Coronary heart disease (CHD) is a complex disease with both environmental and genetic determinants. Each year, CHD kills more Americans than cancer 1,2. Many risk factors are involved, such as hypertension, hypercholesterolemia, diabetes mellitus, obesity, unhealthy diet, protein C deficiency, protein S deficiency, antithrombin III deficiency, elevated clotting factors VIII, IX, XI, and elevated fibrinogen. In addition, a positive family history is a strong independent risk factor for CHD 3.
The development of CHD and myocardial infarction (MI) involves hyperplasia of arterial smooth muscles, the presence of fatty streaks, atheroma formation, plaque rupture, thrombus formation, and vessel occlusion. The presence of a first-degree relative with MI was found to be associated with a seven-fold increased risk for MI 4. Also, it was found that among the underlying causes of CHD, the blood coagulation system is thought to determine the onset of MI through its role in blood clot formation 5. The coagulation cascade is a series of reactions, in which a zymogen (inactive enzyme precursor) and its glycoprotein cofactor are activated to become active components that then catalyze the next reaction in the cascade, ultimately resulting in the cross-linked fibrin. A very important step in thrombus formation is prothrombin’s (PT) conversion to thrombin, which converts fibrinogen to fibrin 6.
Fibrinogen is a protein that is composed of α, β, and γ polypeptide chains and plays a key role in blood clotting 7. Patients with CHD tended to have higher fibrinogen levels than those without the disease 8. The percent mortality rate increased by over seven-fold in those with the highest fibrinogen levels, compared with those with the lowest levels 1. The three polypeptide chains of fibrinogen (α, β, and γ chains) are encoded by three different genes clustered on chromosome 4 in region q28. In-vitro studies have suggested that β-chain synthesis limits the rate of production of mature fibrinogen. Thus, most studies focus on the association of polymorphisms in the fibrinogen β-chain (FGB) gene with MI. The FGB-455 G/A promoter polymorphism was found to be associated with increased plasma fibrinogen levels, but the role of the FGB polymorphisms as a risk factor of MI has been debated 9.
Another component of the coagulation cascade is PT (factor II), which is a blood-clotting protein needed to form fibrin. A common point mutation in the 3′-untranslated region of the PT gene (20210 G/A) has been reported to be associated with elevated plasma PT levels, making the blood more likely to clot and increasing the risk for venous thrombosis by three to five folds. Thus, attention has been paid to investigate whether there is a relationship between this mutation and CHD and MI 10.
The aim of this study was to assess the frequencies of FGB-455 G/A and PT 20210 G/A polymorphisms in CHD Egyptian patients.
Methods
Thirty adult patients with age ranging between 41 and 70 years and angiographically documented CHD, scheduled for elective coronary artery bypass graft surgery, were included in this study. Patients were included irrespective of concomitant risk factors for atherosclerosis such as hypertension, diabetes mellitus, and cigarette smoking.
These patients were compared with 30 age-matched and sex-matched, healthy individuals without any clinical disorders selected from the general population. Coronary angiography was not performed for these individuals and therefore, the presence of atherosclerotic coronaries cannot be excluded. Informed consents were obtained from all the patients and healthy participants according to the guidelines of our ethics committee.
Analysis of polymorphisms
The assay for the identification of FGB-455 and PT 20210 genotypes was based on PCR and reverse-hybridization using CVD StripAssay kit (ViennaLab Labordiagnostika GmbH, Vienna, Austria) 11–13. The procedure included three steps as follows: (a) DNA isolation, (b) PCR amplification using biotinylated primers, and (c) hybridization of amplification products to a test strip containing allele-specific oligonucleotide probes immobilized as an array of parallel lines. The bound biotinylated sequences were detected using streptavidin–alkaline phosphatase and color substrates.
Statistical analysis
The statistical analysis of the results was performed using the program of Statistical Package for Social Science version 11 (Berkeley, California, USA); the analysis was performed using arithmetic mean, SD, Student t-test, χ2-test, and odds ratio (OR). *P-values less than or equal to 0.05 were considered significant, whereas P-values more than 0.05 were considered nonsignificant 14.
Results
The patients included eight women (26.67%) and 22 men (73.33%), with a mean age of 57.5 (±6.80) years, and a range from 41 to 70 years. The 30 control persons included six women (20%) and 24 men (80%), with a mean age of 54.93 (±5.73) years and a range from 45 years to 68 years. There was no significant difference between patients and control individuals with regard to the age and sex distribution (Table 1).
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Table 1: Age and sex difference between patient and control groups
Family history of angina and/or MI was irrelevant in both patient and control groups, and hence, comment on it was excluded. All patients and control participants were smokers, and hence, comment on smoking was excluded. The medical problems in the patient group, obtained by history, clinical examinations, ECG, and echocardiogram, are shown in Table 2.
Table 2: Medical problems in the patient group
The mean value of serum cholesterol
It was significantly higher (P<0.05) in the patient group compared with the control group. The recorded mean value was 259.83 (±27.74) mg/dl in the patient group compared with 152.93 (±15.53) mg/dl in the control group (Table 3) (Fig. 1).
Table 3: Lipid profile of patient and controls groups
Fig. 1: Lipid profile of patient (blue) and control (red) groups. TAG, triacylglycerol.
The mean value of serum triglycerides
It was significantly higher (P<0.05) in the patient group compared with the control group. The recorded mean value was 184.30 (±17.29) mg/dl in the patient group compared with 97.60 (±13.60) mg/dl in the control group (Table 3) (Fig. 1).
The mean value of serum HDL-cholesterol
It was significantly lower (P<0.05) in the patient group compared with the control group. The recorded mean value was 26.00 (±3.02) mg/dl in the patient group compared with 50.73 (±4.97) mg/dl in the control group (Table 3) (Fig. 1).
The mean value of serum LDL-cholesterol
It was significantly higher (P<0.05) in the patient group compared with the control group. The recorded mean value was 123.50 (±15.91) mg/dl in the patient group compared with 93.50 (±9.48) mg/dl in the control group (Table 3) (Fig. 1).
Table 4 shows the results of liver and kidney function tests in addition to some hematological investigations. All parameters showed a nonsignificant difference between the studied groups except the mean total bilirubin, which was significantly higher in the patients as compared with the controls.
Table 4: Biochemical characteristics of the studied groups
Genotypes and alleles of the analyzed polymorphisms
Fibrinogen β-455 gene locus
The GG polymorphism was the genotype found in 20 patients (66.67%), whereas the GA polymorphism was found in nine patients (30%) and the AA polymorphism was found in only one patient (3.33%). Meanwhile, in the control group, the numbers were 24 individuals (80%), six individuals (20%), and none (0%), respectively (Table 5) (Fig. 2).
Table 5: Fibrinogen β-chain-455 genotypes frequencies and significance
Fig. 2: Fibrinogen β-chain (FGB)-455 genotype frequencies of patient (blue) and control (red) groups.
There was no significant difference between patient and control groups regarding the frequency of FGB-455 G/G polymorphism (P=0.243) (Table 5). Also, there was no significant difference between patient and control groups regarding the frequency of FGB-455 G/A polymorphism (P=0.371) or the frequency of FGB-455 A/A polymorphism (P=1.000) (Table 5).
It was found that FGB-455 G/G is more likely to be associated with CHD (OR<1), whereas FGB-455 G/A is less likely to be associated with CHD (OR>1) (Table 5).
The frequency of the A allele was 11 (nine GA and one AA) (18.33%) in the patient group compared with six (six GA and zero AA) (10%) in the control group, which showed a nonsignificant difference (P-value=0.148) (Table 6).
Table 6: Frequency and significance of the A allele of fibrinogen β-chain-455 gene locus in both studied groups
Prothrombin 20210 gene locus
The patient group contained 29 individuals (96.67%) with the GG polymorphism, compared with 30 individuals (100%) in the control group. There was only one patient (3.33%) with the GA polymorphism, compared with none (0%) in the control group (Table 7) (Fig. 3).
Table 7: Genotype frequencies and significance of prothrombin 20210 gene locus of the studied groups
Fig. 3: Prothrombin 20210 genotype frequencies of patient (blue) and control (red) groups.
There was no significant difference between patient and control groups regarding the frequency of the GG polymorphism (P=1.000) or the frequency of GA polymorphism (P=1.000) (Table 7).
Discussion
CHD shows high and increasing rates of morbidity and mortality 2, and its development involves hyperplasia of arterial smooth muscles, the presence of fatty streaks, atheroma formation, plaque rupture, thrombus formation, and vessel occlusion 4. The blood coagulation system is thought to determine the onset of MI through its role in blood clot formation. Searching for genetic variations that may affect this sequential mechanism may be very important for the prediction of the disease occurrence and/or prognosis 5.
Many epidemiological studies have identified the plasma fibrinogen level as a powerful predictor of future CHD. However, fibrinogen is also an acute-phase reactant, and in recent years, the evidence for inflammation as a cause of vascular disease has steadily grown. Investigators have therefore frequently turned to genetic studies that have the potential to provide evidence supporting a pathogenic role of fibrinogen in ischemic heart disease. If genetic polymorphisms causing elevated levels of fibrinogen were shown to be associated with an increased risk of ischemic heart disease, then this would be good evidence for fibrinogen playing a causal role in the disease 15.
However, despite the clear association between fibrinogen levels and ischemic heart disease in both prospective and cross-sectional studies, and the consistent association between genetic polymorphisms of the β-chain and fibrinogen levels, none of the large population-based studies have shown an association between the polymorphisms and ischemic heart disease 8.
Carriers of the A allele of the FGB-455 G/A polymorphism have increased plasma fibrinogen levels, and studies on this polymorphism have been performed mainly among Caucasians. The association between FGB-455 G/A polymorphism and thrombophilic diseases, such as venous thromboembolism and CHD, in the Chinese Han population had been investigated and the presence of the A allele was found to be a greater risk factor in cases than in controls 16.
The present study aimed at elucidating the association of FGB-455 G/A polymorphism with CHD in Egyptian patients. The FGB-455 G/G wild-type polymorphism was found to be the most frequent among the studied CHD Egyptian patients (66.67%) and the control persons (80%). Although it was found to be more likely associated with CHD in Egypt (OR<1); yet, there was no significant difference between the patient group and the control group regarding its frequency (P=0.243).
FGB-455 G/A was found to be less frequent than FGB-455 G/G among the studied CHD Egyptian patients (30%) and control persons (20%). Also, it was found to be less likely associated with CHD in Egypt (OR>1), and there was no significant difference between the patient group and the control group regarding its frequency (P=0.371). No significant difference was found between the patient group and the control group regarding the frequency of FGB-455 AA polymorphism (P=1.000), and it was found to be a rare polymorphism in the studied Egyptian participants (3.33% of the patient group and 0% of the control group). Thus, none of the polymorphisms of the FGB-455 locus differs significantly between patient and control groups (P-values >0.05). Accordingly, none of the three polymorphisms can be considered as a risk factor for CHD in the studied Egyptian population.
The results of this study were in agreement with Carty et al.1, who investigated whether common (≥5% minor allele frequency) variation in the fibrinogen genes (FGA, FGB, FGG) is associated with fibrinogen concentration variations, carotid artery intimal-medial thickening, and a risk of incident MI, ischemic stroke, and cardio vascular disease (CVD) mortality in European-American and African-American descent adults (≥65 years). They found that among European American, minor alleles of FGA3807, FGB1437 (which is FGB-455 G/A polymorphism), and FGG902 were associated with higher fibrinogen levels (P<0.004 for each). The strongest associations were seen for FGB1437; each additional copy of the minor allele was associated with a 13 mg/dl higher fibrinogen level 1. However, whereas fibrinogen genetic variation was strongly associated with fibrinogen levels, there was less evidence of an association with the more complex outcomes of intimal-medial thickening and CVD events 1.
Also Lu et al.17, studied the association between the genetic polymorphisms of the FGB gene and MI in the Chinese Han population. They found that the cases had significantly lower frequencies of FGB-455 G/A polymorphism than the controls; accordingly, they concluded that the FGB-455 G/A polymorphism was not associated with MI in the Chinese Han population after adjustment for the other traditional risk factors.
The most obvious possible explanation for a failure to find a relationship between genotype and disease is that fibrinogen plays no pathogenic role in coronary artery disease. In this scenario, fibrinogen acts as a marker of some other process, most probably inflammatory, which causes ischemic heart disease independently. This is supported by the finding that fibrinogen levels correlate closely with other acute-phase reactants in patients with ischemic heart disease, raising the possibility that fibrinogen is simply an indicator of disease rather than having a causal role 18. A second possible explanation is that fibrinogen does play a pathogenic role in ischemic heart disease, but the effect of genotype is small compared with other modifiers of fibrinogen level. Also, an effect of the genotype may be apparent only in conjunction with some other risk factor such as smoking or the acute-phase reaction and the presence of a polymorphic variation in genes for intermediaries such as IL-6, which is an inflammatory cytokine known to be the main regulator of fibrinogen synthesis 19.
Rather than directly affecting the levels of proteins or the risk of disease, polymorphisms can amplify the effect of environmental or intermediate conditions on the final phenotype. The genetic control of fibrinogen has to be considered together with the environmental factors: fibrinogen genotypes may interact with cigarette smoking, physical activity, use of drugs, hormonal status, and infections in determining the increase in fibrinogen levels and perhaps the risk of ischemic heart disease 20. Thus, in conclusion, it can be said that CVD events are the result of complex interactions among elevated fibrinogen level and cytokines, especially IL-6, which not only increase the fibrinogen level but also stimulate other events incorporated in the CHD pathogenesis such as vascular smooth muscles proliferation and the inflammatory nature of atherosclerotic plaques formation 21,22.
However, the present study does have some limitations. First, the lack of an association between the FGB-455 G/A polymorphism and MI may be due to the relatively small sample size. Second, the study participants were not recruited prospectively. Thus, a selection bias could not be excluded. Another limitation is that the plasma levels of fibrinogen were not determined. However, a larger cohort will ultimately be required to confirm whether or not the results of the present study are reliable.
The results of the present study were opposed by Reiner et al.7 and Chen et al.8, in different studies on different populations, who linked FGB-455 G/A polymorphism with CHD through the effect of the genotype on the circulating fibrinogen level. Reiner et al.7 analyzed the association between common polymorphisms in the fibrinogen genes and the circulating levels of both functional fibrinogen and total fibrinogen in a large, multicenter, biracial cohort of young US adults in European-American and African-American populations, and reported that a common haplotype tagged by the A minor allele of the well-studied FGB-455 G/A promoter polymorphism was confirmed to be strongly associated with increased plasma fibrinogen levels, which is a strong risk factor for the development of CHD. However, Chen et al.8 evaluated the correlation between FGB-455 G/A polymorphism and susceptibility to coronary artery disease in the Chinese population using a meta-analytic approach and found that the combined OR for the nine studies on the susceptibility to coronary artery disease in FGB-455 A allele carriers (G/A and A/A) compared with the FGB-455 G/G wild-type homozygote was 1.75, suggesting the possibility that FGB-455 G/A polymorphism (in particular, allele A) increases the susceptibility to CHD in the Chinese population.
Also, the results of the present study oppose the results of a similar study conducted by Dilley et al.23, who focused on the evaluation of the relationship between Legg–Perthes disease and the FGB-455 G/A polymorphism. Legg-Perthes disease is a pediatric hip disorder characterized by avascular necrosis of the femoral head, and its etiology involves repeated interruptions of the blood supply to the proximal femur. Accordingly, the role of thrombosis in the disease is of interest. They found that individuals who possessed either the G/A or the A/A genotype were over three times more likely to have Legg-Perthes disease compared with those without the polymorphism 23.
Several studies have shown that the A allele of the FGB-455 G/A polymorphism is associated with an elevated plasma fibrinogen concentration and that A instead of G in the FGB-455 position produces a 1.2–1.5-fold increase in the FGB transcription 24–28. The β-chain transcription is a rate-limiting step in the synthesis of the total functional fibrinogen molecule. Increased viscosity and a higher available substrate quantity resulting from an elevated plasma fibrinogen concentration may promote coagulation and act as a risk for small-vessel thrombotic occlusion, thus affecting the phenotype of the cerebral infarction. The results of the study conducted by Martiskainen et al.29, to assess the fibrinogen gene promoter-455 A Allele as a risk factor for lacunar stroke, concluded that stroke patients carrying the A allele of the FGB-455 G/A polymorphism frequently presented with multiple lacunar infarcts, and this association suggests that the A allele may predispose to atherothrombotic events in the cerebrovascular circulation. Their explanation was that the A genotype and the resulting increased fibrinogen concentration in circulation may contribute to the progression of arteriosclerosis, primarily in smaller cerebral arteries with slower blood flow rather than in large vessels 30. In this way, it may predispose one to the development of occlusions in small cerebral arteries and finally to multiple lacunar infarcts 29. Accordingly, the previous studies that oppose the results of the present study can explain their findings.
Inherited abnormalities of the hemostatic system that may predispose one to thrombosis are termed as inherited thrombophilias. One of them is PT 20210 G/A mutation, the prevalence of which in the general population is estimated to be 2.3% for its heterozygous form. However, this mutation is very rare in China. It implies that the variant of the PT gene is probably not the main cause of the thrombophilic diseases, such as venous thromboembolism and CHD, in the Chinese population 16.
PT 20210 G/A polymorphism has also been investigated in the present study, but current evidence does not support the hypothesis that this polymorphism might be a risk factor for CHD. PT 20210 G/A polymorphism was found to be rare among the studied CHD Egyptian patients (3.33%) and absent in the control persons. There was no significant difference between patient and control groups regarding its frequency (P=1.000). Although PT 20210 G/G wild-type was predominant among CHD Egyptian patients (96.67%) and control persons (100%), there was no significant difference between the patient and the control groups regarding its frequency (P=1.000). Although it was found that PT 20210 G/G is more likely to be associated with CHD (relative risk >1) and PT 20210 G/A is less likely to be associated with CHD (relative risk<1), neither polymorphism differs significantly between the patient and the control groups (P-value>0.05). Accordingly, neither polymorphism can be considered as a risk factor for CHD in the Egyptian population studied.
The results of the present study are in agreement with Rahimi et al.31, who assessed the contribution of factor V Leiden 1691 G/A, PT gene 20210 G/A, and methylentetrahydrofolate reductase (MTHFR) 677 C/T mutations in the development of CHD and their association with diabetes. Their results indicated that there was no significant difference between the prevalence of thrombophilic mutations of factor V Leiden, PT 20210 G/A variant, and MTHFR 677 C/T in CHD patients with or without diabetes compared with controls. Also, Özmen et al.32 failed to show any relation between PT gene mutation and increased risk of arterial thrombosis in their study that investigated the prevalence rates of factor V, MTHFR, and PT gene mutations in patients with arterial thrombosis and in healthy controls. Their findings suggested the MTHFR mutation as a synergistic risk factor for thrombosis in patients with factor V mutation, whereas PT gene mutation has no effect on arterial thrombosis as there was only one patient in their study with PT mutation, who was negative for the other two mutations. Thus, it can be proposed that this type of polymorphism may be harmless when alone, but may increase the risk for CHD when associated with other gene(s) polymorphisms. Also, Geng et al.33, in their study to determine whether polymorphisms in the genes for coagulation factors II, V, and VII could predispose an individual to an increased risk of CHD and/or MI in the Chinese, found that factor II (20210 G/A), V (1691 G/A) mutation is absent and may not be a major genetic factor for CHD and/or MI.
Almawi et al.34 studied factor V 1691 G/A, PT 20210 G/A, and MTHFR 677 C/T gene polymorphism in angiographically documented CHD; the results from this study clearly demonstrated a strong association of hyperhomocysteinemia and homozygosity of the MTHFR 677 C/T, but not factor V Leiden or PT 20210 G/A, mutations with confirmed CHD, which is consistent with our results. A mutation in the PT gene was identified to be associated with increased PT levels, but CHD development requires more complex interactions among several risk factors 35.
The results of the present study were opposed by Mugnolo et al.36, who reported a case of MI at a young age in a person with a medical history of repeated thrombophlebitis, who was heterozygous for the PT 20210 G/A mutation and homozygous for factor V Leiden mutation. A coronary angiography revealed the presence of a moderate atherosclerotic plaque (60%) in the left anterior descending coronary artery, which gave rise to suspicion of a relationship between prothrombotic gene mutations and atherosclerosis. Accordingly, they recommended that the genetic screening for inherited thrombophilia, especially in the presence of a strong familiarity or previous venous thrombosis, and the evaluation of atherosclerotic risk factors, may be critical information for primary prevention of arterial thrombosis 36.
Also, Ye et al.37 performed a meta-analysis on seven hemostatic gene polymorphisms in CHD patients; one of them was PT 20210 G/A. The study involved a total of 66 155 CHD cases and 91 307 controls. Their results, which oppose the results of the present study, concluded that the 20210 A variant of the PT gene increases circulating thrombin generation and might be moderately associated with the risk of CHD 37. Thus, whether PT 20210 G/A mutation could be considered as a risk factor for CHD or not remains controversial although it was established that this mutation increases the amount of PT in the blood stream. It is not yet fully understood as to how increased levels of PT act as a risk factor for CHD, but some studies have indicated that extra PT may interfere with the body's natural ability to dissolve clots. About 1–2% of people with European ancestry carry the PT 20210 G/A mutation 38.
However, in the present study, the lipid profile of the patients was significantly higher than that of the controls (P-values=0.00), which ensures the concept that atherosclerosis and CHD are more related to the elevation in serum cholesterol, triglycerides, and the LDL-cholesterol/HDL-cholesterol ratio in addition to an unhealthy lifestyle 39,40. Alber et al.41 investigated the relationship between HDL cholesterol and the prevalence and severity of coronary artery disease (CHD) in 5641 consecutive patients undergoing coronary angiography. They found that HDL-cholesterol was significantly higher in non-CHD persons than in CHD patients (P<0.001). Accordingly, they concluded that HDL-cholesterol is a stronger predictor of CHD than some other major classical risk factors 41. Furthermore, the results of the present study showed a nonsignificant difference between patients and controls with regard to the age (P=0.120) and sex distribution (P=0.307), which again potentiates the value of the significant lipid profile difference between patients and control persons over other traditional risk factors such as age and sex.
Although the present study was conducted on CHD Egyptian patients, yet, its results may be applicable to other CVDs such as lacunar stroke and peripheral arterial ischemia 29,30. This is due to the similar pathogenesis of the diseases and similar involvement of the two genes of the study. This is similar with the results of Banerjee et al.30, who stated that the plasma fibrinogen level is a valuable index for the peripheral arterial ischemia patients with stable intermittent claudications and a high risk of early mortality. Their results also provide further evidence for the involvement of fibrinogen in the pathogenesis of ischemic arterial diseases 30. Also, a meta-analysis, where 41 publications that examined 44 gene polymorphisms were included, was performed by Elias Zintzaras and Nikos Zdoukopoulos 42, on the basis of the similarity among ischemic arterial diseases in the pathophysiological pathways. FGB-455 G/A and PT 20210 G/A polymorphisms were included in that study, and the results concluded that their association with peripheral arterial ischemia remains controversial 42.
Conclusion
Although a specific genotype may be associated, in healthy participants, with modest differences in the levels of a risk factor for thrombosis, this effect may be larger or smaller in subgroups of participants. Documenting such gene–environment interactions is important if genotype information is ever to be used in a clinical or diagnostic setting. Understanding the molecular mechanisms of such interactions is very important for the development of novel therapeutic approaches to reduce the risk of MI. Future studies should be designed specifically to investigate interactions between potential genetic polymorphisms and acquired risk factors.
Acknowledgments
Conflicts of interest
There are no conflicts of interest.
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