ß-Fibrinogen Gene Polymorphism (C₁₄₈T) Is Associated With Carotid Atherosclerosis

Results of the Austrian Stroke Prevention Study

From the Institute for Medical Biochemistry (H.S., P.B., G.M.K.) and the Departments of Neurology (R.S., K.N., S.H., B.R.) and Internal Medicine (M.S., V.W.), Karl-Franzens University, Graz, Austria.

Correspondence to Helena Schmidt, MD, Institute for Medical Biochemistry, Karl-Franzens University Graz, Harrachgasse 21, A-8010 Graz, Austria. E-mail helena.schmidt{at}kfunigraz.ac.at

	Abstract

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Abstract—Polymorphisms at the ß-fibrinogen locus have been shown to be associated with plasma concentration of fibrinogen and coronary heart disease. The effect of the genetic heterogeneity of fibrinogen on carotid atherosclerosis has not been determined so far. We examined the influence of the C₁₄₈T polymorphism on carotid disease in a large cohort of middle-aged to elderly subjects without evidence of neuropsychiatric disease. This polymorphism is located close to the consensus sequence of the interleukin-6 element and may represent a functional sequence variant. The genotype of 399 randomly selected, neurologically asymptomatic individuals, aged 45 to 75 years, was determined by denaturing gradient gel electrophoresis. Carotid atherosclerosis was assessed by color-coded duplex scanning and was graded on a five-point scale ranging from 0 (=normal) to 5 (=complete luminal obstruction). The C/C, C/T, and T/T genotypes were noted in 226 (56.6%), 148 (37.1%), and 25 (6.3%) individuals, respectively. The T/T genotype group demonstrated higher grades of carotid atherosclerosis than did the C/C and C/T genotypes (P=.003). Logistic regression analysis created a model of independent predictors of carotid atherosclerosis that included apolipoprotein B (odds ratio [OR], 1.17/10 mg/dL), age (OR, 2.46/10 years), lifetime tobacco consumption (OR, 1.03/1000 g), presence of the ß-fibrinogen promoter T/T genotype (OR, 6.17), plasma fibrinogen concentration (OR, 1.05/10 mg/dL), and cardiac disease (OR, 1.80). These data suggest that the ß-fibrinogen promoter T/T148 genotype represents a genetic risk factor for carotid atherosclerosis in the middle-aged to elderly.

Key Words: fibrinogen • genetics • atherosclerosis • carotid arteries

	Introduction

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There are numerous studies describing an association between plasma fibrinogen levels and coronary heart disease,^{1 2 3 4 5 6} stroke,^{5 6 7} and carotid atherosclerosis.^{8 9 10 11 12 13 14 15 16 17 18} However, it is unclear whether the elevations in plasma fibrinogen level are a causal factor in the development of atherosclerosis or only an epiphenomenon of the atherogenic process.

Fibrinogen concentration is controlled by genetic and environmental factors, including smoking, obesity, use of contraceptives, trauma, and lack of exercise, which have been reported to elevate fibrinogen concentrations.^{19 20 21} Fibrinogen level also increases with age and in the presence of diabetes mellitus, hypertension, or lipid abnormalities.^{5 6}

The estimate of heritability for fibrinogen is in the range of 30% to 50%, depending on the study design.^{22 23} Theoretically, any gene coding for proteins involved in fibrinogen metabolism may have an impact on the genetic regulation of the plasma fibrinogen level. The synthesis of the Bß chain has been shown to be the rate-limiting step in the formation of fibrinogen.²⁴ The 5' region of the ß gene contains binding sites for several trans-acting factors, which largely control expression of the gene.^{25 26 27} Several polymorphisms have been identified within this region.^{28 29 30} One of them, the C₁₄₈T polymorphism, is located close to an interleukin-6–responsive element and may affect fibrinogen gene expression, mainly in response to the acute-phase reaction.²⁸ There are some population-based studies that have investigated the effect of polymorphisms in the promoter region of the ß-fibrinogen gene on fibrinogen level and the risk of coronary atherosclerosis.^{30 31 32} Their results are controversial. To our knowledge, this is the first investigation on the effect of the C₁₄₈T polymorphism on carotid atherosclerosis.

	Methods

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Study Population
Individuals aged 45 to 75 years and stratified by gender and 5-year age groups were randomly selected from the official register of residents of the city of Graz, Austria. They were all white and of central European origin. They received a written invitation to participate in the Austrian Stroke Prevention Study, a single-center, prospective, follow-up study in our community. The study had been approved by the Medical Ethics Committee of the Karl-Franzens University of Graz. Written, informed consent was obtained from all study participants. The rationale and design of the Austrian Stroke Prevention Study have been previously described.³³ In brief, the objective of the study was to examine the frequency of cerebrovascular risk factors and their effects on carotid atherosclerosis as well as on cerebral morphology and function in the elderly. The inclusion criteria for the study were no history of neuropsychiatric disease and a normal neurological examination. From a total of 8193 individuals invited between September 1991 and March 1994, a sample of 2794 subjects agreed to participate, of whom 1998 individuals fulfilled the inclusion criteria. All study participants underwent a structured clinical interview, a physical and neurological examination, three blood pressure readings, electrocardiography, and echocardiography as well as laboratory investigations including blood cell count and a complete blood chemistry panel. Every fourth study participant was then invited to enter phase II of the Austrian Stroke Prevention Study, which included Doppler sonography, magnetic resonance imaging, single-photon emission computed tomography, and neuropsychological testing. Since 1993 we also performed ß-fibrinogen promoter genotyping in all phase II attendees. The current study cohort consists of those 399 individuals who underwent both carotid duplex scanning and assessment of the ß-fibrinogen polymorphism. There were 204 women and 195 men. The mean±SD age of this cohort was 60.1±6.0 years.

Vascular Risk Factors
The diagnosis of major risk factors for stroke, including arterial hypertension, diabetes mellitus, and cardiovascular disease, relied on the individual's history and appropriate laboratory findings. A detailed description of the definition of these risk factors is given elsewhere.^{34 35}

Study participants were defined as current smokers, ex-smokers, or never-smokers. For current smokers and ex-smokers, information was obtained about the daily number of items smoked and the smoking duration in years. The data on the amount of tobacco were converted to grams of tobacco consumed during their lifetime by using the following conversion factors: 1 cigarette=1 g, 1 cheroot=3 g, and 1 cigar=5 g. Body mass index was calculated as weight in kilograms divided by the square of height in meters squared. The regular use of estrogen replacement therapy was recorded among all female study participants.

For measurements of hematocrit, blood was obtained from a large antecubital vein without stasis. Lipid status, including the level of triglycerides, total cholesterol, LDL cholesterol, HDL cholesterol, lipoprotein(a), apolipoprotein B, and apolipoprotein A-I, was determined for each study participant. Triglycerides and total cholesterol were enzymatically determined by using commercially available kits (Uni-Kit III "Roche" and MA-Kit 100 "Roche," Hoffman –La Roche). HDL cholesterol was measured by the use of the TDx REA cholesterol assay (Abbott). LDL cholesterol was calculated by the equation of Friedewald. The lipoprotein(a) concentration was determined by the electroimmunodiffusion method using a reagent kit containing monospecific anti-lipoprotein(a) antiserum and the Rapidophor M3 equipment (Immuno AG). The levels of apolipoprotein B and A-I were assessed by an immunoturbidimetric method utilizing polyclonal antibodies and a laser nephelometer (Behringwerke AG). The plasma fibrinogen concentration was measured according to the Clauss method³⁵ by using the recommendations and reagents of Behringwerke AG.

Isolation of DNA and Genotype Analysis
High-molecular-weight DNA was extracted from peripheral whole blood by using Qiagen genomic tips (Qiagen Inc) according to the protocol of the manufacturer. Genotyping was performed by denaturing gradient gel electrophoresis (DGGE). This procedure is routinely used in our laboratory and is preferred over restriction enzyme digestion because it detects point mutations within the amplified DNA fragment with high sensitivity and does not require further processing of polymerase chain reaction (PCR) products.^{36 37} A 417-bp-long fragment containing part of the promoter region and exon 1 (from -263 to +114 nucleotides) of the ß-fibrinogen gene and a 40-bp-long GC clamp serving as an artificial high-melting-point domain for DGGE was amplified by using two oligonucleotides (5'-GC clamp CTC TTT GAG GAG TGC CCT AAC TTC C-3' and 5'-TGT CGT TGA CAC CTT GGG ACT TAA C-3'). PCR was performed on 1 µg of genomic DNA in a buffer containing 10 mmol/L Tris (pH 8.3), 50 mmol/L KCl, 1.5 mmol/L MgCl₂, 0.2 mmol/L of each dNTP, 0.5 µmol/L of each primer, and 1 U of Taq DNA polymerase in a final volume of 50 µL. After 5 minutes at 94°C, amplification was carried out in 40 cycles consisting of 1 minute at 94°C, 1 minute at 52°C, and 2 minutes at 72°C on a Mastercycler (Eppendorf). A final elongation step was performed for 10 minutes at 72°C. Amplification was assessed by electrophoresing 5 µL of the PCR product on a 1.5% agarose gel stained with ethidium bromide.

The melting domain map for the DGGE analysis was calculated with the MELT87 computer algorithm.³⁸ PCR products were analyzed on 8% polyacrylamide gels containing a 20% to 50% linearly increasing denaturing gradient (100% denaturant is equivalent to 7 mol/L urea and 40% [vol/vol] deionized formamide). Electrophoresis was performed at 100 V at 60°C for 16 hours in TAE buffer (40 mmol/L Tris acetate, 1 mmol/L EDTA, pH 7.5). Gels were stained with ethidium bromide and examined under UV illumination. DNA samples with known genotypes were used initially to determine the position of the bands of the different genotypes. The C/C genotype corresponded to the lower band; the T/T genotype, the higher band; and the C/T genotype, a four-band pattern resulting from the two homoduplexes and two heteroduplexes.

Sequencing of the control DNA samples used as standards for the DGGE analysis was performed on a model 373A automated DNA sequencer (Perkin Elmer/Applied Biosystems Inc) and applying the dye terminator cycle sequencing ready reaction kit (model No. 402079, Perkin Elmer/Applied Biosystems).

Carotid Artery Duplex Scanning
A color-coded device (Diasonics, VingMed CFM 750) was used to determine atherosclerotic vessel wall abnormalities of the carotid arteries. All B-mode and Doppler data were transferred to a Macintosh personal computer for postprocessing and storage on optical disks. The imaging protocol involved scanning of both common and internal carotid arteries in multiple longitudinal and transverse planes and has been previously described.^{33 34} The examinations were performed by one experienced physician (S.H.). Image quality was assessed and graded into good (common and internal carotid arteries clearly visible and internal carotid arteries detectable for a distance >2 cm), fair (common and internal carotid arteries sufficiently visible and internal carotid arteries detectable for a distance of 2 cm), and poor (common and internal carotid arteries insufficiently visible or internal carotid arteries detectable for a distance <2 cm). Examinations of poor quality were excluded from further analysis. Measurements of maximal plaque diameter were done in longitudinal planes, and the extent of atherosclerosis was graded according to the most severe visible changes in the common and internal carotid arteries as follows: 0=normal, 1=vessel wall thickening (<1 mm), 2=minimal plaque (one 2 mm), 3=moderate plaque (two 3 mm), 4=severe plaque (>3 mm), and 5=lumen completely obstructed. Assessment of the intrarater reliability of this score was done in 50 randomly selected subjects and yielded a kappa value of 0.83.

Statistical Analysis
We used the Statistical Package for the Social Sciences (SPSS/PC+) for data analysis. Categorical variables among the three ß-fibrinogen genotypes were compared by the ² test. Assumption of a normal distribution for continuous variables was tested by Lilliefors statistics. Normally distributed continuous variables were compared by one-way ANOVA, whereas the Kruskal-Wallis test was used for comparison of nonnormally distributed variables. To assess the relative contribution of the three ß-fibrinogen genotypes on the presence of carotid atherosclerosis, we used multiple logistic regression analysis. The sonographic score was dichotomized as normal (grade 0) or abnormal (grades 1 to 5). Odds ratios (ORs) and 95% confidence intervals (CIs) with and without adjustment for age were calculated from the ß coefficients and their SEs. We used first-order interaction terms to evaluate whether or not the association between ß-fibrinogen genotype and the presence of carotid atherosclerosis was modified by plasma fibrinogen level, lifetime tobacco consumption, or use of hormone replacement therapy.^{19 20 21} These factors were considered because previous studies demonstrated that polymorphisms at the ß-fibrinogen locus may influence the plasma concentration of fibrinogen and that this may in turn be modified by smoking and hormone therapy. We used forward stepwise regression to create a model of independent predictors of carotid disease. At each step, each variable not in the model was assessed as to its contribution to the model, and the most significant variable was added to the model. This process continued until no variable not in the model made a significant (P<.05) contribution. ORs and 95% CIs were calculated from the ß coefficients and their SEs.

	Results

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Using DGGE between nucleotides -263 and +114 in the ß-fibrinogen gene, we were able to detect only one sequence variation. This sequence alteration was identical to the known C₁₄₈T polymorphism. The overall allele frequency of the T148 allele was found to be .248, similar to what has been observed in other countries in Europe.^{34 35} The C/C, C/T, and T/T ß-fibrinogen promoter genotypes were noted in 226 (56.6%), 148 (37.1%), and 25 (6.3%) study participants, respectively, and these values were in Hardy-Weinberg equilibrium.

Table 1 compares demographic variables and risk factors among the three genotypes. As shown in this table, there were no statistically significant differences among the three groups. The plasma fibrinogen concentration was almost equal in the three subsets. There were also no differences in fibrinogen level among the C/C, C/T, and T/T ß-fibrinogen promoter genotypes in current smokers (306.4±85.8 mg/dL, 300.6±62.5 mg/dL, and 299.7±95.0 mg/dL, respectively; P=.92) and in the subset of 55 women who were on estrogen replacement therapy (282.3±69.6 mg/dL, 277.4±56.0 mg/dL, and 288.0±46.0 mg/dL, respectively; P87).

The quality of carotid duplex examinations was good in 389 (97.5%) and fair in 8 (2.0%) subjects. Only 2 (0.2%) studies were of poor quality and were thus excluded from further analysis. As shown in Table 2, subjects carrying the T/T ß-fibrinogen promoter genotype had atherosclerotic carotid abnormalities more commonly than did their counterparts with either the C/C or C/T genotype with very similar sonographic findings. Table 2 demonstrates that the most striking differences between the T/T genotype and the two other genotypes were seen with respect to the extremes of the duplex score. Normal findings occurred in only 12.0% of T/T carriers but in 46.4% of C/C and 45.9% of C/T carriers. By contrast, grade 4 atherosclerotic changes were noted in 20.0% of subjects with the T/T genotype but in only 4.0% and 4.7% of those with the C/C and C/T genotypes, respectively. There were no individuals with grade 5 changes. Overall, atherosclerotic carotid abnormalities were recorded in 53.6% of the C/C and 54.1% of the C/T carriers but in 88.0% of the T/T carriers, and this difference was statistically significant (P=.003). The unadjusted and age-adjusted ORs for abnormal sonographic findings in the T/T genotype relative to the other two genotypes was 6.29 (1.91 to 20.71 95% CI; P=.003) and 5.97 (1.77 to 20.15 95% CI; P=.005), respectively. On the basis of our finding that the T/T genotype subset was, on average, slightly older than those with the C/C and C/T genotypes, we repeated our analyses after matching the investigational groups for age to avoid overreliance on statistical adjustment. For 22 individuals of the T/T group, we were able to randomly select 3 individuals of the C/C and C/T groups each of whose ages were ±2 years of that of a given T/T carrier. The ages of the matched C/C, C/T, and T/T subgroups were 62.6±6.1, 62.9±6.4, and 62.8±6.5 years, respectively, (P=.97), and there were no significant between-group differences in demographics and vascular risk factors. As in the entire cohort, T/T carriers demonstrated a higher frequency and severity of carotid atherosclerosis (P=.01). In this age-matched subset of study participants, the ORs for the presence of carotid artery disease associated with the T/T genotype was 4.38 (1.28 to 15.04 95% CI; P=.02). The interaction terms T/T genotypexplasma fibrinogen, T/T genotypexlifetime tobacco consumption, and T/T genotypexuse of oral contraceptives were not associated with evidence of carotid atherosclerosis in the total study group. The respective ORs were 1.001 (0.98 to 1.02 95% CI; P=.92), 1.0002 (0.99 to 1.001; P=.65), and 0.50 (0.42 to 0.67; P=.64). When we used forward stepwise regression analysis to create a model of predictors of atherosclerotic changes in the carotid arteries, the T/T genotype remained significantly and independently associated with evidence of abnormal sonographic findings. Apolipoprotein B entered the model first, age second, lifetime tobacco consumption third, the T/T ß-fibrinogen promoter genotype fourth, plasma fibrinogen fifth, and cardiac disease sixth (Table 3). All other variables, including male sex, hypertension, diabetes mellitus, fasting blood glucose level, current and former smoking status, body mass index, other lipid fractions, and hematocrit did not enter the model.

	Discussion

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In the current study, we have demonstrated an association between the T/T148 genotype at the ß-fibrinogen locus and the presence of carotid atherosclerosis in a neurologically asymptomatic, randomly selected population aged 45 to 75 years. T/T carriers tended to be older than their counterparts with the C/C or C/T genotype, but the ß-fibrinogen promoter polymorphism remained significantly related with vessel wall status even after adjustment for age by either multivariate logistic regression analysis or analysis of subgroups of study participants closely matched for age. We found that not only the T/T genotype but also plasma fibrinogen concentration are independent predictors of carotid atherosclerosis in addition to the well-established risk factors age, apolipoprotein B, lifetime tobacco consumption, and cardiac disease. The C₁₄₈T polymorphism was not correlated with plasma fibrinogen concentrations in our study participants.

There are at least 10 polymorphisms present in the ß-fibrinogen gene.²⁹ Of particular interest are sequence alterations in the 5'-flanking region of the gene, because this region contains several regulatory elements that control gene expression under different conditions.^{25 26 27} The increased fibrinogen synthesis during the acute-phase reaction is due to a higher transcriptional rate and is mainly mediated by interleukin-6.³⁹ Anderson et al²⁶ characterized two distinct sequence elements required for maximal induction of transcription by interleukin-6. One of them is similar to the interleukin-6 responsive-element core sequence of the rat ₂-macroglobulin gene promoter and lies between nucleotides -137 and -143. The other is a CAAT-enhancer binding protein (C/EBP) binding site between nucleotides -124 and -133. The C₁₄₈T polymorphism lies in the direct vicinity of these regions. It is thought to modulate acute-phase fibrinogen response by altering the binding of hepatic nuclear proteins to this part of the DNA.⁴⁰ Thus, this polymorphism may represent a functional sequence variant. Some evidence for such a mechanism has been given in a study by Montgomery et al,⁴¹ who reported that the acute rise in fibrinogen concentration after physical activity in young men is influenced by the G₄₅₅A (ß HaeIII) polymorphism, which is tightly linked with the C₁₄₈T polymorphism.^{28 29} We have found that subjects homozygous for the T148 allele have a 6.17-fold increased risk for carotid atherosclerotic abnormalities when compared with subjects with the C/C or C/T genotype after adjustment for age. Because this trend was seen to occur independently of fibrinogen concentration, we speculate that a transient, fibrinogen promoter genotype–dependent rise of fibrinogen levels in response to repeated extrinsic and intrinsic stimuli might play a role in the etiology of carotid atherosclerosis, irrespectively of the possible atherogenic effects of baseline fibrinogen concentrations. We most likely measured baseline fibrinogen values, as our study participants were clinically normal at the time of the examination. We found that tobacco consumption in all subjects and use of oral contraceptives in women did not modify the association of the ß-fibrinogen promoter genotype and carotid atherosclerosis. It is therefore unlikely that these two factors were extrinsic stimuli in the present study cohort.

There have been several studies that have addressed the question of the extent of variation in the ß-fibrinogen gene and its influence on the baseline plasma content of fibrinogen. In the ECTIM Study, the G₄₅₅A (ß HaeIII) polymorphism was shown to be significantly associated with the level of fibrinogen.³¹ In the EARS Study, the association between the G₄₅₅A (ß HaeIII) polymorphism and plasma fibrinogen was present, but this relation was affected by sex, hormonal status, and smoking habits.³² In a recent report from the ECTIM Study, the presence of the BclI polymorphism in the 3'-region, which is also tightly linked with the C₁₄₈T polymorphism,²⁹ was shown to be highly correlated with the severity of coronary atherosclerosis in myocardial infarction patients. The BclI polymorphism has also been implicated by Fowkes et al⁴² in occlusive peripheral artery disease. Similar to our results, they found that the polymorphism was associated with the presence of atherosclerosis without influencing plasma fibrinogen levels. A recent study by Carter et al⁴³ has investigated the effect of the arginine-to-lysine substitution at position 448 on fibrinogen levels and the risk for stroke. They observed an association between the Bß448 polymorphism and baseline fibrinogen levels in male patients only, but similar to our results, not in male control subjects or females. Regarding the effect of genotype on stroke, they found that the polymorphism was associated with a lower risk in females. The authors therefore speculated that genetic variations at the ß-fibrinogen locus might modulate the risk for stroke through different mechanisms in males and females. The participants of the EARS Study were young men and women (aged 18 to 26 years) and those of the ECTIM Study, young to middle-aged men (25 to 64 years). The study population investigated by Fowkes et al⁴² and Carter et al⁴³ consisted of men and women with an age range of 55 to 74 years and 68 to 82 years, respectively, close to the age range of our study population. It is conceivable that the effect of the polymorphism on fibrinogen level and its dependence on environmental factors and sex disappears with advancing age and therefore was not detectable in older study populations like those examined by Fowkes, Carter, and us.

Another explanation for the controversial results on the association between genotype and fibrinogen level in the different studies may be the high intraindividual variation in fibrinogen level^{44 45 46} as measured by the Clauss method.³⁵ This method has been used not only by us but also by all major previous investigations. The Clauss method is a functional assay measuring the level of clottable fibrinogen with a reported batch error of 5% to 7%.^{44 45 46} Using this method, Rosenson et al⁴⁴ examined the intraindividual variation in fibrinogen concentration over a 6-week period. They found a coefficient of variation of 17.8%, comprising both biological fluctuations as well as methodological variations. On the basis of their calculations, at least four measurements are required for an accurate fibrinogen concentration assessment in a given individual, which is not practicable for risk assessments in population studies. In population-based studies, the ability of the investigation to detect fibrinogen concentration–associated effects is mainly dependent on sample size. According to Rosenson et al,⁴⁴ our sample was large enough to detect a possible association between fibrinogen concentration and carotid atherosclerosis in general. Nevertheless, the limited number of subjects with the T/T genotype might have led to an underestimation of the effect of genotype on fibrinogen level in this subset. However, also on the basis of our data, we cannot exclude the possibility that the C₁₄₈T polymorphism per se is not a functional sequence variant but is in linkage disequilibrium with another important, yet-undefined sequence alteration in the ß-fibrinogen gene or in another gene in its neighborhood.

In summary, our study demonstrates the first evidence of a significant association between the T/T148 genotype at the ß-fibrinogen gene and carotid atherosclerosis. The results should be interpreted with caution, given the small number of T/T homozygotes. A larger cohort will ultimately be required to confirm whether or not this association is real.

	Acknowledgments

 
This project was partly supported by the Austrian Research Foundation Project P11691 MED (to G.M.K.) and by the Franz Lanyar Stiftung of the Karl-Franzens University (to H.S.), Graz, Austria. The excellent technical assistance of Johann Semmler and Anita Gradert is appreciated.

Received October 13, 1997; accepted November 26, 1997.

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