β Fibrinogen Gene Polymorphisms Are Associated With Plasma Fibrinogen and Coronary Artery Disease in Patients With Myocardial Infarction
The ECTIM Study
Background Polymorphisms of the β fibrinogen gene have been shown to affect plasma fibrinogen levels and the risk of peripheral arterial disease. We now present the results of a detailed analysis of the β fibrinogen gene in relation to plasma fibrinogen and to the severity of coronary artery disease (CAD) in patients with myocardial infarction (MI) in the ECTIM Study.
Methods and Results Ten polymorphisms of the β fibrinogen gene, including five new polymorphisms identified by single-strand conformation polymorphism analysis, and one polymorphism in the 3′ flanking region of the α fibrinogen gene were investigated in 565 patients with MI and 668 control subjects. The polymorphisms were in tight linkage disequilibrium and the genotype frequencies were similar in patients with MI and control subjects. In the multivariate analysis, only two polymorphisms, β Hae III (P<.0003) and β-854 (P<.01), were independently associated with plasma fibrinogen. The significant association between β fibrinogen polymorphisms and plasma fibrinogen was present in smokers but not in nonsmokers. In French MI patients, the number of coronary arteries with >50% stenosis was estimated by angiography and used as a criterion for severity of CAD. Presence of the less frequent allele of the β Bcl I (P<.0003) and of other polymorphisms was positively associated with the severity of CAD.
Conclusions Genetic variants of the β fibrinogen gene are associated with an increased plasma level of fibrinogen, especially in smokers. The association with CAD appears to be the consequence of an increased risk of MI in subjects with severe CAD who carry the predisposing β fibrinogen genotypes.
Human fibrinogen is composed of three pairs of nonidentical polypeptide chains, denoted Aα, Bβ, and γ, in a bilaterally symmetrical arrangement and connected by disulfide bounds. After cleavage by thrombin, fibrinogen forms fibrin monomers, which can polymerize into a fibrin clot. Fibrinogen can be directly integrated in arteriosclerotic lesions, where it is converted to fibrin. There is evidence indicating that fibrinogen as well as fibrin and its degradation products accumulate in the atherosclerotic plaque1 and that this accumulation is proportional to the level of plasma fibrinogen.2 Fibrinogen also influences platelet aggregability through its effect on specific receptors and blood viscosity. Considering all these effects, it is not surprising that plasma fibrinogen is a risk factor for CHD,3 4 5 6 7 8 stroke,4 9 and peripheral arterial disease.10 However, whether fibrinogen is a causal or secondary factor in the development of atherosclerosis and its complications remains a subject of debate. It has been reported that a parental history of early MI is associated with an elevation of plasma fibrinogen in young adults,11 thus suggesting that plasma fibrinogen level could be an inherited risk factor for CHD. Demonstration of an association between variants of the fibrinogen genes and CHD would strongly support a causal role of fibrinogen in coronary atherosclerosis or its complications.
The three chains of fibrinogen are encoded by different genes, denoted α, β, and γ, that are grouped in a cluster of approximately 50 kb on the long arm of chromosome 4.12 The process responsible for the coordination of the expression of the three genes is poorly understood; however, the β chain appears to play a limiting role in the production of the other two components of fibrinogen.13 The expression of the β gene is largely controlled by the interaction of trans-acting factors with sequences located in the 5′ region of the gene.14
Several polymorphisms of the β fibrinogen gene have been characterized and investigated in relation to plasma fibrinogen level.15 An Hae III polymorphism (β Hae III) located in the promoter region of the gene is associated with plasma fibrinogen level16 and a Bcl I polymorphism (β Bcl I) located in its downstream region is related to the presence of peripheral atherosclerosis.17 In a previous analysis of the ECTIM study data,18 we confirmed a significant association between the β Hae III polymorphism and plasma fibrinogen level, but no significant contribution to the risk of MI could be detected. We now report the results of an extensive analysis of polymorphisms of the β fibrinogen gene in relation to plasma fibrinogen and to the severity of CAD in patients with MI.
The study population has been described previously.19 Men aged 25 to 64 years were recruited between 1989 and 1991 from four WHO/MONICA registers, one in Northern Ireland (Belfast) and three in France (Lille, Strasbourg, and Toulouse). Patients with a previous history of CHD (angina pectoris or MI) were not excluded from the study. The subjects had to be residents of the region in which they were recruited, their parents had to have been born in this same region, and their four grandparents had to have been born in Europe. Case subjects were recruited into the study 3 to 9 months after the event and had to satisfy the WHO criteria for definite acute MI (category I). Control subjects were randomly recruited from the same areas as the case subjects, and stratification by age was used to approximately match the age distribution of the control subjects with that of case subjects. In the present analysis, all control subjects with CHD (prevalent case subjects in the random samples) were excluded, and only those patients and control subjects who were genotyped for all genetic polymorphisms investigated were included in the analyses (182 patients and 168 control subjects in Belfast and 383 patients and 500 control subjects in France). Furthermore, as a consequence of transportation or storage problems, plasma fibrinogen levels were measured in only 90.1% of study participants.
All subjects completed a series of questionnaires including, among other items, medical history and smoking habit. Cigarette consumption in case subjects was defined as the daily consumption just before the MI, and in control subjects, the daily consumption at the time of the examination. Those consuming at least one cigarette daily were considered smokers. A coronary angiography was available for 93% of the French case subjects and 18% of the Irish case subjects. Since a strong selection bias could be expected in the Irish sample, the angiography results are reported only in French case subjects. The reading of coronary angiographies was performed in each recruitment center. As central reading was impossible, the number of major arteries with >50% stenosis was the only information recorded to assess the degree of CAD. Associations between gene polymorphisms and CAD were analyzed globally and in each center to assess their consistency.
Plasma fibrinogen level was assessed centrally by the thrombin time method as previously described18 and is provided in mg/dL (g/L×100).
Analyses of Known Polymorphisms
Genomic DNA was prepared from white blood cells by phenol extraction. Four RFLPs (α Taq I, β HindIII, β Ava II, and β Bcl I) were genotyped after amplification of relevant DNA regions by PCR and digestion with the appropriate restriction enzymes as previously described.20 The β HindIII and β Ava II polymorphisms were analyzed only in a subsample of 100 individuals, because the genotypes were identical to those already available for β Hae III.
Search for Novel Polymorphisms by PCR/SSCP and Sequencing
To search for novel polymorphisms of the β fibrinogen gene, 11 individuals who smoked >10 cigarettes per day, with plasma levels of fibrinogen >440 mg/dL, and heterozygous or homozygous for the less frequent allele of the β Hae III polymorphism were selected from the ECTIM study. The coding sequence12 and 1500 bp in the upstream region21 of the gene were studied by PCR amplification22 followed by SSCP analysis.23 The target sequence was divided into 13 overlapping fragments of 200 to 400 bp. Each fragment was amplified by use of appropriate amplimers (Table 1⇓) with 0.3 μCi of α-dCTP-32P. Some PCR fragments were restricted overnight by addition of 2 or 5 U of the appropriate enzyme to yield fragments between 150 and 300 bp in length.24 Thereafter, products were diluted twofold in a solution containing 95% formamide, 10 mmol/L EDTA, 0.05% bromphenol blue, and 0.05% xylene cyanol. After denaturation at 95°C for 5 minutes, the samples were placed on ice and 4 μL was loaded onto nondenaturing 8% acrylamide gels (acrylamide to bis-acrylamide ratio of 39:1). Two different conditions were used for electrophoresis: gel containing 0% glycerol at 4°C and gel with 7.5% glycerol at room temperature but cooled with a fan. The gels were dried and autoradiographed overnight.
Samples exhibiting a polymorphism by SSCP analysis were reamplified by PCR with unlabeled primers. PCR products were then purified by precipitation with 4 mol/L ammonium acetate and isopropanol. Sequencing was performed by the Sanger method25 in 20 cycles of PCR with [γ32P]dATP end-labeled primer by use of a direct sequencing kit (GIBCO-BRL). The polymorphisms in the 5′ flanking region of the gene have been assigned positions according to the sequence published by Huber et al.21
The polymorphisms were characterized in all individuals included in the ECTIM study by allele-specific oligonucleotide hybridization.26 Each allele was detected after preincubation of the membranes for 2 hours with 50 pmol of unlabeled probe specific for the other allele, followed by incubation for 4 hours with 10 pmol of the labeled probe specific for the allele. The allele-specific oligonucleotides are listed in Table 2⇓.
To simplify the presentation, the three French populations were pooled because no significant heterogeneity could be detected between them. Data were analyzed with SAS statistical software (SAS Institute Inc). Statistical tests were performed on log-transformed plasma fibrinogen levels. Plasma fibrinogen levels were compared between groups by ANOVA (SAS-PROC GLM) or by multiple regression analysis when independent ordinal variables were tested, taking into account the country of origin of the subjects and their age. Genotype frequencies were compared between case subjects and control subjects by logistic regression analysis (SAS-PROC LOGISTIC). Homogeneity of the results according to country or cigarette consumption was tested by introducing the relevant interaction terms into the model. The relation between coronary score and genotypes was tested by logistic regression analysis with the score coded as an ordinal (1, 2, 3) response variable. This model assumes proportionality of odds ratios, and no significant deviation from this assumption was observed.
Hardy-Weinberg equilibrium was tested by a χ2 test with 1 df. Pairwise linkage disequilibrium coefficients were estimated in the control sample. Coefficients are reported as the ratio of the unstandardized coefficients to their minimal/maximal value (|D′|)27 ; the sign added in front of the coefficients indicates whether the linkage disequilibrium is negative (Table 3⇓).
Systematic Search for Polymorphisms of the β Fibrinogen Gene
Twenty-two alleles of the β fibrinogen gene, 12 of them carrying the β Hae III cutting site at position β-455, were analyzed by use of PCR/SSCP. By screening the entire coding sequence and 1500 nucleotides upstream of the gene, six variants were identified and further characterized by sequencing (Figure⇓).
Three variants were found in the 5′ flanking region of the gene, at positions β-249 (C→T), β-854 (G→A), and β-1420 (G→A). A silent C→T transition affecting codon 345 (β C345) in exon 7, a G→A transition affecting codon 448 (β C448) in exon 8, and an I/D polymorphism of four bases (TTTG) in intron 6, 12 bp before the intron 6/exon 7 junction (β I6 [I/D]), were also identified by PCR/SSCP. The already reported28 β C448 variant predicts an Arg→Lys change on the fibrinogen β-chain. These 6 polymorphisms and the β Hae III, β Bcl I RFLPs, and α Taq I RFLP downstream of the α fibrinogen gene (Figure⇑) were analyzed in all participants of the ECTIM study.
Associations Between Polymorphisms
The β C345 (C→T), β C448 (G→A), and β I6 (I/D) genotypes in the 3′ part of the gene were completely concordant. Although unambiguous haplotypes cannot be deduced from a combination of genotypes at linked loci when more than one locus is heterozygous, the most likely explanation for the observed complete concordance is that the three loci define two main haplotypes, C-G-I (the most frequent) and T-A-D. The β Hae III, β HindIII, and β Ava II RFLPs in the 5′ part of the β fibrinogen gene were also strongly associated: complete concordance of genotypes for the three polymorphisms was observed for the first 100 subjects investigated, and as a result, the β HindIII and β Ava II RFLPs were not studied further. As shown in Table 3⇑, the β Bcl I, β C448, and β-1420 polymorphisms were in strong positive linkage disequilibrium with the β Hae III polymorphism, all with |D′| values >0.90. Conversely, the other polymorphic sites, α Taq I, β-249, and β-854, were in negative linkage disequilibrium with the other polymorphisms.
Genotype Frequencies in Patients and Control Subjects
The genotype distributions for the different polymorphisms in Belfast and France are reported in Table 4⇓. The genotype frequencies in the control groups were similar in the two populations as well as in the subpopulations in France (not shown) and did not deviate from Hardy-Weinberg expectations. No case-control difference was observed except for α Taq I (P<.005) and β-249 (P<.03) in Belfast.
Association of Polymorphisms With Plasma Fibrinogen Level
Plasma fibrinogen level was significantly associated with the β Bcl I (P<.015), β C448 (P<.004), β Hae III (P<.002), and β-1420 (P<.003) polymorphisms (Table 5⇓), and the mean increase of plasma fibrinogen level was approximately proportional to the number of less frequent alleles (codominant effect). The associations were not heterogeneous across country; however, they appeared stronger (but not significantly so) in the case subjects than in the control subjects. The simultaneous effects of the polymorphisms on plasma fibrinogen were tested by stepwise regression analysis. As reported in Table 6⇓, only two polymorphisms, β Hae III (P<.0003) and β-854 (P<.01), were independently associated with plasma fibrinogen. These independent associations could also be deduced from the fact that plasma fibrinogen was significantly associated with the β-854 polymorphism in β Hae III/11 homozygotes (P<.02) and with the β Hae III polymorphism in β−854/11 homozygotes (P<.0005). In these analyses, polymorphisms were tested as 0, 1, 2 ordinal variables (according to number of less frequent alleles). When the analyses were repeated after genotypes 12 and 22 were pooled, very similar results were obtained (data not shown).
Interaction Between Polymorphisms and Cigarette Smoking on Plasma Fibrinogen
The percentage of individuals regularly smoking at least one cigarette per day was 45.8% in case subjects before MI and 30.1% in control subjects. A significant interaction between the number of cigarettes smoked per day and the β Hae III polymorphism on plasma fibrinogen level was observed (P<.03). Similar significant interactions were also found for the β Bcl I (P<.03), β C448 (P<.03), and β-1420 (P<.05) polymorphisms. As shown in Table 6⇑, when patients and control subjects were categorized according to smoking status, no significant association could be detected between the β Hae III polymorphism and plasma fibrinogen in nonsmokers, whereas in smokers a significant association was present. Among smokers, the association between the β Hae III polymorphism and plasma fibrinogen was apparently of greater magnitude in case subjects than in control subjects, although this heterogeneity was not statistically significant. In case subjects who were smokers, the difference in plasma fibrinogen levels between genotypes 11 and 12+22 for β Bcl I (341 versus 384 mg/dL, P<.0005), β C448 (340 versus 380 mg/dL, P<.001), and β Hae III (338 versus 380 mg/dL, P<.0002) were of similar magnitude, and after adjustment on any of these polymorphisms, no other polymorphism remained significantly associated with plasma fibrinogen.
Association of Polymorphisms With Coronary Artery Stenosis
In French MI patients, the number of coronary arteries with >50% stenosis was estimated by angiography and used as a criterion for the severity of CAD. Severity of CAD was positively correlated with age in the entire group and with plasma fibrinogen and hypolipidemic treatment in those with no previous history of CHD but was unrelated to body mass index and plasma levels of HDL cholesterol, LDL cholesterol, or VLDL cholesterol, apolipoprotein AI, or apolipoprotein B (data not shown). In patients with no previous history of CHD, the percentage of smokers was greater in those with three affected arteries than in those with less severe lesions, but this excess was not statistically significant (Table 7⇓). The presence of the less frequent allele of the β Bcl I (P<.0003), β C448 (P<.002), β Hae III (P<.006), and β-1420 (P<.01) polymorphisms was associated with more severe CAD (Table 8⇓). When the effects of all these polymorphisms were analyzed by stepwise logistic regression analysis, only β Bcl I remained significantly associated with the severity of CAD. The increasing frequency of the β Bcl I/2 allele in patients with one, two, or three stenosed coronary arteries, respectively, was consistently observed in the three French centers: 8 (23%), 2 (15%), and 2 (67%) in Lille; 17 (20%), 17 (30%), and 17 (50%) in Strasbourg; 19 (28%), 13 (34%), and 11 (48%) in Toulouse. The association between the β Bcl I polymorphism and CAD was similar in patients with or without a previous history of CHD.
As a consequence of this association, patients with three-vessel lesions were more frequently carriers of the β Bcl I/2 allele than control subjects (P<.05) (Table 8⇑). The association between the β Bcl I polymorphism and degree of coronary stenosis was not significantly heterogeneous in smokers and nonsmokers.
The purpose of this investigation was to evaluate the impact of β fibrinogen gene polymorphisms on the variability of plasma fibrinogen, the risk of MI, and CAD in patients with MI. A systematic search for variants of this gene, including its flanking regions, was undertaken on a selected sample of individuals, most of them carrying the less frequent β Hae III allele, which has previously been shown to be associated with the level of plasma fibrinogen.16 18 This approach was used to increase the chance of finding one or several putative functional variants on this particular β Hae III allele.
Polymorphisms of the β Fibrinogen Gene Define Very Conserved Haplotypes
All identified polymorphisms were tightly associated; this was especially the case for two groups of three polymorphisms at both ends of the β fibrinogen gene that were in complete association. The two groups of polymorphisms were also tightly associated with each other and with the β Bcl I and β-1420 polymorphisms, as shown by the similar genotype frequencies and by the strong positive pairwise linkage disequilibrium that existed between them (Table 3⇑). This indicates a low rate of recombination within the β fibrinogen gene and its vicinity, even at fairly large distances encompassing intron sequences, and suggests the existence of two ancestral haplotypes. Extensive study of polymorphisms of several genes has shown, as for the β fibrinogen gene, that two or more predominant haplotypes differing at several sites are frequently observed.29 30 A possible explanation for these observations is that the conserved haplotypes have evolved and accumulated variation in different isolated populations and passed through severe bottlenecks and/or environmental selection. In this situation, we might anticipate that a particular haplotype may not be functionally characterized by a single variant but by the co-occurrence of several functional variants that might affect protein expression or structure and interact with each other in term of effects.
Polymorphisms of the β Fibrinogen Gene Affect Plasma Fibrinogen in Smokers
As a consequence of the tight association existing among most of the β fibrinogen gene polymorphisms, their respective effects on plasma fibrinogen, MI, and CAD were very similar and could hardly be differentiated. When the simultaneous effects of the polymorphisms on plasma fibrinogen level were investigated, only two of them, β Hae III and β-854, remained independently associated with plasma fibrinogen (P<.003 and P<.01, respectively). It is interesting that the effect of β-854 was not significant in univariate analysis but only became significant after adjustment for β Hae III. The less frequent allele of β-854 was almost always present on the most frequent allele of β Hae III (negative linkage disequilibrium, ±|D′|=−0.87). It is not likely that these polymorphisms characterize a single functional haplotype, because the effect of β-854 on plasma fibrinogen was significant in β Hae III/11 homozygotes (P<.02) and the effect of β Hae III was significant in β-854/11 homozygotes (P<.0005). It is plausible then that both polymorphisms define or are linked to two functional sites in the 5′ flanking region of the β fibrinogen gene.
Our results show that the association between polymorphisms of the β fibrinogen gene and plasma fibrinogen is observed only in cigarette smokers. Cigarette smoking is an important risk factor for CHD and peripheral arterial disease, and it is the major known determinant of plasma fibrinogen.31 It has been postulated that the effect of cigarette smoking on plasma fibrinogen is an acute-phase response mediated by cytokines such as IL-6.16 Three IL-6 response elements have been described within the β fibrinogen promoter by footprinting, mobility shift assay, and mutagenesis.32 The most distal one contains a CTGGGAA motif at positions −143 to −137, which is known to be used by IL-6–regulated genes. Another site, slightly more proximal and located at positions −132 to −124, appears to react with different nuclear proteins, including members of the C/EBP family, and might play an important role in the constitutive expression of the β fibrinogen gene. Finally, another sequence corresponding to a hepatocyte nuclear factor-1 binding site is located at positions −91 to −79 and is also involved in the stimulation induced by IL-6. We were unable to identify any polymorphism near the last two sites. On the other hand, the most distal IL-6 response element is very close to the HindIII polymorphic locus located at position −148, and recent evidence suggests that this polymorphism influences the interaction between nuclear proteins and this response element.33 34 It is therefore possible that the β HindIII polymorphism, which is tightly or completely associated with the β Hae III polymorphism, affects the interaction between this regulator site and transcription factors induced or modulated by cigarette smoking.35 The β HindIII polymorphism would then be the functional locus that explains the interaction of cigarette smoking and polymorphisms of the β fibrinogen gene on plasma fibrinogen.
The β Bcl I polymorphism located in the downstream region of the β fibrinogen gene has previously been shown to be associated with the risk of peripheral arterial disease.17 This association was apparently not mediated by the influence of the polymorphism on plasma fibrinogen concentration. This suggested that a structural variant of fibrinogen could be responsible for the association between β Bcl I and peripheral atherosclerosis, thus implying that β Bcl I was only a marker for a functional variant that affects the sequence of the protein. This functional variant could be the G→A substitution at position β C448. This polymorphism predicts an Arg→Lys change, 13 amino acid residues before the carboxyl terminus of the β fibrinogen chain. This region has no well-defined functional properties; however, the possibility that the function of the protein is affected by this polymorphism cannot be excluded and warrants further investigation. The β I6 (I/D) polymorphism, which is completely associated with β C448, is characterized by the presence or absence of a TTTG sequence located before a stretch of 10 pyrimidine residues preceding the AGG site at the intron 6/exon 7 junction. Whether this polymorphism might influence the processing of the β fibrinogen mRNA is not clear, but this possibility cannot be excluded in the absence of experimental evidence.
Polymorphisms of the β Fibrinogen Gene Are Associated With MI in Patients With Severe CAD
The distributions of α Taq I and β-249 genotypes were significantly different in case subjects and control subjects in Belfast (P<.005 and P<.025, respectively) but not in France. As the two polymorphisms were not associated with plasma fibrinogen or CAD, it is reasonable to assume that the associations observed in Belfast were spurious. The genotype frequencies of the other polymorphisms did not differ between case subjects and control subjects in Belfast or France as well as in the entire study population. Conversely, in the group of French patients for whom a coronary angiography was available (93% of French MI patients), a highly significant association was observed between the severity of CAD and the β Bcl I polymorphism.
Fibrinogen and fibrin accumulate in the atherosclerotic plaque and stimulate smooth muscle cell proliferation1 ; furthermore, thrombus organization is involved in the progression of atherosclerosis.36 Functional variants of the β fibrinogen gene, by affecting fibrinogen production or properties, might thus be causally related to the development of atherosclerosis. This could explain the relation between polymorphisms of the β fibrinogen gene and the severity of coronary stenosis in patients with MI. However, our results are not entirely in concurrence with this hypothesis, because β fibrinogen genotypes predisposing to atherosclerosis were not more frequent in patients with MI than in control subjects in the entire ECTIM study. Another possibility to explain the present results is that genotypes of the β fibrinogen gene do not contribute to the evolution of coronary stenosis but are involved in its complications. Indeed, if a factor predisposes patients with severe coronary atherosclerosis to MI, a higher prevalence of this factor would be expected in MI patients with severe CAD than in MI patients with less severe coronary lesions. This hypothesis is compatible with our data showing a significant excess (P<.05) of carriers of β Bcl I/2 in MI patients with three stenosed coronary arteries. Plaque rupture and thrombus formation are two important contributors to the complications of coronary atherosclerosis. Given the tight link existing between fibrinogen and inflammation and the important role apparently played by inflammation in coronary plaque disruption,37 a variant of the β fibrinogen gene, by affecting fibrinogen production or properties, could predispose to plaque rupture and, as a consequence of the role of fibrinogen in the coagulation cascade, such a variant could also affect thrombus formation. Further studies will be needed to test these hypotheses.
In conclusion, among 11 variants of the β fibrinogen gene that were investigated in the ECTIM study, 8 were mutually very tightly or completely associated. This suggests the existence of two ancestral haplotypes of the β fibrinogen gene. The less frequent allele of these polymorphisms, whose frequency is ≈20%, was associated with an increased plasma level of fibrinogen, especially in smokers and patients with MI, and with the degree of CAD assessed by coronary angiography in patients with MI. We surmise that this latter association is not the consequence of a causal effect of fibrinogen on the development of atherosclerosis but is probably due to a higher risk of MI in patients with severe atherosclerosis who carry the predisposing β fibrinogen genotype.
Selected Abbreviations and Acronyms
|CAD||=||coronary artery disease|
|CHD||=||coronary heart disease|
|ECTIM||=||Etude Cas-Temoins sur l’Infarctus du Myocarde|
|MONICA||=||Monitoring Trends and Determinants in Cardiovascular Disease|
|PCR||=||polymerase chain reaction|
|RFLP||=||restriction fragment length polymorphism|
|SSCP||=||single-strand conformation polymorphism|
|WHO||=||World Health Organization|
This work was supported by grants from the Squibb laboratory, the Sandoz laboratory, the Mutuelle Generale de l’Education Nationale, the British Heart Foundation, INSERM, the Institut Pasteur-Lille, and the Groupement de Recherches et d’études sur les Génomes (GREG). We thank C. Souriau and F. Defranoux for technical assistance and P. Ducimetière and L. Tiret for helpful discussion of the manuscript.
- Received May 17, 1995.
- Revision received September 11, 1995.
- Accepted September 14, 1995.
- Copyright © 1996 by American Heart Association
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