© Borgis - New Medicine 3/2003, s. 61-64
Krzysztof Chizynski1, Tadeusz Pietrucha2
The –455G/A fibrinogen genetic polymorphism is associated with a risk of coronary artery disease only in population of young 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: An elevated fibrinogen level is an independent CAD risk factor. Low fibrinogen plasma concentration is associated with low levels of coronary risk even when total or low-density lipoprotein cholesterol is high. Several polymorphisms on the b fibrinogen gene have been characterised and investigated in relation to plasma fibrinogen level. However, further studies have examined the relationship between fibrinogen genotype and atherosclerosis. From the clinical point of view, the most important are the 455-G/A and the 148-C/T polymorphisms. In this paper, we present the association of the 455-G/A Fg genotype with CAD and the impact of the 455-G/A Fg polymorphisms on younger and elderly CAD patients.
Material and methods: The study group enrolled 314 patients aged 33 to 84 yrs. (average age 54 yrs.), with angiographically concerned CAD. The control group numbered 41 patients aged 29 to 74 yrs. (average age 52 yrs.) without CAD (normal coronary arteries in angiography). Genotypes and polymorphism were determined by polymerase chain reaction amplification of genomic DNA using the following allele specific primers; sense: 5´-AGA ATT TGG GAA TGC AAT CTC TGC TAC CT -3´ and antisense: 5´-TCC TCA TTG TCG TTG ACA CCT TGG GAC -3´. After Hae III enzyme digestion, the following genotypes were detected: G/G: 575 bp, 383 bp, 343 bp, A/A: 958 bp, 343 bp and G/A: 958 bp, 575 bp, 383 bp, 343 pz. PCR products were run by electrophoresis in a 3% polyacrylamide gel.
Results: Among 314 subjects with CAD enrolled in the study, 133 (42.4%) had a genotype G/G, 153 (48.7%) had G/A and 28 (8.9%) had AA. Among 41 subjects without CAD enrolled in the study, 23 (56.1%) had genotype G/G, 17 (41.5%) had G/A and 1 (2.4%) had AA. Genotype 455-G/A Fg polymorphism was not related to the presence of CAD. Further analysis was performed after dichotomy dividing genotypes into A+ (A/A and G/A genotypes) and A- (G/G genotypes). In the case of elderly subjects, differences in genotypes (A+, A-) and alleles (A, G) distribution were observed. The elderly subjects´ A+ genotype contained no CAD risk factor. In the case of the younger subjects with CAD, a higher frequency of A+ genotypes (59% vs. 40.6%; p <0.05) and A alleles (34.1% vs. 21.9%; p <0.05) was observed. The odds ratio for having CAD among the younger levelled at 2.10 (0.99-4.46) (p <0.05) in cases of subjects with A+ genotype and 1.85 (0.99-3.44) (p <0.05) in subjects with A allele.
Conclusion: In the case of the Polish population, the 455-G/A Fg genotype is a risk factor for coronary disease only among subjects aged less than 65 yrs.
Introduction
There are numerous studies describing the association between plasma fibrinogen levels and coronary artery disease (CAD). Elevated fibrinogen level is an independent CAD risk factor (1, 2). Low fibrinogen levels are associated with low levels of coronary risk even when total or low-density lipoprotein cholesterol is high (3). Levels of fibrinogen increase with age (4, 5, 6, 7, 8) and are higher in women (5, 9, 10, 11, 12). Fibrinogen level increases in smokers (5, 10, 13, 14, 15, 16, 17), among diabetic subjects (4, 18), subjects with hypertension (5, 10, 15), and is strongly correlated with obesity (4, 5, 8, 17, 19, 20) and dyspipidaemia.
The three chains of fibrinogen are encoded by different genes, denoted a, b and g, which are grouped in a cluster of approximately 50 kb on the long arm of chromosome 4 (21). The process responsible for the coordination of the expression of the three genes is poorly understood; however, the b chain appears to play a limiting role in the production of the other two components of fibrinogen (21). For fibrinogen, several polymorphisms on the b fibrinogen gene have been characterised and investigated in relation to plasma fibrinogen level (22, 23, 24, 25). However, fewer studies have examined the relationship between the fibrinogen genotype and atherosclerosis (21, 23, 26). From the clinical point of view the most important are the 455-G/A and the 148-C/T polymorphisms (21, 22, 27, 28, 29).
In this paper, we present the association of 455-G/A Fg genotype with CAD and the impact of the 455-G/A Fg polymorphisms on young and elderly CAD patients.
Material and methods
The study group numbered 314 patients aged 33 to 84 yrs. (average age 54 yrs.), with angiographically related CAD.
The control group numbered 41 patients aged 29 to 74 yrs. (average age 52 yrs.), without CAD (normal coronary arteries in angiography).
Additionally, separate analyses were performed among 74 elderly patients (aged over 65 yrs.), (65 subjects with and 9 without CAD) and among 281 younger ones (below 65 yrs.) (249 subjects with and 32 without CAD).
DNA extraction and 455-G/A Fg 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 genomic DNA using the following allele specific primers (30); sense: 5´-AGA ATT TGG GAA TGC AAT CTC TGC TAC CT-3´ and antisense: 5´-TCC TCA TTG TCG TTG ACA CCT TGG GAC-3´. After Hae III enzyme digestion the following genotypes were detected: G/G: 575 bp, 383 bp, 343 bp, A/A: 958 bp, 343 bp and G/A: 958 bp, 575 bp, 383 bp, 343 pz. PCR products were run by electrophoresis in a 3% polyacrylamide gel.
Statistical analysis
Results are expressed as mean and standard deviations for normally distributed data. Differences between groups were tested with unpaired Student´s T-tests. The significance of differences between the frequencies of 455-G/A Fg genotypes and alleles was estimated by a chi2 test with Yates´ correction (31).
Results
Among 314 subjects with CAD enrolled in the study, 133 (42.4%) had genotype G/G, 153 (48.7%) had G/A and 28 (8.9%) had AA. Among 41 subjects without CAD, 23 (56.1%) had genotype G/G, 17 (41.5%) had G/A and 1 (2.4%) had AA. As shown in Table 1, genotype 455-G/A Fg polymorphism was not related to the presence of CAD.
Additional analysis was performed on 74 elderly patients (over 65 yrs.), (65 subjects with and 9 without CAD) and on 281 younger ones (below 65 yrs.) (249 subjects with and 32 without CAD). As shown in Table 2, among the 74 older subjects with CAD, 27 (41.5%) had a genotype G/G, 29 (44.6%) had G/A and 9 (13.9%) had A/A. Among the 9 elder subjects without CAD, 4 (44%) had a genotype G/G, 5 (56%) had G/A and none of them (0%) had A/A. In the younger group (subjects <65 yrs.), among 281 subjects with CAD, 102 (41%) had a genotype G/G, 124 (49.8%) had G/A and 23 (9.2%) had A/A. In the younger group, among 32 subjects without CAD, 19 (59.4%) had a genotype G/G, 12 (38.6%) had G/A and 1 (3.0%) had A/A.
Further analysis was performed after dichotomy dividing genotypes into A+ (A/A and G/A genotypes) and A- (G/G genotypes). As shown in Table 3, in elderly subjects there were differences in genotype (A+, A-) and allele (A, G) distribution. In elderly subjects A+ genotype was not a CAD risk factor. Among younger subjects with CAD, a higher frequency of A+ genotypes (59% vs. 40.6%; p <0.05) and A alleles (34.1% vs. 21.9%; p <0.05) was observed. The odds ratio for having CAD among the younger was 2.10 (0.99-4.46) (p <0.05) in subjects with A+ genotype and 1.85 (0.99-3,44) (p <0.05) in subjects with A allele.
Table 1. 455-G/A Fg genotypes and alleles related to CAD.
| 455-G/A Fg polymorphism | CAD
n = 314 | Without CAD
n = 41 | Significance |
| genotypes | A+ (A/A+G/A) | 181 (57.6%) | 18 (43.9%) | p = 0.095 |
| A- (G/G) | 133 (42.4%) | 23 (56.1%) | |
| alleles | A | 209 (33.3%) | 19 (23.2%) | p = 0.060 |
| G | 419 (66.7%) | 63 (76.8%) | |
| A+ (A/A+G/A) vs A-(G/G) | OR = 1.74 (0.91-3.33) | | p = 0.095 |
| A allele vs G allele | OR = 1.65 (0.98-2.79) | | p = 0.060 |
Table 2. 455-G/A Fg genotypes and alleles in elderly and younger groups.
| 455-G/A Fg polymorphism | Subjects > 65
yrs. | Subjects < 65 yrs. |
| CAD n = 65 | Without CAD n = 9 | CAD n = 249 | Without CAD n = 32 |
| genotypes | A/A | 9 (13.9%) | 0 (0%) | 23 (9.2%) | 1 (3.0%) |
| G/A | 29 (44.6%) | 5 (56%) | 124 (49.8%) | 12 (38.6%) |
| G/G | 27 (41.5%) | 4 (44%) | 102 (41%) | 19 (59.4%) |
Table 3. Odds ratio for having CAD in elderly and younger subjects with 4G+ genotype.
| 455-G/A Fg polymorphism | Subjects > 65 yrs. | | Subjects < 65
yrs. | |
| CAD n = 65 | Without CAD n = 9 | CAD n = 249 | Without CAD n = 32 |
| genotypes | A+(A/A+G/A) | 38 (58.5%) | 5 (56%) | 147 (59%) | 13 (40.6%)* |
| A-(G/G) | 27 (41.5%) | 4 (44%) | 102 (41%) | 19 (59.4%)* |
| alleles | A | 47 (36.2%) | 5 (27.8%) | 170 (34.1%) | 14 (21.9%)* |
| G | 83 (63.8%) | 13 (72.2%) | 328 (65.9%) | 50 (78.1%)* |
| | OR (95% CI) | | OR (95% CI) | |
| A+(A/A+G/A) vs A-(G/G) | - | | OR = 2.10 (0.99-4.46)* | |
| A allele vs G allele | OR = 1.47 (0.49-4.38) | | OR = 1.85 (0.99-3,44)* | |
* p < 0.05
Discussion
Fibrinogen and fibrin accumulate in the atherosclerotic plaque and stimulate smooth muscle cell proliferation (32); furthermore, thrombus organisation is involved in the progression of atherosclerosis (33). Family studies suggest that up to 50% of variation in plasma fibrinogen levels may be hereditary (34) and an increasing proportion of such a variation can be explained by fibrinogen gene polymorphisms. Fibrinogen polymorphisms, by affecting fibrinogen production, might thus be causally related to the development of atherosclerosis and CAD (21, 22, 35, 36, 37, 38, 39). However, there are papers in which no correlation between polymorphism and fibrinogen level (26, 40) has been stressed. Explaining the above problem, ethnic differences are taken into account, similar to the differences in genes and alleles distribution (22). Demaat et al. (41) found that subjects with A/A genotype (–455G/A Fg polymorphism) fibrinogen levels were higher (3.9 g/l) than in subjects with G/A (3.3 g/l) or G/G (3.1 g/l) genotypes. Also, an important role is played by such risk factors as cigarette smoking. In smoking subjects the –455G/A Fg polymorphism influence on fibrinogen level is higher than in non-smokers (36, 39, 42). Smoking subjects with A allele have the highest fibrinogen levels (39, 42). From the presented papers (21, 35, 40, 43, 44, 45) it is not generally possible to make a statement on the presence of the A allele and its correlation with CAD or MI. In our study, carried out among the Polish population, similar to the results of the Copenhagen City Heart Study (44), Gardemann et al. (43), van der Bom et al. (46) and Lee i et al., (40) study, the A allele contained no CAD risk factor. But when the study population was divided into younger subjects (<65yrs.) and older ones (> 65 yrs.), in younger subjects with A allele the odds ratio for having CAD was 1.85 (0.99-3,44) (p <0.05). Our study demonstrated that in the case of the Polish population, A allele is a CAD risk factor only among subjects younger than 65 yrs.
Conclusion
In the case of Polish population, the 455-G/A Fg genotype is a risk factor for coronary disease only among subjects aged less than 65 yrs.
Piśmiennictwo
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