Polymorphisms of some increased cardiovascular risk related genes in acute coronary syndromes and their relation to coronary artery disease severity

Background

Cardiovascular disease, especially coronary artery disease (CAD), is the primary cause of mortality and mortality worldwide. Coronary artery disease has a multifactorial pathogenesis, which involves environmental and inherited risk factors. Patients with CAD are admitted to hospital most frequently with acute coronary syndromes [1]. Acute coronary syndromes (ACS) are the most mortal clinical manifestation of CAD. A thrombus on a ruptured atherosclerotic plaque is the main cause of ACS [2].

The role of haemostatic markers as factors predisposing to thrombus formation and atherosclerosis was investigated, and several genetic mutations affecting coagulation proteins were suggested as likely inherited risk factors for CAD [3, 4]. In this study, -fibrinogen 455G/A, factor V 1691G/A, 1299H/A and glycoprotein (Gp) IIb/IIIa PL A1/A2 polymorphisms were the subjects of investigation. These polymorphisms are responsible for some alterations in the coagulation system and are proposed as risk factors for CAD, especially in younger populations or in association with traditional cardiovascular risk factors, such as smoking [5].

Aim

Therefore this study was designed to investigate the frequencies of the mentioned polymorphisms in two major groups of ACS (ST elevation and non-ST elevation), to investigate the relationship between ACS subtypes and mentioned polymorphisms, and to investigate the relation between these polymorphisms and the angiographic severity of coronary atherosclerosis.

Material and methods

The study consisted of 45 consecutive patients who were admitted to our hospital with ACS and then underwent coronary angiography (CAG). Subjects were excluded if they had a history of CAD, revascularization therapies, malignancy, bleeding diathesis or hypercoagulable state, or received medications such as aspirin, clopidogrel or warfarin which affect the coagulation system. The platelet counts and prothrombin times of patients were assessed and patients with platelet counts below 150 000/ml or above 400 000/ml or with an INR (international normalised ratio) higher than 1.5 were also excluded. The current study was approved by the local ethics committee and all participants gave informed consent.

The diagnoses of ACS were made according to ACC/AHA guidelines [6]. There were two groups in the study: the ST elevation (STE) group consisted of patients with ST elevation myocardial infarction (STEMI), and the non-ST elevation group (NSTE) consisted of patients with unstable angina (USAP) and non-ST elevation myocardial infarction (NSTEMI).



Genetic analysis



DNA was isolated from whole blood using standard procedures. The genotype analyses were made by polymerase chain reactions (PCR) using CVD Strip Assay (Viennalab GmBH, Vienna, Austria). Three genotypes were determined for each gene: G/G, G/A and A/A for -fibrinogen 455; G/G, G/A and A/A for factor V 1691; H/H, H/A and A/A for factor V 1299; A1/A1, A1/A2 and A2/A2 for Gp IIb/IIIa PL. To compare the frequencies of polymorphisms between the groups, the mutant allele was used in statistical analyses. The mutant allele was A for -fibrinogen 455G/A, factor V 1691G/A and 1299H/A polymorphisms and A2 for Gp IIb/IIIa PL A1/A2 polymorphism.

Selective coronary angiography was performed with the standard technique with the Integris H 5000 model monoplane cardiac angiography device (Philips Medical Systems Nederland). The angiograms recorded on CD media were evaluated by two experienced observers via Image View DICOM Player computer program. All atherosclerotic lesions were investigated and the following angiographic parameters were calculated to define the severity of coronary atherosclerosis in patients: the number of diseased vessels with stenosis higher than 10% (DV  %10), the number of diseased vessels with stenosis higher than 50% (DV  %50), and total number of atherosclerotic lesions with a lumen narrowing equal to or higher than 70% (TLC, total lesion count). Furthermore, the atherosclerotic lesions were classified according to the ACC/AHA lesion classifications and numbers of type A, B and C lesions were calculated [7]. Also Gensini and Extent scores were calculated to establish the severity of coronary atherosclerosis as reported previously [8-10].



Statistical analysis



Student t and Mann-Whitney U tests were used to compare means and c2 tests to compare proportions. All probability values were two-sided and a p value below 0.05 was accepted as significant. SPSS Version 15.0 for Windows (SPSS Inc., Chicago, IL, USA) was used to perform all statistical calculations.

Results

Forty-five patients were included preliminarily in the study, 23 in the STE group and 22 in the NSTE group, but DNA of 3 patients from the STE group and 7 from the NSTE group could not be assessed. Data of a total of 35 patients were entered in the statistical analyses. The mean age was 61.65 ±11.5 years. The NSTE group consisted of 15 patients (9 males, 6 females) and the STE group consisted of 20 (13 males, 7 females). The diagnosis of patients in the NSTE group was NSTEMI in 4 and USAP in 11; in the STE group, anterior MI in 13 and inferior MI in 7.

The baseline characteristics were not different between groups (Table 1). The angiographic parameters, Gensini and extent scores were also not significantly different (Table 2). The proportions of patients with mutant alleles were similar in the two groups for all polymorphisms (Table 3).

Fifteen patients (42.8%) from all 35 had a -fibrinogen 455A allele. The angiographic parameters, Gensini and extent scores were not different among the patients with a 455A allele and patients without a 455A allele. The same comparisons were made for the other three polymorphisms, but the differences were not statistically significant (Table 4).

Discussion

Every year approximately 1.7 million ACS patients are admitted to hospitals in the USA. Of those, only one-quarter present with acute STEMI; three-quarters (approximately 1.4 million patients) present with USAP or NSTEMI [11]. Angiographic studies showed that luminal obstructions by thrombus in culprit coronary arteries are significantly different according to the presence of ST segment elevation. Acute STEMI is most commonly caused by acute total thrombotic occlusion of a coronary artery, whereas USAP/NSTEMI is usually associated with severe coronary obstruction but no total occlusion of the culprit coronary artery [12-15]. The question why the thrombus occludes the culprit artery totally in some patients and not totally in others is not clearly answered. This may be partially explained by the fact that former studies that investigated the formation of thrombi in acute MI used the old MI classification: Q and non-Q [16]. The atherosclerotic plaques that are associated with thrombosis and a total occlusion, located in infarct-related vessels, are generally more complex and irregular than those in vessels not associated with STEMI. Coronary arterial thrombi responsible for STEMI are approximately 1 cm in length in most cases, adhere to the luminal surface of an artery, and are composed of platelets, fibrin, erythrocytes and leukocytes [17]. Observational data suggest that NSTEMI is seen more commonly in elderly patients and patients with a prior MI.

This study showed that the frequencies of the -fibrinogen 455G/A, factor V 1691G/A, 1299H/A and Gp IIb/IIIa PL A1/A2 polymorphisms were not different between two major groups of ACS. These findings suggest that these polymorphisms have no effects on the relative magnitude of thrombus responsible for ACS or the severity of occlusion of the coronary artery related ACS.

Many prospective studies have demonstrated a relationship between fibrinogen levels and thrombotic arterial diseases such as MI and ischemic stroke [18-20]. The AA genotype in 455 G/A polymorphism on the gene encoding the  chain of fibrinogen is associated with higher fibrinogen levels. The studies investigating the relationship between the 455A allele and MI reported conflicting results [5, 21]. There are previously no data about the frequencies of -fibrinogen 455 G/A polymorphism in subgroups of ACS, similarly for the other three polymorphisms: factor V 1691G/A, 1299H/A and Gp IIb/IIIa PL A1/A2. This study demonstrates for the first time the frequencies of these polymorphisms in subgroups of ACS. A meta-analysis investigating the relation between MI and multiple genetic risk factors found that the 455A allele is associated with lower incidence of MI [22]. So the relation between CAD and -fibrinogen 455G/A polymorphism is not clear yet.

Factor V 1691G/A (or Leiden mutation) and 1299H/A polymorphisms are responsible for active protein C resistance: a hypercoagulable state [23-26]. Two meta-analyses showed that MI risk is increased 1.3 fold in patients carrying the 1691A allele [27]. The elevation of the risk did not reach statistical significance in another meta-analysis [22]. Three studies reported that the frequency of the 1691A allele was higher in patients who experienced an acute MI without coronary atherosclerosis [27-29]. The patients carrying factor V 1299A allele had no higher risk for MI in a study, but the risk of MI increased 4.4 fold in association with smoking compared to non-smokers [30]. There are no data reporting the frequencies of factor V 1691G/A and 1299H/A polymorphisms in the two groups of ACS.

Glycoprotein IIb/IIIa is the primary receptor on the surface of platelets for fibrinogen binding during haemostasis. The sensitivity of platelets to aggregation is increased in patients with PL A2 allele in Gp PL A1/A2 polymorphism on the gene encoding these receptor proteins [31]. Some of the studies investigating the relation between PL A2 allele and arterial thrombosis reported positive results [32-34]. A study performed with patients with MI at younger ages reported that the PL A2 allele increased the risk of MI 1.8 fold and the elevation of risk was 13.7 fold in association with smoking [35]. Similarly, there are no data reporting the frequency of Gp PL A1/A2 polymorphism in the two groups of ACS.

The 455A allele in beta fibrinogen 455 G/A polymorphism has been shown to increase the progression of atherosclerotic plaques [36]. Some clinical properties such as age, gender and diabetes are associated with more severe atherosclerosis. The present study found no relation between the angiographic severity of coronary atherosclerosis and -fibrinogen 455G/A, factor V 1691G/A, 1299H/A and Gp IIb/IIIa PL A1/A2 polymorphisms.



Limitation



Because of the relatively small population, multivariate analyses could not be performed in this study.

Conclusions

We were not able to find any evidence for associating ACS subtype and angiographic severity of coronary atherosclerosis with -fibrinogen 455G/A, factor V 1691G/A, 1299H/A and Gp IIb/IIIa PL A1/A2 polymorphisms. These findings suggest that these polymorphisms have no effects on the relative magnitude of thrombus responsible for ACS or the severity of the occlusion of the coronary artery related ACS. Future studies with larger groups may reveal whether these genetic alterations have a significant impact on ACS.

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