© Borgis - New Medicine 3/2003, s. 65-68
Krzysztof Chizynski1, Tadeusz Pietrucha2
4G/5G genetic polymorphism in the promoter of the plasminogen activator inhibitor-1 (PAI-1) gene is associated with a risk of coronary artery disease, but only in younger people
1 Institute of Cardiology, Medical University of Lodz, Poland
Head: prof. Janusz Zaslonka, MD, PhD
2 Laboratory of Medical Biotechnology, Department of Medical Biochemistry,
Medical University of Lodz, Poland
Head: Tadeusz Pietrucha, MD, PhD
Summary
Objective: Suppression of fibrinolysis by high circulating levels of plasminogen activator inhibitor-1 (PAI-1) may contribute to an increased risk of coronary disease. The PAI-1 gene, which contains nine exons and eight introns distributed over approximately 12.3 kb of DNA, has been localised to 7q21.3-22 of chromosome 7. The highest PAI-1 levels have been observed in 4G/4G homozygous individuals, and in this group an increased risk of CAD has been expected. In this paper, we present evidence for the association of the PAI-1 promoter genotype with CAD, and the contribution of PAI-1 4G/5G polymorphism in young and elderly CAD patients.
Material and methods: The study group included 514 patients (aged 31 to 86 yrs.; mean 55 yrs.), with angiographically-concerned CAD. The control group was 67 patients (aged 26 to 76 yrs.; mean 52 yrs.), without CAD (normal coronary arteries in angiography). Genotypes were determined by a polymerase chain reaction amplification of the genome DNA using the following allele specific primers: 5´-GTC TGG ACA CGT GGG GG-3´ for the insertion allele (5G) and 5´-GTC TGG ACA CGT GGG GA-3´ for the deletion allele (4G).
Results: PAI-1 promoter 4G5G polymorphism was related to the presence of CAD with an increased frequency of the 4G/4G genotype (p = 0.018), and decreased frequency of the 5G/5G genotype (p <0.0001), in subjects with coronary disease. The CAD odds ratio in subjects with the 4G/4G genotype was 2.08 (1.13-3.80) (p = 0.018) and in the subjects with 4G allele genotype (4G/5G and 4G/5G) was 2.84 (1.70-4.73) (p <0.0001). An additional analysis was performed on 121 elderly patients (over 65 yrs.), (111 subjects with and 10 without CAD) and in 460 younger ones (below 65 yrs.) (403 subjects with, and 57 without CAD). In elderly subjects there was no difference in the distribution of genotypes (4G+, 4G-) and alleles (4G, 5G). In elderly subjects with the 4G+ genotype there was no increased risk of CAD. In younger subjects with CAD, a higher frequency of the 4G+ genotypes (78.4% vs. 54.4%; p <0.0001) and 4G alleles (57.1% vs. 37.7%; p <0.0001) was observed. The odds ratio for having CAD was 3.05 (1.72-5.40) (p <0.0001) in the younger subjects with 4G+ genotype, and 2.20 (1.47-3.29) (p <0.0001) in the younger subjects with 4G allele.
Conclusion: Plasminogen activator inhibitor-1 promoter 4G/5G genotype is a risk factor for coronary disease in a Polish population, but only in younger people.
Introduction
High circulating levels of plasminogen activator inhibitor-1 (PAI-1) may contribute to an increased risk of coronary disease by suppression of fibrinolysis. Elevated PAI-1 levels have been found in patients with coronary artery disease (CAD) and in patients with previous myocardial infarction (MI) (1, 2, 3, 4, 5, 6). Increasing levels of PAI-1 were found in with conjunction a worsening angiographic extent of coronary disease in the ECAT study (7). Elevated PAI-1 levels have been found to predict a recurrence of myocardial infarction in young male subjects with hyperlipidaemia (2). The PAI-1 level increasing with age (8) is strongly correlated with the level of triglycerides (9, 10, 11, 12), insulin resistance (13, 14), obesity (9, 15, 16, 17, 18, 19), smoking (8, 9), diabetes (3), hypertension (20, 21, 22) and low high-density lipoproteins cholesterol level (8).
The PAI-1 gene, which contains nine exons and eight introns distributed over approximately 12.3 kb of DNA, has been localised to 7q21.3-22 of chromosome 7 (23, 24, 25). PAI-1 gene polymorphisms, f.i. –844 (G/A) polymorphism, +9785 G/A polymorphism, +11053 G/T polymorphism, and +11319 polymorphism have been described (23, 24). The most popular is guanosine –675 insertion/deletion polymorphism, where one allele has a sequence of four guanosines (4G) and the other has five guanosines (5G). The highest PAI-1 levels have been observed in 4G/4G homozygous individuals, and in this group an increased risk of CAD was expected (25, 26, 27, 28).
In this paper, we present evidence of the association between the PAI-1 promoter genotype and CAD, and the contribution of PAI-1 4G/5G polymorphism in young and elderly CAD patients.
Material and methods
The study group included 514 patients (aged 31 to 86 yrs., mean 55 yrs.), with angiographically confirmed CAD.
In the control group there were 67 patients (aged 26 to 76 yrs., mean 52 yrs.), without CAD (normal coronary arteries in angiography).
An additional analysis was performed on 121 elderly patients (over 65 yrs.), (111 subjects with and 10 without CAD), and in 460 younger ones (below 65 yrs.) (403 subjects had CAD and 57 without CAD).
DNA extraction and 4G/5G polymorphism detection
Peripheral venous blood samples were drawn and the white blood cells were separated. DNA was isolated by the modified phenol extraction method.
Genotypes and polymorphism were determined by polymerase chain reaction amplification of the genome DNA using an allele-specific primer (29); for the insertion of the 5G allele: 5´-GTC TGG ACA CGT GGG GG-3´, and for the deletion of 4G allele: 5´-GTC TGG ACA CGT GGG GA-3´, each in a separate reaction together with the common downstream primer 5´-TGC AGC CAG CCA CGT GAT TGT CTA G-3´. To verify the occurrence of DNA, amplification in the absence of the allele on the genome DNA a control upstream primer 5´-AAG CTT TTA CCA TGG TAA CCC CTG GT-3´ was used. Thermal cycling conditions were as follows: 45 sec. at 94°C, 45 sec. at 65°C, 1 min. and 15 sec. at 72°C, repeated for 30 step cycles. The PCR products were run by electrophoresis in a 3% agarose gel and visualised by ethidium bromide staining.
Statistical analysis
The results are expressed as a mean and standard deviation for normally-distributed data. Differences between groups were tested with unpaired Student´s T-tests. The significance of differences between the frequencies of 4G/5G PAI-1 genotypes and alleles was estimated by a chi2 test with Yates´ correction (30).
Results
Among 514 subjects with CAD enrolled in the study, 109 (21.2%) had genotype 5G/5G, 223 (43.4%) had 4G/5G and 182 (35.4%) had 4G/4G. Among 67 subjects without CAD enrolled in the study, 29 (43.3%) had genotype 5G/5G, 24 (35.7%) had 4G/5G and 14 (21.0%) had 4G/4G. As shown in Table 1, the genotype at the PAI-1 promoter 4G5G polymorphism was related to the presence of CAD with an increased frequency of the 4G/4G genotype (p = 0.018) and an underrepresentation of the 5G/5G genotype (p <0.0001) in subjects with coronary disease. CAD odds ratio in 4G/4G homozygous subjects was 2.08 (1.13-3.80) (p = 0.018) and in subjects with 4G allele genotype (4G/5G and 4G/5G) was 2.84 (1.70-4.73) (p <0.0001). The allele frequencies were 5G = 42.9%, 4G = 57.1% in the study group and 5G = 61.2%, 4G = 38.8% in the control group (p <0.0001).
Table 1. PAI-1 promoter 4G/5G genotypes and alleles in relation to CAD.
| 4G/5G PAI-1 polymorphism | CAD
n = 514 | Without CAD
n = 67 | Significance |
| genotypes | 4G/4G | 182 (35.4%) | 14 (21.0%) | p = 0.018 |
| 4G/5G | 223 (43.4%) | 24 (35.7%) | p = 0.239 |
| 5G/5G | 109 (21.2%) | 29 (43.3%) | p < 0.0001 |
| alleles | 4G | 587 (57.1%) | 52 (38.8%) | p < 0.0001 |
| 5G | 441 (42.9%) | 82 (61.2%) | |
| 4G/4G vs 4G/5G+5G/5G | OR = 2.08 (1.13-3.80) | p = 0.018 |
| 4G+ (4G/4G+4G/5G) vs 4G- (5G/5G) | OR = 2.84 (1.70-4.73) | p < 0.0001 |
| 4G/4G vs 5G/5G | OR = 3.46 (1.80-6.64) | p < 0.0001 |
| 4G allele vs 5G allele | OR = 2.10 (1.46-3.02) | p < 0.0001 |
The additional analysis was performed in 121 elderly patients (over 65 yrs.), (111 subjects with and 10 without CAD) and in 460 younger ones (below 65 yrs.) (403 subjects had CAD and 57 had no CAD). As shown in Table 2, among 111 elderly subjects with CAD, 22 (19.8%) had a genotype 5G/5G, 51 (46.0%) had 4G/5G and 38 (34.2%) had 4G/4G. Among 10 elderly subjects without CAD, 3 (30%) had a genotype 5G/5G, 5 (50%) had 4G/5G and 2 (20%) had 4G/4G. In the younger group (subjects <65 yrs.), among 403 subjects with CAD, 87 (21.6%) had a genotype 5G/5G, 172 (42.7%) had 4G/5G and 144 (35.7%) had 4G/4G. In the younger group, among 57 subjects without CAD, 26 (45.6%) had genotype 5G/5G, 19 (33.3%) had 4G/5G and 12 (21.1%) had 4G/4G.
Table 2. PAI-1 promoter 4G/5G genotypes and alleles in elderly and younger groups.
| 4G/5G PAI-1 polymorphism | Subjects > 65 yrs. | Subjects < 65
yrs. |
| CAD n = 111 | Without CAD n = 10 | CAD n = 403 | Without
CAD n = 57 |
| genotypes | 4G/4G | 38 (34.2%) | 2 (20%) | 144 (35.7%) | 12 (21.1%) |
| 4G/5G | 51 (46.0%) | 5 (50%) | 172 (42.7%) | 19 (33.3%) |
| 5G/5G | 22 (19.8%) | 3 (30%) | 87 (21.6%) | 26 (45.6%) |
Further analysis was performed after dividing genotypes into 4G+ (4G/4G and 4G/5G genotypes) and 4G-(5G/5G genotypes). As shown in Table 3, in elderly subjects there were differences in genotypes (4G+, 4G-) and alleles (4G, 5G) distribution. The was no increasing CAD risk in elderly subjects with the 4G+ genotype. In the younger subjects with CAD, a higher frequency of the 4G+ genotypes (78.4% vs. 54.4%; p <0.0001) and the 4G alleles (57.1% vs. 37.7%; p <0.0001) was observed. Odds ratio for having CAD was 3.05 (1.72-5.40) (p <0.0001) in the younger subjects with the 4G+ genotype, and 2.20 (1.47-3.29) (p <0.0001) in the younger subjects with the 4G allele.
Table 3. Odds ratio for having CAD in elderly and younger subjects with 4G+ genotype.
| 4G/5G PAI-1 polymorphism | Subjects > 65
yrs. | Subjects < 65 yrs. |
| CAD n = 111 | Without CAD n = 10 | CAD n = 403 | Without CAD n = 57 |
| genotypes | 4G+ (4G/4G+4G/5G) | 89 (80.2%) | 7 (70%) | 316 (78.4%) | 31 (54.4%)* |
| 4G- (5G/5G) | 22 (19.8%) | 3 (30%) | 87 (21.6%) | 26 (45.6%)* |
| alleles | 4G | 127 (57.2%) | 9 (45%) | 460 (57.1%) | 43 (37.7%)* |
| 5G | 95 (42.8%) | 11 (55%) | 346 (42.9%) | 71 (62.3%)* |
| 4G+(4G/4G+4G/5G) vs 4G-(5G/5G) | OR (95% CI) | | OR (95% CI) | |
| | 1.63 (0.65-4.10) | | OR = 3.05 (1.72-5.40)* | |
* p <0.0001
Discussion
Raised plasma levels of plasminogen activator inhibitor-1 (PAI-1) are reported to be associated with an increased risk of CAD (31). The level of PAI-1 is determined in part by environmental influences (3, 8, 9, 10, 11, 12, 20, 21, 22), but some genetic variations are reported to explain the individual variations in plasma level (32, 33). In 1995 Erikson et al. (27) reported that the 4G allele of 4G/5G polymorphism in the PAI-1 promoter is associated with a higher plasma activity. The prevalence of the 4G allele is significantly higher in patients with myocardial infarction before the age of 45 years than in the population-based controls in the Swedish population (27). The 4G/5G polymorphism in the PAI-1 promoter was not an MI risk factor in the ECTIM study (25), or in the Japanese population (34). A PAI-1 circulating level is strongly associated with the 4G/4G genotype (25, 35). In subjects with the homozygous 4G/4G genotype, an increased risk of CAD has been observed, but the results of studies performed in various ethnic groups and populations are different (25, 26,27, 28).
This is the first study to report the frequency of PAI-1 promoter 4G/5G genotypes in a Polish population. Our study demonstrated that in the Polish population, the 4G allele is the CAD risk factor. The odds ratio for having CAD was 2.08 for the 4G/4G genotype and 2.84 for genotype with 4G allele (4G/4G and 4G/5G genotypes) in the whole group. Because there are some reports stating that 4G/5G promoter PAI-1 polymorphism is a risk factor only in younger subjects (25, 36), an additional analysis was performed in subjects older than 65 yrs. and in subjects below this age.
In the Ye et al. study (25), approximately 30% of individuals were homozygous for the 4G allele. In our study the distribution of the 4G/4G, 4G/5G and 5G/5G genotype among subjects who developed CAD was respectively 34.2%, 46% and 19.8% in the older group and 35.7%, 42.7% and 21.6% respectively, in the younger group. In the Polish population the PAI-1 promoter 4G/5G genotype was not a risk factor for CAD in elderly patients, probably because the presence of the 4G allele in these patients may influence an individual´s fibrinolytic capacity only slightly, perhaps by interaction with other environmental risk factors. In the younger patients with the 4G+ genotype (4G/4G and 4G/5G genotypes) the odds ratio for having CAD was 3.05. The major risk factors influencing the PAI-1 level were lower in this group.
Conclusion
Plasminogen activator inhibitor-1 promoter 4G/5G genotype is a risk factor for coronary disease in the Polish population, but only in younger adults.
Piśmiennictwo
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