© 1995 American Heart Association, Inc.
Articles |
European Atherosclerosis Research Study: Genotype at the Fibrinogen Locus (G-455-A ß-Gene) Is Associated With Differences in Plasma Fibrinogen Levels in Young Men and Women From Different Regions in Europe
Evidence for Gender-Genotype-Environment Interaction
From the European Atherosclerosis Research Study (EARS) group. See "Acknowledgments" for a list of participating individuals and centers.
Correspondence to Prof Steve E Humphries, The Centre for Genetics of Cardiovascular Disorders, Department of Medicine, The Rayne Institute, 5 University St, London WC1E 6JJ, UK.
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Abstract |
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Abstract The European Atherosclerosis Research Study (EARS) compares genetic and environmental factors in the offspring of fathers with myocardial infarction before the age of 55 years (designated cases) and control subjects from five different regions of Europe. Genotype was determined for a G-A polymorphism 455 bp upstream from the start of transcription of the ß-fibrinogen gene. In 585 cases and 1106 control subjects, the relative frequency of the A allele was similar (0.223 and 0.217, respectively), with small and nonsignificant differences in frequency observed among the five regions. Because of evidence for an interaction between a number of factors and genotype in the determination of plasma fibrinogen levels (in particular among female cases who reported use of oral contraceptives), the data were analyzed without adjusting for covariates except for age and region, and analyses were carried out excluding women taking oral contraceptives (n=297). In agreement with previous reports, in all regions the A allele was associated with elevated plasma fibrinogen levels, with the strongest and most consistent effects being seen in men. In nonsmokers, after adjusting for the effects of age and region, male cases and control subjects with genotype A/A had mean fibrinogen levels 0.49 and 0.33 g/L higher, respectively, than those with genotype G/G, whereas those with genotype G/A had intermediate levels (P<.01). In female nonsmokers there was a similar but smaller and nonsignificant effect (A/A levels higher than G/G by 0.12 and 0.07 g/L, respectively). In both men and women, there was significant evidence for an interaction between genotype and smoking in the determination of fibrinogen levels, with genotype A/A being associated with the highest mean levels in nonsmoking individuals and the lowest mean levels in smoking individuals (interaction between smoking and genotype in men, P<.02). Part of the explanation for this difference may be that individuals with genotype A/A reported significantly lower consumption of tobacco per day than individuals with other genotypes (1.41 versus 2.87 g/d, P=.035), and this difference was observed consistently in both genders, in all regions, and in cases and control subjects. Overall, the data show that the G-455-A substitution is a strong and consistent predictor of plasma fibrinogen levels in all regions of Europe sampled but that the strength of the association is affected by factors such as gender, hormonal status, and smoking. In particular, the fibrinogen levels in individuals with genotype A/A (5% of the sample) who were smokers, who were female offspring of cases, or who used oral contraceptives show strong evidence of interaction, the mechanism of which is unknown. Although the G-455-A genotype itself is not a strong predictor of parental history of myocardial infarction, the data confirm the importance of a gene-environment interaction in determining plasma fibrinogen levels in individuals in Europe and thus risk of ischemic heart disease.
Key Words: gene:environment interaction • fibrinogen gene polymorphism • smoking • oral contraception • offspring study
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Introduction |
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It is now widely accepted that plasma fibrinogen levels are strongly associated with and an independent predictor of vascular disease, including ischemic heart disease (IHD), in both men and women. The effects on both IHD and stroke have been demonstrated in large prospective studies in Europe1 2 3 4 5 and the United States6 and in many case-control studies.7 8 9 The prospective Northwick Park Heart Study (NPHS) estimated that an increase of 1 SD in plasma fibrinogen levels (0.6 g/L) was associated with an increase in IHD risk of 84% over the next 5 years in middle-aged men,3 and the Framingham prospective study reported the predictive effect to be of similar magnitude in men and women and to be maintained in all age cohorts.6 Elevated fibrinogen in an individual could either be due to the presence of predisposing factors or be the consequence of the presence of atherosclerosis; evidence in favor of the former has come from a recent report from the European Atherosclerosis Research Study (EARS) group.10 This study compared the young (mean age, 23 years) offspring of fathers with myocardial infarction (MI) before the age of 55 years (designated cases) and control subjects from five different regions of Europe11 and has shown that fibrinogen levels were statistically significantly higher in the healthy male cases than in the control subjects (2.38 versus 2.29 g/L, P<.01), although this effect was not seen in women.10 This article explores possible genetic reasons for this gender and case-control difference.
The fibrinogen level observed in the plasma of an individual at any particular time is determined by the interaction between a number of specific environmental factors experienced by the individual and his or her genetic makeup. Fibrinogen levels increase in both men and women with age and obesity,12 13 14 15 16 and use of oral contraceptives, pregnancy, and menopause12 16 17 18 19 are also associated with raised fibrinogen levels. Diabetics have higher levels than nondiabetics,20 and levels have been reported to be higher in middle-aged individuals with reduced growth in fetal life and infancy.21 In one study of elderly individuals, fibrinogen levels were reported to be significantly higher during colder weather.22 Fibrinogen levels are not affected greatly by dietary factors23 but are significantly increased by smoking,12 15 16 which is the single major environmental factor determining fibrinogen levels in individuals in the general population. It has been suggested that a large part of the relation between smoking and IHD is mediated through the rise of fibrinogen levels.24 Finally, fibrinogen is an acute-phase protein, whose plasma levels rise rapidly after injury or infection.25 In one small study of 14 healthy individuals, the degree of within-individual variation for fibrinogen was estimated to be at least twice that for cholesterol.26 This is confirmed by other larger studies13 ; thus, a single measure of an individual's plasma fibrinogen level will result in a significant underestimate of the true relation between fibrinogen and, for example, subsequent risk of disease.
There is less information on the relative contribution of genetic
variation to the determination of plasma fibrinogen levels in the
general population. One study using path analysis in families of
healthy individuals and smokers estimated a heritability of
0.5,27 whereas two studies in twins reported a lower
heritability of 0.3.28 29 It is likely that variation at
the gene locus coding for the fibrinogen protein may contribute to the
genetic component determining plasma fibrinogen levels. Fibrinogen
consists of two of each of three polypeptide chains, A
, Bß, and
,30 joined together by disulfide bonds. Each chain is
encoded by a separate gene, which is in a cluster on chromosome
4.31 Variation in this gene region, as detected using DNA
polymorphisms, has been reported to be associated with differences in
plasma fibrinogen levels in some but not all studies of healthy
individuals9 28 31 32 33 34 35 and in some but not all studies of
patients with peripheral arterial disease36 37 or
premature MI.9 35 In a study from Scotland, genotype was
an independent predictor of risk of peripheral arterial
disease,37 but this was not observed in the ECTIM study of
MI survivors and control subjects from Belfast and
France.9
Of the polymorphisms, the G-A substitution in the 5' flanking region of the ß-fibrinogen gene appears to be associated with the most consistent differences in plasma fibrinogen levels in both smokers and nonsmokers in studies from the United Kingdom,32 from Sweden,35 and in healthy men and MI survivors in the ECTIM study.9 We therefore investigated the association between this G-A substitution and plasma fibrinogen levels in the individuals recruited for EARS. The study design allowed the comparison of the effect of genetic and environmental factors in healthy young men and women from different countries in Europe who have a father with documented IHD at a young age and those who do not.11 The effect of the G/A genotype on fibrinogen levels was thus compared in individuals with and without a family history of early IHD and in different genetic and environmental backgrounds, with particular focus on the effect of smoking and use of oral contraceptives.
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Methods |
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Subjects and Measures
A detailed description of the design of EARS is presented elsewhere.11 Briefly, students from 14 universities throughout Europe, aged between 18 and 26 years whose fathers had a verified heart attack before the age of 55, were recruited for the study and represent the cases. Two age- and sex-matched control subjects for each case were recruited by computer selection from the same university population. Fasting venous blood was collected, and details of lifestyle—ie, smoking, alcohol consumption, dietary intake, and physical exercise—as well as personal and family history and physiological measurements were taken using standardized questionnaires and protocols. Details of these protocols have been described elsewhere.11 Measurements of lipids and clotting factors were carried out as described.10 38 The within- and between-coefficients of variation of the laboratory measurements of fibrinogen have been reported10 and were 2% and 4%, respectively.
Isolation of DNA and Genotype Analysis
DNA was isolated using the "salting-out"
procedure.39 Samples were isolated in London and Reus, and
genotyping for the G-A substitution was carried out entirely in London.
The polymerase chain reaction (PCR) was carried out in a total volume
of 50 µL, containing 0.5 µg genomic DNA, 200 ng of each primer
(5'-CTCCTCATTGTCGTTGACACCTTGGGAC-3' and
5'-GAATTTGGGAATGCAATCTCTGCTACCT-3'), 200 µmol/L dNTP, 10 mmol/L
Tris-HCl (pH 8.3), 50 mmol/L KCl, 1.5 mmol/L MgCl2, 0.05%
W1, 0.01% (wt/vol) gelatin, and 1 U Taq polymerase. The
solution was overlaid with 50 µL liquid paraffin and underwent 1
cycle of 3 minutes at 95°C, 1 minute at 55°C, and 2 minutes at
72°C, followed by 29 cycles of 1 minute at 95°C, 1 minute at
55°C, and 1 minute at 72°C. PCR products (20 µL) were
electrophoresed on a 1% agarose gel and denatured in 0.5 mol/L NaOH
and 1.5 mol/L NaCl for 40 minutes. Denatured DNAs were then transferred
to two Hybond N+ nylon membranes (Amersham) by placing a membrane on
each side of the agarose gel and blotting overnight. Two
allele-specific oligonucleotides
(5'-AAAGGGGCCATTAAAAT-3' and
5'-ATTTTAATAGCCCCTTTT-3') were phosphorylated at their 5' ends with
[32P]dATP (Amersham) and T4 polynucleotide kinase (BRL
Ltd). Membranes were prehybridized individually in a solution
containing 5x SSPE, 5x Denhardt's, and 0.5% sodium dodecyl sulfate
at 4°C for 10 minutes. The labeled allele-specific oligonucleotide
probe was then added and hybridized at 4°C for 1 hour. Membranes were
washed together in 5x SSC and 0.1% sodium dodecyl sulfate at 46°C
for 5 minutes. Autoradiographs were prepared by exposing films (Konica
Corp) to membranes at -70°C for 2 to 12 hours. Genotyping was
determined blindly by two independent observers. Any discrepancies
between observers were resolved by repeat amplification and genotyping.
Data were compiled on an ASCII file and sent to INSERM U258 (Paris) for
analysis.
Statistical Analysis
Data were analyzed using the SAS statistical software package
(SAS Institute Inc). Because of technical difficulties in DNA isolation
from some centers (Naples, Innsbruck, and Zurich) or because of low
yields of DNA or contamination with inhibitors of PCR, genotypes were
not obtained for 303 samples (97 cases, 206 control subjects). Because
of difficulties with sample transport, fibrinogen measurements were not
available in 255 individuals, including all those from Zurich and 44%
of those from Naples. In total, a sample of 1559 subjects had both
genotype and fibrinogen measured. Since such technical difficulties
were not related to status or fibrinogen level, there should be no
consistent bias to the subsequent analysis. As previously
reported,11 the 14 recruitment centers were grouped into
five regions for analysis on the basis of IHD mortality rates and
geographic and linguistic proximity: Finland (Helsinki and Oulu) and
Great Britain (Bristol and Glasgow) and Northern (Gothenburg, Aarhus,
Hamburg), Middle (Innsbruck, Ghent, Zurich), and Southern (Bordeaux,
Naples, Barcelona, Reus) Europe. Observed numbers of each genotype were
compared with those expected if the sample were in Hardy-Weinberg
equilibrium using 
2 analysis. Allele frequencies in
different groups were compared by gene counting and 2
analysis. Statistical significance for this and all other tests was
taken at a value of P<.05. Since the skewness of the
distribution of fibrinogen in this sample was small,10 no
transformation was performed for the analyses. Plasma fibrinogen level
was compared between genotype groups using ANOVA, with adjustment for
the stratification criteria of the study (age, gender, region, and
status). The homogeneity of the association of genotype with fibrinogen
level among different levels of a covariate (eg, smoking) was tested
using interaction terms involving genotype and the considered
covariate. For several of these covariates (smoking and use of oral
contraceptives), there was evidence for interaction effects with
genotype, and thus ANOVAs were also performed separately for smokers
and nonsmokers and for women using and not using oral contraceptives,
men, women, and cases and control subjects. To estimate the magnitude
of the effect associated with the A allele, genotype was coded 0/1/2 as
an ordinate variable, assuming a codominant effect. A model assuming
additive effects of alleles with no dominance deviation (codominant
model) was fitted to the data, and no significant deviation from this
hypothesis was observed. Allele effects were thus estimated assuming a
codominance model.
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Results |
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Genotype was determined by PCR amplification and allele-specific oligonucleotide melting as shown in Fig 1
. For all
regions and in both cases and control subjects, genotype distribution
was that expected from Hardy-Weinberg proportions. Table 1 presents the number of genotypes obtained and the
relative allele frequencies in cases and control subjects in the five
regions. The frequency of the A allele varied from 0.179 in cases from
Finland to 0.246 in cases from Britain. The mean frequency was 0.222 in
women and 0.214 in men and slightly higher in cases than in control
subjects (0.223 versus 0.217, respectively), although none of these
differences were statistically significant.
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The association between genotype and fibrinogen level was next examined
separately in men and women and by status (cases and control subjects);
Table 2 presents the data. As shown previously,
fibrinogen levels were significantly higher in cases than control
subjects and in women than men.10 The covariates having a
significant independent effect in women were body mass index, use of
oral contraceptives, and smoking and in men were body mass index and
status. Exploratory statistical analysis had shown evidence for an
interaction between genotype and some covariates; for this reason, data
are presented after adjustment only for the stratification criteria
of the study, ie, age, sex, status, and region. In the control
subjects, the A allele was associated with elevated levels in both
genders, with a larger difference observed between the G/G and A/A
genotypes in men (0.27 g/L, 11%) than in women (0.11 g/L, 4.3%). In
the cases, the A allele was associated with elevated levels in men (A/A
versus G/G, +0.25 g/L, 10.9%), but in women, although those with
genotype G/A had elevated levels, those with genotype A/A had lower
levels (A/A versus G/G, -0.14 g/L, -5.5%; genotypexstatus,
P=.07). This pattern of lower levels in A/A female cases was
consistent in the five regions (data not shown).
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To explore possible explanations for this interaction with genotype, we
examined the relation between fibrinogen level and a number of
covariates in women with different genotypes. In women, a major
determinant of plasma fibrinogen level is use of oral contraceptives,
and as expected, women who reported use of oral contraceptives had
higher levels (2.64 versus 2.53 g/L, P=.003). In women not
reporting use (Table 3), there was a moderate
association between the A allele and elevated fibrinogen level in both
cases and control subjects, with those with genotype G/A having levels
0.06 to 0.10 g/L higher than those with genotype G/G. However, the
levels in those with genotype A/A were not correspondingly higher, and
overall, the effect of genotype did not reach statistical significance.
In the group using oral contraceptives, there was significant evidence
for an interaction between genotype and status; control subjects with
genotype A/A had among the highest and cases with genotype A/A among
the lowest fibrinogen levels. Although these effects are striking, the
numbers of individuals in these groups are small, and the significance
of the genotypexstatus interaction in the group taking oral
contraceptives is essentially due to the opposite effect between those
with genotype G/G and G/A in cases and control subjects. The borderline
significance of the interaction term in women shown in Table 2
thus
appears to be primarily due to the effect in those using oral
contraceptives, because there is no interaction in the group not using
oral contraceptives. Therefore, although based on small numbers,
following these analyses, the data from women taking oral
contraceptives were excluded in all further analyses to avoid examining
three-way interactions.
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The association of the A allele with elevated fibrinogen level was
consistent over the five regions of Europe studied (test of
homogeneity, P=.22), and the data are presented in Fig 2. Those with one or more A alleles had adjusted
fibrinogen levels 0.07 to 0.22 g/L higher than those lacking an A
allele; only in Great Britain were the mean levels not higher, and this
was the smallest sample.
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Smoking is known to be a major determinant of plasma fibrinogen level,
and the relation between genotype and fibrinogen level was examined in
men and women who reported as smokers or nonsmokers (Table 4). Although for both genders there was no evidence of a
significant interaction between genotype and status, the data are
presented for cases and control subjects separately and adjusted
only for age and region. For the nonsmokers, both female and male
control subjects with genotype A/A had elevated fibrinogen levels
compared with those with genotype G/G, with a larger difference
observed in men than women (0.33 g/L, 14.6% and 0.07 g/L, 2.8%,
respectively). In both male and female cases, the difference between
the G/G and A/A genotype groups was larger (0.49 and 0.12 g/L,
respectively), although the interaction was not significant. By
contrast, in both men and women who were smokers, although those with
genotype G/A had levels slightly elevated over those with genotype G/G,
those with genotype A/A had the lowest levels; although the number of
subjects was small, this effect was seen in both cases and control
subjects (male control subjects, -0.10 g/L, -4.5%; female control
subjects, -0.08 g/L, -3.3% lower than the G/G group). After the data
from the cases and control subjects were combined, this interaction in
men between genotype and smoking was statistically significant
(P=.02). Among nonsmokers, the effect of the A allele was
estimated assuming a codominant effect as +0.15 g/L in men
(P<.0001) and +0.04 g/L in women (P=NS); the
interaction of the effect with gender was significant
(P=.04). Data were also available for reported tobacco use
for each individual based on the number and type of cigarettes smoked.
As shown in Table 5, this value was consistently lower
in individuals with genotype A/A compared with those with the G/G and
G/A genotypes. These differences, which were significant overall, were
consistent in individuals divided into cases and control subjects, men
and women, and by region. In all regions, fewer individuals with
genotype A/A reported use of cigarettes compared with those with other
genotypes (overall, 15.2% versus 26.9%, P<.01). Overall,
17.4% of individuals reported that they had stopped smoking, with no
significant difference between the three genotype groups.
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Of the other measured factors, there was no evidence for interactions that were consistent between men and women between genotype and body mass index, blood pressure, alcohol consumption, physical activity, or measures of plasma fatty acids, glucose, or plasma lipid traits in the determination of fibrinogen levels.
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Discussion |
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EARS compares a sample of young healthy men and women selected from different regions in Europe with the aim of comparing genetic and environmental factors in the offspring of fathers with and without early IHD (cases and control subjects). Because of the size of the study and the extent of the information available on lifestyle factors, it is possible to explore evidence for genotype-environment interaction. The data presented here focus on the relation between the G/A genotype at -455 bp of the ß-fibrinogen gene and fibrinogen levels in these different groups of individuals. This genotype was chosen because of previous association with fibrinogen levels in healthy men and in MI survivors.9 32 33 34 35 In this, the largest study of healthy men and women reported to date, results strongly confirm the association reported by others9 33 34 between the A-455 allele and higher plasma fibrinogen levels. However, although gene-environment interactions were generally hypothesized, the specific interactions detected were not proposed a priori, and multiple testing may have resulted in some chance findings, which will need to be confirmed.
The prevalence of the A allele was similar in all European regions studied, showing no evidence for a north-south gradient, and the frequency was similar in cases and control subjects. This is not surprising because the development of IHD is multifactorial, and variation at any single gene is likely to make only a small contribution to overall risk in the general population. For example, it has been estimated that variation at the apolipoprotein E locus, as determined by the three common isoforms, explains only 2% to 3% of the population risk of IHD, although this polymorphism explains 8% to 10% of the population variance of levels of low-density lipoprotein,40 a well-known risk factor for atherosclerosis. In addition, even if the frequency of any particular allele is significantly higher in the fathers with early IHD, the study has a limited power to detect a frequency difference in offspring. The power of the EARS design to detect such frequency differences has recently been examined,41 and assuming a codominant model of effect on risk and a frequency of the ß-fibrinogen A allele among control subjects of 0.2, the size of the present sample would have a 90% power to detect a difference in frequency between cases and control subjects if the odds ratio for IHD associated with the A allele is 1.55 or greater. This would correspond to an allele frequency in the affected fathers of 0.28, and because no such frequency difference was detected, this implies that the relative risk, if any, associated with this genotype is likely to be less than 1.55 in this group of individuals.
Although the G/A genotype was not a significant predictor of case-control status, it was strongly associated with fibrinogen level in the men from all five regions studied. In the male offspring of both cases and control subjects, the effect associated with genotype was similar, with those nonsmokers with genotype A/A having levels roughly 16% (0.37 g/L) above those with genotype G/G (cases and control subjects combined). By extrapolation from NPHS,3 this higher fibrinogen level would be predicted to increase the future risk of IHD of A/A individuals by roughly 32%, although the subjects of the NPHS were older than those of EARS (40 to 64 years versus 18 to 26 years) and results are not directly transposable. In this sample of men, G/A genotype explained 2% of the sample variance, with other significant measured factors including body mass index and case-control status explaining an additional 2.6%. Because of the large within-individual variability in fibrinogen level over time, these are underestimates of the size of the true effects but are likely to be an accurate estimate of the relative contributions associated with genotype G/A and measured environmental factors on the variance of fibrinogen levels in healthy men. The effect of genotype G/A seen here is similar in size to that previously reported in healthy men by other researchers (eg, G/G versus A/A difference of 0.31 g/L33 and 0.17 g/L9 ). By contrast, in women the effect of genotype G/A was much smaller, with the A/A group being only 0.11 g/L higher than the G/G group in control subjects. Overall, significant measured factors (body mass index, tobacco, oral contraception) explained 9.0% of the variance in women, with only 0.3% of the variance explained by genotype.
It is possible to propose a molecular mechanism for the effect on fibrinogen level associated with genotype G/A. It has been shown using pulse-chase experiments and more recently by transfection of the Bß chain cDNA into HepG2 cells that synthesis of the Bß chain is the rate-limiting step in fibrinogen formation42 43 44 ; it is therefore plausible that changes in the transcription rate of the ß-fibrinogen gene will alter the production rate of the protein. The region up to 150 bp upstream of the start of transcription has been shown to contain the information necessary for liver-specific transcription of the ß-fibrinogen gene,45 whereas a region between -150 and -82 bp is responsible for interleukin-6 induction.46 Farther upstream a sequence between -2900 and -1500 has been shown to be necessary for dexamethasone induction.46 Because of the location of the G/A variable site at -455 bp, it is possible that the substitution may itself be a functional change, which affects the affinity of a nuclear protein involved in the control of transcription and the gene.47 However, recently it has been found that the A-455 sequence change is in complete allelic association in all Caucasian populations studied to date, with a C-148-T change48 located close to the consensus sequence of the interleukin-6 element.49 Fibrinogen transcription is markedly increased by cytokines such as interleukin-6 that are induced in response to injury,50 51 52 with one likely source of such cytokines being from macrophages recruited to the lungs as a result of damage from smoking. This raises the possibility that the G-A change is acting as a neutral marker for the functional C-T change, and to test this hypothesis, experiments are in progress to insert this gene fragment into the appropriate vector to test promoter strength.
Since the effect associated with the G-A substitution is smaller in women than men, even though plasma fibrinogen levels are 9% higher in women, this implies that hormones or other gender-specific factors modulate the transcription of the gene. Gender-specific effects associated with polymorphisms in apolipoprotein genes have been reported in several studies, with, in general, the effects of genetic factors being larger in men than women.53 54 The effect of hormones on the association between genotype G/A and fibrinogen level was examined further in women who reported use of oral contraceptives. The raising effect associated with the A allele is small but consistent in both cases and control subjects who do not use oral contraceptives but is variable in women who report use of oral contraceptives. Compared with the G/G groups, cases with genotype G/A have higher levels and control subjects have lower levels; by contrast, cases with genotype A/A have the lowest and control subjects among the highest levels. Although it would be possible to propose a molecular mechanism, with hormones having an allele-specific effect on transcription, the numbers are small and the explanation of a chance observation or sample bias cannot be ruled out. A recent study examining the effects on plasma lipid levels associated with polymorphisms in the apolipoprotein genes also found evidence suggestive of interaction with oral contraceptive use,52 with polymorphisms being associated with larger effects on lipid traits in those women using oral contraceptives. In EARS, it has previously been noted that the case-control differences in levels of low-density lipoprotein cholesterol and apolipoprotein B were more marked in women using oral contraceptives than in those who did not.55
Most of the effect of interaction between genotype and status observed
in women was explained by this effect of oral contraceptive use; thus,
all subsequent analyses were carried out excluding women using oral
contraceptives. There was then no significant evidence for
heterogeneity of genotype effects in cases and control subjects in
either gender. However, after the data from cases and control subjects
were combined, there was significant evidence of an interaction between
genotype and smoking. In both men and women nonsmokers, the pattern
observed was consistent with a codominant raising effect associated
with the A allele, with an estimated effect of +0.15 g/L in men but a
significantly smaller effect (+0.04 g/L) in women, and with those with
genotype G/A having intermediate levels. For the smokers, in both men
and women, those with genotype G/A had elevated levels but those with
genotype A/A had significantly lower levels than observed in the G/G
group, with statistical evidence for interaction in men
(P<.02). However, when only genotypes G/G and G/A are
considered, the interaction term is no longer significant, in
accordance with previous reports showing no interaction with genotype
G/A and smoking.9 33 Since the interaction observed here
is due to the particularly low fibrinogen levels in the A/A smokers,
one explanation for this could be that for unknown reasons or by
chance, people with genotype A/A and a high fibrinogen level have
stopped smoking and are now in the group of nonsmokers, as is suggested
by the data in Table 5 showing that the prevalence of smokers and use
of tobacco are lower in the genotype A/A group. The possibility of some
sample bias cannot be ruled out if, for example, those individuals who
smoke heavily declined to participate in the study. This appears
unlikely, because the frequency of individuals with genotype A/A was
not significantly lower than expected (81 predicted subjects from the
allele frequency and 92 observed).
Overall, these studies confirm the importance of the G-A substitution in determining plasma fibrinogen levels in individuals in different regions of Europe and particularly in men. Because of the consistency and magnitude of the effect, it can be predicted that this single DNA sequence change is associated with a small but clinically relevant risk of IHD in men. If further such sequence changes can be detected in the fibrinogen genes or other genes that determine levels of risk factors for IHD, such information will be useful in determining management strategy for individuals. In particular, there appear to be important effects of interaction between such genetic variation, gender-specific factors, and environmental factors such as smoking and use of oral contraceptives. Understanding the molecular mechanism of such interaction will add significantly to the usefulness of DNA-based tests for identifying individuals at risk of IHD.
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Acknowledgments |
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This work was supported by a grant from the European Economic Community and by the British Heart Foundation (RG16 and 87/78). We thank Gina Deeley for assistance in preparation of the manuscript.
The European Atherosclerosis Research Study (EARS) included the following participants and centers:
EARS project leader: J. Shepherd, Glasgow, UK. EARS project management group: F. Cambien, Paris, France; G. De Backer, Ghent, Belgium; M.-M. Galteau, Nancy, France; D. St. J. O'Reilly, Glasgow, UK; M. Rosseneu, Brugge, Belgium; and L. Wilhelmsen, Göteborg, Sweden. EC COMAC–epidemiology liaison officer: T. Sorensen, Copenhagen, Denmark.
The EARS group, collaborating centers, and their associated investigators: Austria: C. Sandholzer, C. Duba, H.-G. Kraft, H.-J. Menzel, Institute for Medical Biology and Genetics, University of Innsbruck; recruitment center and laboratory. Belgium: G. De Backer, S. De Henauw, D. De Bacquer, A. Bael, Department of Hygiene and Social Medicine, State University of Ghent; recruitment center. Belgium: M. Rosseneu, C. Labeur, N. Vinaimont, Department of Clinical Chemistry, University Hospital St Jan, Brugge; laboratory. Denmark: C. Gerdes, O. Faergeman, Medical Department I, Aarhus Amtssygehus, Aarhus; recruitment center and laboratory. Finland: C. Ehnholm, National Public Health Institute, Helsinki; recruitment center and laboratory; R. Elovaino, J. Peräsalo, The Finnish Student Health Service. Finland: A. Kesaniemi, Department of Internal Medicine, University of Oulu; recruitment center; P. Palomaa, The Finnish Student Health Service; recruitment center. France: F. Cambien, L. Tiret, R. Agher, V. Nicaud, R. Rakotovao, INSERM U258, Unité de Recherche d'Epidémiologie Cardiovasculaire, Hôpital Broussais, Paris; EARS Data Centre and recruitment center. France: M.-M. Galteau, S.M. Visvikis, Centre de Médecine Préventive, Nancy; EARS Central Laboratory. France: J.C. Fruchart, J.M. Bard, P. Lebel, Service de Recherche sur les Lipoprotéines et l'Athérosclérose (SERLIA), INSERM U325, Institut Pasteur, Lille; laboratory. France: L. Bara; Laboratories de Thrombose Expérimentale, Paris; laboratory. France: C. Bady, J. Beylot, A. Lindoulsi, L. Tiret, UFR de Santé Publique, Bordeaux; recruitment center. Germany: U. Beisiegel, A. Jorge, M. Papanikolaou, I Medizinische Klinik Universitätskrankenhaus, Hamburg; recruitment center and laboratory. Italy: E. Farinaro, C. Cortese, M. Liguori, F. De Lorenzo, Institute of Internal Medicine and Metabolic Disease, University of Naples; recruitment center. The Netherlands: L.M. Havekes, P. de Knijff, IVVO-TNO Health Research, Gaubius Institute, Leiden; laboratory. Spain: S. Sans, T. Puig, Programma CRONICAT, Hospital Sant Pau, Barcelona; recruitment center. Spain: J. Ribalta, J. Balanya, P.R. Turner, L. Masana, Unitat Recerca Lipids, Universitat Barcelona, Reus; recruitment center and laboratory. Sweden: L. Wilhelmsen, I. Wallin, S. Johansson, Department of Medicine, Ostra Hospital, University of Göteborg, Göteborg; recruitment center. Switzerland: F. Gutzwiller, B. Marti, M. Knobloch, P. Anliker, Institute of Social and Preventive Medicine, University of Zurich; recruitment center. United Kingdom: D. Stansbie, H. Denton, S. Plumridge, Department of Chemical Pathology, Bristol, Royal Infirmary; recruitment center. United Kingdom: J. Shepherd, D. St J. O'Reilly, G.W. Tait, G.M. Hamilton, Institute of Biochemistry, Royal Infirmary, Glasgow; recruitment center and laboratory. United Kingdom: S. Humphries, P. Talmud, S. Ye, University College London School of Medicine, London; laboratory.
Received October 12, 1994; accepted October 31, 1994.
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