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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:246-251.)
© 1997 American Heart Association, Inc.

Articles

Polymorphisms of Factor V, Factor VII, and Fibrinogen Genes

Relevance to Severity of Coronary Artery Disease

X.L. Wang; J. Wang; R.M. McCredie; D.E.L. Wilcken

the Department of Cardiovascular Medicine, University of New South Wales, Prince Henry/Prince of Wales Hospitals, Sydney, Australia.

Correspondence to Professor David Wilcken, Department of Cardiovascular Medicine, Clinical Sciences Building, Prince Henry Hospital, Little Bay, NSW 2036, Australia. E-mail X.L.Wang@unsw.EDU.AU.

	Abstract

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We explored the associations between GA mutations of factor V and factor VII genes and the Hae III polymorphism of the fibrinogen gene and the severity of coronary artery disease (CAD), as assessed angiographically in 545 white Australian patients (388 male and 157 female) aged 65 years. We also assessed the relations with other potentially atherogenic variables. Elevated fibrinogen levels were associated with more severe CAD (P<.05), but none of the factor V, factor VII, and fibrinogen DNA variants were predictive of CAD severity, as assessed by the number of significantly diseased vessels (>50% luminal obstruction). The rare allele frequencies of factor V (A allele), factor VII (M2 allele), and fibrinogen (H2 allele) were .025, .114, and .201 for men and .022, .077, and .169 for women, respectively, and were not different from those in healthy whites. In the patient population, there was a strong, positive association between lifetime smoking dose (in pack-years) and circulating fibrinogen levels (r=.184, P=.001). This association was stronger than that between current smoking habit and fibrinogen and is consistent with a dosage effect. However, there was no significant contribution of fibrinogen genotype to fibrinogen levels in this patient population. We conclude that elevated fibrinogen levels are associated not only with the occurrence of CAD but also with more severe CAD and that measurement of DNA variants of the factor V, factor VII, and fibrinogen genes that we assessed may not provide information in predicting CAD severity in addition to that obtained by measuring circulating levels of the relevant clotting factors. There is, moreover, a positive dosage effect (in pack-years) of smoking on circulating fibrinogen levels.

Key Words: coronary artery disease • factor V mutations • factor VII • fibrinogen

	Introduction

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Several lines of evidence suggest that increased coagulability and reduced fibrinolytic capacity are associated with an increased risk of CAD.^{1 2 3 4 5 6 7 8 9} Among the many well defined factors involved in the coagulation and fibrinolytic cascades, levels of fibrinogen and factor VII have been consistently shown to be associated with an increased CAD risk in both prospective and retrospective studies.^{3 4 5 6 7} Although environmental influences such as smoking may alter the circulating levels and activities of fibrinogen and factor VII,^{6 10 11 12} genetic contributions are also significant.^{11 12 13 14 15 16 17 18 19 20 21} An Hae III RFLP at -453 bp of the promoter region of the ß-fibrinogen gene has been reported to explain as much as 3% of the variance in circulating levels of fibrinogen; the presence of the restriction site is associated with increased concentrations.^{12 17 18 19} A GA mutation in the codon for amino acid 353 of factor VII accounts for as much as 40% of the variation in factor VII levels; the rare mutant allele (A) is related to low factor VII activity.^{13 14 15 16} Factor V is another important coagulation factor. It has recently been shown that a GA mutation at nucleotide 1691 of the factor V gene produces an amino acid change of Arg₅₀₆Gln, which results in increased active factor V levels and a tendency to thrombosis by enhancing resistance to cleavage by activated protein C.^{22 23 24} This point mutation has been identified in some patients with idiopathic venous thromboembolism and has been postulated to be related to an increased risk of MI.^{22 23 24 25 26}

Although thrombus formation is usually responsible for acute occlusion of atherosclerotic coronary arteries, old thrombus also forms an integral part of advanced atherosclerotic lesions.²⁷ Thus, increased coagulability and reduced fibrinolytic capacity may be related not only to an increased occurrence of MI but also to the severity of coronary atherosclerosis. However, circulating levels of coagulation factors may not necessarily reflect the local densities and activities of these factors. Furthermore, circulating levels and activities of coagulation factors may be affected by diet, exercise, and drug therapy,^{6 10 21} and measurement of these levels at any one time may not be indicative of the true coagulation status. Genetic variants at candidate loci that are known to regulate the production of relevant levels may therefore be more reliable predictors of both circulating and local effects.

Our previous studies have shown that polymorphisms at various loci, including those of apo E and lipoprotein lipase, predict the severity of CAD directly in addition to their effects on circulating lipoprotein levels.^{28 29} Thus, we hypothesized that polymorphisms and mutations at loci for factor V, factor VII, and fibrinogen genes could also be directly predictive of the severity of CAD. To explore this hypothesis, we studied the distributions of genotypes of these polymorphisms among patients with angiographically defined CAD and assessed the relation between genotype frequencies and disease severity, as defined by the number of significantly stenosed (>50% luminal obstruction) major coronary arteries.

	Methods

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Patients
We studied male and female white patients aged 65 years or less who had been consecutively referred to the Eastern Heart Clinic at Prince Henry Hospital for coronary angiography over a 16-month period in 1994 and 1995. We excluded only those patients who were shown to have significant left main CAD (>50% luminal obstruction) because it was difficult to categorize this small proportion (5%) of the total patient group within the classification system we used (see below). Written consent was obtained from every patient after each was given a full explanation of the study, which had been approved by the Ethics Committee of the University of New South Wales.

A 4-mL venous blood sample was drawn into an EDTA sample tube before the angiogram and after at least a 6-hour fast. The blood sample was centrifuged within 2 hours, and plasma and cellular components were stored separately at -70°C in aliquots until analysis.

Determination of Polymorphism in the ß-Fibrinogen Gene and Factors V and VII Gene Point Mutations
DNA was extracted from the frozen, cellular blood component by a salting-out method adapted from that described by Miller et al³⁰ for whole frozen blood. The extracted DNA was stored at 4°C until analysis. The factor V GA mutation at nucleotide position 1691 was detected as described by Bertina et al.²² The method is a PCR based on Mnl 1 digestion, since the GA mutation results in loss of the Mnl 1 cleavage site. The genotypes are designated as GG, GA, and AA.

The GA mutation in the codon for amino acid 353 of exon 8 in the factor VII gene is responsible for the replacement of Arg by Gln and was determined as described by Green et al.¹³ In this method, the relevant fragment of DNA is amplified by PCR and digested with Msp I. The GA mutation results in loss of the recognition site and is designated the M2 allele and the wild-type as the M1 allele.

The Hae III polymorphic marker located at -453 bp of the ß-fibrinogen promoter region was determined as described by Thomas et al.¹² The mutant type is associated with loss of the restriction site and designated the H2 allele and the wild-type as the H1 allele.

Biochemical Analysis
TC, HDL-C, and TG levels were measured for each patient by the hospital's clinical chemistry department using standard enzymatic methods. LDL-C levels were calculated with the Friedewald formula. We also measured plasma fibrinogen levels in those patients who were not receiving warfarin or heparin therapy at the time of study. Fibrinogen levels were measured by the "derived" method from the clotting curve of prothrombin time on automated photo-optical coagulometers.³¹

Documentation of CAD Severity
The severity of coronary stenosis was determined by the number of significantly stenosed coronary arteries as follows. The angiograms were assessed by two cardiologists who were unaware that the patients were to be included in the study. Each angiogram was classified as revealing either no coronary lesion with >50% luminal stenosis or as having one, two, or three major epicardial coronary arteries with >50% luminal obstruction. We also used the Green-Lane coronary scoring system, which provides a numerical value for lesion severity and takes account of the amount of myocardium supplied by an affected vessel; the maximum score is 15.

Documentation of Other Medical Conditions
Relevant medical history was obtained for each patient by using a questionnaire with a standardized choice of answers to be selected during the interview. We recorded the presence or absence (yes/no) of a history of MI, hypertension requiring treatment, diabetes, and angina pectoris. The presence or absence of CAD among first-degree relatives (parents and siblings) and the age at first onset were recorded for a quantitative assessment of family history of premature CAD. We recorded the presence and severity of angina according to whether each patient was experiencing no angina, stable angina, or unstable angina before and during the current hospitalization. All those patients who were classified as having unstable angina had an increase in pain frequency as well as rest pain. The lifetime smoking dose in pack-years was recorded as described previously.³²

Statistical Analysis
We determined whether or not the distributions of genotypes were in Hardy-Weinberg equilibrium by ² analysis as described by Emery.³³ The frequencies of alleles and genotypes among different subgroups were compared by the ² test. A log-linear analysis was also used to evaluate genotypic effects on CAD severity while other independent variables were controlled.

With a value of P<.05 that was used to reject the null hypothesis with a power of .80, the sensitivities of the present study to detect statistical significances were set when the differences in marker prevalences between groups for factor V, factor VII, and fibrinogen were >0.030, 0.057, and 0.07, respectively. The power calculation was based on the method of McNeil.³⁴

	Results

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Genetic Characteristics of the Patients
The demographic information for the 545 patients is shown in Table 1. As expected, men tended to have more unfavorable lipoprotein variables and smoking habits relevant to CAD risk. Body mass index was not different in male and female patients.

View this table: [in this window] [in a new window]   Table 1. Characteristics of Patients in the Study

The rare-allele frequencies of these three mutations are listed in Table 1. Genotype distributions of all three genes were in Hardy-Weinberg linkage equilibrium and are shown in Table 2 for men and women separately. There was no statistically significant association between genotype and sex, although the rare-allele frequencies tended to be higher in male patients (Table 1). There was also no statistical difference in the distribution of genotypes among smokers and nonsmokers. The lifetime smoking dose was also not different among patients with different genotypes. None of these genotypes was associated with levels of TC, HDL-C, TG, or LDL-C; body mass index; or age.

View this table: [in this window] [in a new window]   Table 2. Genotype Distributions for Factor V, Factor VII, and Fibrinogen Among All Patients

CAD Severity and Genotypes at Fibrinogen, Factor V, and Factor VII Loci
As shown in Table 2, there was no consistent relation between the point mutations for factors V and VII and the fibrinogen polymorphism and the number of significantly diseased vessels in either male or female patients. In a log-linear analysis in which sex, past history of MI, and family history of premature CAD were controlled as independent variables and age, lipid variables, lifetime smoking dose, and fibrinogen levels were controlled as covariates, the genotypes at these three loci were still not statistically associated with the number of significantly diseased vessels.

Because there were only a few patients homozygous for the rare alleles of factor VII and fibrinogen genes, we grouped all those with one or two rare alleles into single groups (n=101 for factor VII and n=191 for fibrinogen) and assessed the relation of each group to the number of significantly diseased vessels. There was still no significant association between genotype and CAD severity. The power of detecting any relation between genotypes for fibrinogen, factor VII, and factor V and significant CAD or no CAD in men and women with 95% confidence varied from 71% to 96%, with the genotype frequencies found and the number of patients included in the study. The same statistical analyses were also conducted for smokers and nonsmokers separately; no significant relation was detected. However, because of the smaller number of patients in each subgroup, there was reduced power in the subgroup analyses.

When coronary scores were compared among patients with different genotypes as a measure of CAD severity, there was also no relation between CAD severity and the genotype as shown in Table 3. This lack of association was confirmed in an ANOVA after controlling for other variables, particularly smoking.

View this table: [in this window] [in a new window]   Table 3. Coronary Severity Scores (Mean±SEM) and Genotypes of Factor V, Factor VII, and Fibrinogen Among Patients With One or More Significantly Diseased Vessels

In this patient population, we observed a linear increase in fibrinogen levels in patients with zero (3.52±0.09 g/L), one (3.69±0.09 g/L), two (3.78±0.11 g/L), and three (3.82±0.10 g/L) significantly diseased vessels, and the levels for patients with triple-vessel disease were significantly higher than those in patients with no significantly diseased vessels (P<.05). Furthermore, fibrinogen levels were significantly elevated in smokers, as has been found in other studies.^{6 10 11 12} The positive linear relation between fibrinogen level and lifetime smoking dose (in pack-years) was statistically significant in men (r=.185, P=.013) and women (r=.288, P=.01). This association remained significant after controlling for sex, CAD severity, and fibrinogen genotype. However, fibrinogen genotypes were not associated with fibrinogen levels in this patient population (Table 4), even after controlling for CAD severity, sex, and smoking.

View this table: [in this window] [in a new window]   Table 4. Association Between Fibrinogen Genotypes and Fibrinogen Levels

Medical Conditions and Genotypes at Fibrinogen, Factor V, and Factor VII Loci
The distributions of genotypes among patients with or without a past history of MI were not statistically different. There were also no associations between genotypes for the three factors and hypertension, diabetes, or family history of premature CAD. We also compared genotype distributions among patients with no angina, stable angina, and unstable angina. Again, there were no statistically significant relations.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
We undertook the present investigation of white Australians aged 65 years or less and living in Sydney who were studied consecutively to establish a diagnosis of CAD and to document its severity. This patient group is therefore representative of Australians of European origin in this age group who are judged by cardiologists to require coronary angiography, and our results refer specifically to this population. They confirm that smoking increases circulating fibrinogen levels in the Australian CAD population, as has been extensively reported for other populations.^{6 10 11 12} We have also shown that lifetime smoking dose has a strong, linear association with levels of circulating fibrinogen (P<.001). The more or the longer the smoking history, the higher are fibrinogen levels. This relation was much stronger than that between present smoking status and fibrinogen and is indicative of a causal relation between lifetime smoking dose and elevated fibrinogen levels. Although some studies have reported that the fibrinogen genotypes we assessed are associated with fibrinogen levels, the documented contribution is small (3%)^{12 14} and may be "masked" by other factors, eg, sex and smoking.^{10 11 12 14 15 16 17 18} The lack of a statistically significant association between fibrinogen genotypes and levels in our patients is another example of this environmental effect, since there was a high proportion of smokers in this patient series. We did not measure levels or activities of factors V and VII, since it has been extensively documented that base changes at both loci are strongly associated with circulating levels.^{13 14 15 16} We explored the possibility that these DNA variants could be directly associated with CAD severity in our current study.

As reported in most European studies,^{3 5 6 7 8 9} an elevated fibrinogen level was also a predictor of severe CAD in this white Australian population. Patients with triple-vessel disease had fibrinogen levels that were 8.5% higher than those without significantly diseased vessels (P<.05). However, while increased coagulation and reduced fibrinolysis are reported to be associated with an increased risk of MI, the DNA variants of factor V, factor VII, and fibrinogen genes that we tested were not associated with the severity of CAD in our patients. This is supported by first, the lack of a statistically significant difference in the distributions of rare alleles among patients with different numbers of significantly diseased vessels and second, the fact that coronary severity scores were not different among patients with different genotypes. Although the study has less power (71%) in detecting any relation between factor V genotype and CAD severity, it nevertheless has sufficient power to detect whether genotypes of factor VII and fibrinogen together are directly related to CAD severity. Our study therefore suggests that DNA variants of factor V, factor VII, and fibrinogen genes may provide no more information in predicting CAD than do circulating levels of clotting factors, such as fibrinogen. Even though a high proportion of patients in our study were either exsmokers or current smokers and this fact may affect the findings, DNA variants at these loci were not associated with CAD severity in a major way; effects, if present, are likely to be minimal.

In relation to CAD occurrence, we have no data for genotype distributions in the local (control) population. However, genotype distributions in this white Australian patient population are not significantly different from those observed in other white populations. We found no patient homozygous for the factor V Arg₅₀₆Gln mutation, and the percentage of heterozygotes was the same as that reported by Marz et al²⁵ (4%) in a control population and far less than the 9% heterozygosity among 224 CAD patients reported in the same study.²² However, the heterozygosity prevalence in our patients was the same as that in the American Physicians' study (n=14 916),³⁵ and these authors also reported no difference in heterozygosity prevalence between men with (.061) and without (.060) MI.

The frequency distribution of the GA mutation in the factor VII gene in our patient population (M2, .115 for male patients) was very close to that of healthy white English men as reported by Green et al¹³ (allele frequency for the rare M2 allele of .10). Furthermore, the allele frequency for the rare fibrinogen allele (H2, .201 for male patients) was also not different from that reported by Thomas et al¹² in 292 healthy white men (.190). We therefore concluded that factor V and factor VII gene point mutations and the base substitution at the promoter region of the fibrinogen gene are not more prevalent in our CAD patients than in healthy populations.

In a recent study, Behague et al³⁶ reported that the fibrinogen Hae III polymorphism was associated with the number of significantly diseased vessels among 358 French MI patients. Although the percentage of patients with one or two rare alleles increased linearly with one (29.8%), two (37.4%), and three (48.3%) significantly diseased vessels, the control population in that study also had quite a high percentage of patients with rare alleles (37.7%). Behague et al³⁶ also found that after stepwise logistic regression analysis, Bcl I, another polymorphic marker at the ß-fibrinogen gene, was the only significant predictor of CAD severity. However, even for the Bcl I polymorphism, the percentage of control patients who had rare alleles (32.4%) in that study was higher than in those with one (23.0%) and two (29.9%) significantly diseased vessels. When we used the same approach to analyze the results for our CAD patients with a past history of MI, the percentage of patients with rare alleles for the fibrinogen Hae III polymorphism increased with increasing disease severity—no (29.6%), one (34.3%), two (35.6%), and three (41.4%) significantly diseased vessels, although this relation was not statistically significant. Therefore, any direct association between the fibrinogen gene DNA polymorphism and CAD severity is, at best, only weak.

In conclusion, our study of a representative white Australian population assessed for CAD showed that while elevated fibrinogen levels are predictive of CAD severity, measurements of point mutations in factor V, factor VII, and fibrinogen genes provide no additional information in predicting the occurrence or severity of CAD.

	Selected Abbreviations and Acronyms

 

CAD = coronary artery disease HDL-C = HDL cholesterol LDL-C = LDL cholesterol MI = myocardial infarction PCR = polymerase chain reaction RFLP = restriction fragment length polymorphism TC = total cholesterol TG = triglyceride

	Acknowledgments

 
This work was supported by grants from the National Health and Medical Research Council of Australia. We thank Lily Fenech, Shelly Brown, Steven Brouwer, Dr Chao Jun Ma, Dr Greg Cranny, and all nurses in the Eastern Heart Clinic for their assistance in clinical data collection and Dr Sheng Xiang Liu and Ah Siew Sim for their laboratory assistance. We are also most grateful to the cardiologists in the Department for allowing us to study their patients.

Received June 13, 1996; revision received September 24, 1996;

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