C, 30 s of annealing at 56°C, and 90 s of extension at 70°C. Buffers and polymerase were as previously reported.12
The genotypes were denominated as follows: (1) 5′F7 polymorphism, alleles A2 (decamer insertion) and A1 (absence of decamer); (2) 353R and 353Q polymorphism, alleles M1 and M2, respectively; and (3) variable number of tandem repeats in intron 7 (IVS7), alleles a (7 monomers), b (6 monomers), c (5 monomers), and d (8 monomers).
Haplotype frequencies and the coefficients of gametic linkage disequilibrium were calculated by likelihood methods in accordance with Terwilliger and Ott.24 Standardized correlation coefficients were calculated as described in Chakravarti et al.25
One-way analyses of covariance (using age as the covariate) and two-way analyses of variance using the general linear model procedure were carried out on values of FVIIa, FVIIc, and VIIAg to test the null hypothesis that the spread of such values is not associated with genetic variation within the FVII locus.
Data were log-transformed to normalize distributions and to stabilize variances. BMDP software was used. The percentage of genotype-based variance in FVII levels was estimated as described by Sing and Davignon.26 The allele distribution of FVII genotypes was as expected for a sample in Hardy-Weinberg equilibrium.
FVII genotypes and allelic frequencies were determined in 737 subjects from Leiden, Murcia, Oslo, Paris, and Rome (Table 1⇑).
The frequency of the A2 and M2 alleles, those previously found to be associated with reduced FVII levels,12,13 were significantly lower (Table 1⇑) in the most northern city than in the southern ones, with a frequency of the A2 allele ranging from about 9% in Oslo to 15% to 16% in Murcia and Rome. The frequency of subjects carrying at least one A2 or M2 allele ranged from about 30% (Rome and Murcia) to 19% (Oslo). Most of the subjects carrying the rare c allele of the IVS7 polymorphism were from Northern countries.
FVIIa, FVIIc, and FVIIAg mean values were studied in 500 of the 737 genotyped subjects (Table 1⇑). The association between each pair of FVII variables was observed (correlation coefficients, FVIIa/FVIIc 0.72, FVIIa/VIIAg 0.46, FVIIc/FVIIAg 0.61).
Age exerted a significant effect (P<.0001) on FVIIa, FVIIc, and FVIIAg with F values of 16, 24, and 8, respectively. No statistically significant difference was observed in the ages of subjects carrying different genotypes. Mean values of FVIIa and FVIIAg did not differ significantly in males and females. FVIIc was, however, higher in females (P=.02), as a result of a major increase in levels observed in postmenopausal women aged 51 to 75 years (P=.006).
When the distribution of FVII levels was compared over the five centers (Table 1⇑), the mean FVIIa levels were significantly higher in Paris than in any of the other centers, and mean FVIIAg in Oslo was lower than in any of the other centers. The highest mean FVIIc value, found in Murcia, differed significantly from the lowest one (Leiden).
To define the contribution of genetic variation to FVII levels, subjects were grouped by genotypes determined by a single polymorphism, by couples (Fig 1⇓) and by all of them. Most genotypes determined by a single marker (Table 2⇓) correlated with highly significant statistical differences in the mean FVII values, except the IVS7 genotypes with the mean FVIIAg levels. The strongest association found was between FVIIa and the 353R/Q or 5′F7 polymorphisms. Subjects carrying the A1A1 and M1M1 genotypes had mean FVIIa levels (86 and 85 mU/mL, respectively) four or five times as high as subjects homozygous for the A2 or M2 alleles (20 and 16 mU/mL, respectively).
The phenotypic variation associated with the 353R/Q and 5′F7 polymorphisms, which showed a strong allelic association (Δ, .87), contributed about one third of the variance of FVIIa, ranging from 27% in Leiden to 48% in Paris (Table 3⇓). A low contribution of the IVS7 polymorphism was observed (mean 4.5%).
Although the linkage disequilibrium between the 5′ F7 and 353R/Q polymorphisms was very high, the large number of subjects investigated enabled us to find genotypes with uncoupled A2 and M2 alleles (ie, genotypes 2, 3, and 5 in Fig 1⇑) and to investigate differences in the associated FVII phenotypes.
Combining all markers grouped around 95% of the population in 11 genotypes (I through XI in Table 2⇑), each found in at least four control subjects. Although some groups held only a few individuals, informative differences in FVII levels were detected. Genotypes differing by one A2 and one M2 allele (genotypes II and III or V and XI) showed significant differences for FVIIc and FVIIa values. Even the presence of one additional A2 allele (I/IV, I/VIII, II/VI, II/VIII, IV/VI, and VIII/IX) was associated with lower FVII values. A small contribution of the IVS7 alleles (I/II, II/IV) with a constant 5′F7 and 353R/Q background was also observed.
Genetic variation associated with the polymorphisms studied in conjunction (Table 3⇑) was shown to contribute up to 32% of the total variance in FVIIa values and up to 26.8% of that in FVIIc values (Table 3⇑).
The independence of the effects of the gene polymorphisms and their additive contribution were tested (Table 4⇓) in an analysis of variance model.
The differences in FVII activity levels and in FVII genotypes previously reported in various populations,15,19,20 and the almost complete absence of information on FVIIa levels in those studies, led us to design a systematic multicenter study aimed at investigating European geographical differences in FVII genotypes and phenotypes, particularly in FVIIa levels. Because an uneven distribution of the risk for acute myocardial infarction in Europe has been reported, our study of a control population is an indispensable reference for studies of the FVII genotype–phenotype relationship in patients with heart disease.3
The present study also differs from previous ones in that the evaluation of the FVII genotype–phenotype relationship was conducted with the use of complex FVII genotypes determined by three polymorphisms (eight alleles) in different functional regions of the gene.
We established that the contribution of FVII genotype to plasma FVIIa levels, which has a priming role in the activation of the coagulation cascade, is very high (Tables 1⇑ and 3⇑), approaching half the variation in one center, and much higher than that to either FVIIAg or FVIIc. These data indicate that part of the considerable interindividual variation in FVIIa levels17 may be attributed to genetic variation. The importance of this observation is increased when we take into account the intraindividual variation in FVIIa levels and the intra- and interassay variation of the FVIIa assay, which usually make a single determination of the level of FVIIa inadequate as an estimate of the true mean value for an individual. The interpretation of FVIIa levels on an individual basis is assisted by knowledge of the genotype. For instance, 120 mU/mL of FVIIa may be considered a frequent level in subjects with genotype I or II but very rare in subjects with genotype III or V (Table 2⇑). These data further strengthen the FVII genotype as a candidate for the study of genetic components in cardiovascular disease.
The 5′F7 and 353R/Q polymorphic systems contribute to a similar extent to the total phenotypic variance (Table 3⇑), a finding potentially explained by the presence of high linkage disequilibrium between these two markers. However, the selection of particular genotypes (Table 2⇑ and Fig 1⇑) in which A2 and M2 were not coupled showed statistically significant differences in FVII phenotypes that clearly indicate the independent contribution of each allele to lowering levels of FVII, particularly those of FVIIa. This observation was confirmed by the two-way analysis of variance (Table 4⇑). A previous investigation13 of the marker that most efficiently predicts FVII levels indicated that the A2 allele had a larger lowering effect on FVIIc and FVIIAg levels. In our study, however, the two-way analysis of variance indicated a slightly higher contribution to FVIIc and FVIIAg on the part of the 353R/Q polymorphism (Table 4⇑). Our data also define this polymorphism as a major contributor to FVIIa levels. A mean 27% decrease in FVIIa is associated with the presence of a single A2 allele, which contains an insertion of a decanucleotide in the 5′ region of the FVII gene. Because this insertion has been demonstrated in vitro to reduce transcription from the FVII promoter,27 and thus probably acts by modulating the biosynthesis of the FVII zymogen, its independent association with plasma FVIIa highlights the importance of the “FVII mass” in determining FVIIa levels. Although the contribution of the IVS7 polymorphism is lower than that of the other two (Tables 3⇑ and 4⇑), the selection of complex genotypes (Table 2⇑, genotypes I and II) makes it nonetheless statistically significant, which may suggest small differences in splicing efficiency, caused by the IVS7 variations.
Significant differences were found in the frequency of FVII gene polymorphisms between the northernmost center and the southern ones. The number of heterozygous subjects carrying at least one of the alleles strongly associated with lower FVII levels was 40% lower in Oslo than in Rome or Murcia. The intermediate allelic frequencies observed in Paris and Leiden (Table 1⇑) may suggest the presence of a north–south gradient for the A2, M2, and a alleles. A study of the relationship between the higher incidence of acute myocardial infarction in northern European countries and the higher frequency in these countries of particular FVII genotypes would be highly interesting.
A significant proportion of the FVII phenotypic variance (Table 1⇑) was associated with the FVII genotype in all centers. The unchanged proportion when FVII levels were adjusted for triglyceride and phospholipid levels28–30 (data not shown) and for age confirmed the genotype–phenotype relationship.
Because common blood sampling and handling protocols were followed in all centers and all FVII assays were centralized, it may be supposed that methodological differences will have had a negligible influence on mean FVII values. The impact of the differences in the frequency of the FVII genotypes (5% to 10%) on the mean FVII level in each center was barely detectable, however, suggesting the presence of other environmental and/or genetic factors, not in linkage with the FVII gene, that mask the effect of intragenic components.
Our data indicate that the FVII genotype is a major predictor of FVIIa levels and suggest that a study of the contribution of FVII genetic components in the development of cardiovascular disease would be of great interest.
Selected Abbreviations and Acronyms
|aPTT||=||activated partial thromboplastin time|
|FVIIa||=||activated factor VII|
|FVIIAg||=||factor VII antigen|
|FVIIc||=||factor VII coagulant activity|
|PCR||=||polymerase chain reaction|
|PNP||=||pooled normal plasma|
This work was carried out within the framework of the European Union Concerted Action on the role of the Tissue Factor Pathway in Ischemic Heart Disease, BMH1-CT94–1292 (CLOTART). The authors wish to thank Paolo Ferraresi for his technical assistance and David Holmes for amending the text.
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