|Year : 2014 | Volume
| Issue : 3 | Page : 98-102
Fibrinogen -455G/A promoter polymorphism in acute ST elevation myocardial infarction in Egyptian patients
Nadia I Sewelam1, Akram A Ahmed2, Hanan A Alwakeel MD 1, Mohamed Y Khaled2
1 Department of Clinical and Chemical Pathology, Faculty of Medicine, Cairo University, Cairo, Egypt
2 Department of Critical Care, Faculty of Medicine, Cairo University, Cairo, Egypt
|Date of Submission||14-Jul-2014|
|Date of Acceptance||27-Jul-2014|
|Date of Web Publication||31-Dec-2014|
Hanan A Alwakeel
Clinical and Chemical Pathology Department, Faculty of Medicine, Cairo University, 12351, Cairo
Source of Support: None, Conflict of Interest: None
Background Coronary artery diseases (CAD) are complex disorders resulting from interactions of different predisposing factors.
Aim to evaluate the role of -455 G/A Fibrinogen polymorphism (rs1800790) in Egyptian patients with CAD.
Subjects and Methods Plasma fibrinogen levels were assayed and PCR-RFLP was used to evaluate fibrinogen -455 G/A polymorphism in CAD. Patients with stable CAD and STEMI compared to a control group with negative stress Tc 99m sesta MIBI myocardial perfusion scan.
Results 137 patients were studied. No significant difference between the patients and controls as regards different genotypic distribution of -455 G/A Fibrinogen polymorphism. However, the AA genotype was more prevalent among the control group. No significant correlation between the genotypic variants and the fibrinogen levels was detected.
Conclusion The A allele of fibrinogen -455 gene promoter may confer some protection against CAD, however, GG genotype maybe more common among Egyptian CAD patients.
Keywords: fibrinogen, myocardial infarction, promoter polymorphism
|How to cite this article:|
Sewelam NI, Ahmed AA, Alwakeel HA, Khaled MY. Fibrinogen -455G/A promoter polymorphism in acute ST elevation myocardial infarction in Egyptian patients. Egypt J Haematol 2014;39:98-102
|How to cite this URL:|
Sewelam NI, Ahmed AA, Alwakeel HA, Khaled MY. Fibrinogen -455G/A promoter polymorphism in acute ST elevation myocardial infarction in Egyptian patients. Egypt J Haematol [serial online] 2014 [cited 2020 Feb 20];39:98-102. Available from: http://www.ehj.eg.net/text.asp?2014/39/3/98/148225
| Introduction|| |
Acute coronary syndromes are a major cause of emergency medical care and hospitalization in the whole world. Smoking, hypercholesterolemia, hypertension, and diabetes mellitus are established risk factors  ; however, still genetic predisposition is questionable and there is no sufficient evidence to incriminate any of the prothrombotic gene polymorphisms as an established predisposing factor. In developing countries, it is challenging to investigate genotype correlation with acute coronary syndromes ,,,, .
The role of plasma fibrinogen as a central protein of the coagulation system has been suspected for many years and recently documented by experimental and clinical evidence that human plaques are rich in fibrinogen ,,,,, . Early epidemiological evidence has associated high levels of plasma fibrinogen with cardiovascular diseases ,, , and consequently fibrinolytic drugs until date are considered an integral therapy in acute coronary syndromes  .
Fibrinogen beta chain gene polymorphisms have been suggested by several authors as a contributing risk factor. However, most of the conducted studies did not prove the superiority of any polymorphism over another as a risk factor ,,,,,,,, .
We aimed to study fibrinogen -455G/A promoter polymorphism (rs1800790) in acute ST elevation myocardial infarction (STEMI) patients and stable angina patients in comparison with a healthy control group and to correlate different genotypic variants of this polymorphism with measured fibrinogen levels in different studied groups.
| Patients and methods|| |
This was a prospective, cross-sectional, pilot single-center study, conducted on patients admitted or subjected to myocardial perfusion imaging in the Critical Care Center, Cairo University during the time period from February 2011 to August 2012. This study was approved by the ethical committee of the Faculty of Medicine, Cairo University.
Seventy-nine STEMI patients were intended for inclusion; 40 patients were excluded because of refusal of inclusion or administration of anticoagulants before presentation to the Critical Care Department.
Patients scheduled for stress-rest technetium-99m ( 99m Tc) sestaMIBI (methoxy isobutyryl isonitrite) myocardial perfusion study (MPS) with abnormal MPS were included as stable angina patients. Ninety-eight patients subjected to myocardial perfusion imaging were intended for inclusion. Interpretation of MPS of the included patients sorted out 37 patients with negative MPS upon maximal exercise (the control group) and 61 patients with positive MPS (the stable angina group) ([Figure 1]).
|Figure 1 A flowchart showing included participants. MPS, myocardial perfusion study; STEMI, ST elevation myocardial infarction.|
Click here to view
Patients who were above 70 years of age or presented with cardiogenic shock and/or patients refusing the perfusion were excluded from the study. Consents were taken from all participants before being involved in this study.
All patients included in the study were subjected to history taking with special emphasis on the risk factors of coronary artery disease (CAD). Samples were withdrawn from each participant to determine fibrinogen level and for DNA extraction for detection of fibrinogen -455G/A polymorphism using PCR and restriction fragment length polymorphism (RFLP). Samples were transferred and stored in the Clinical Pathology Department where they were processed by other investigators blinded to the clinical state of the patients.
Patients with STEMI were subjected to full clinical examination on admission to the Critical Care Unit and to 12-lead ECG and were subjected to primary percutaneous coronary intervention (PCI) by experienced operators within 90 min of admission. Samples needed for the study were withdrawn on admission with routine baseline laboratory investigations before administration of any anticoagulant or antiplatelet therapy. Laboratory data and clinical data were gathered by the statistician and further analyzed.
99m Tc sestaMIBI myocardial perfusion imaging
Patients were subjected to 99m Tc sestaMIBI myocardial perfusion imaging.
- Ischemia provocation stress testing using:
- Treadmill exercise stress test using the Bruce protocol to reach 85% at least from the target heart rate or termination of exercise upon development of chest pain, ECG changes, or serious arrhythmias. Patients with exercise terminated because of physical fatigue were excluded.
- Pharmacologic stress test using either dipyridamole given at a dose of 0.56 mg/kg body weight over 4 min or dobutamine infusion in incremental doses of 5, 10, 20, and 40 μg/kg/min for 5 min each.
- A dose of 15-20 mCi of 99m Tc sestaMIBI was injected during the peak of exercise or after 2 min of dipyridamole infusion or at the maximum dose of dobutamine tolerated. Another similar dose of 99m Tc sestaMIBI was injected on a separate day at rest to acquire the rest images.
- Stress images were acquired after 45 min and rest images were acquired after 90 min and up to 6 h of injection. Single photon emission computed tomography image acquisition was performed using a double-head Siemens Symbia gamma camera (Philips, Medical Solution, Inc. IL, USA). A total of 20 frames were acquired 30 s each over 120° arc to get two studies (stress-rest) demonstrated as a comparison display showing the classical short axis, vertical long axis, and horizontal long axis slices.
- Myocardial perfusion scans were interpreted and revised by two experienced investigators blinded to the laboratory results.
Determination of fibrinogen level
Withdrawal of 2 ml venous blood on sodium citrate was performed for quantitative determination of fibrinogen level by the clotting method of Clauss  using Fibri-Prest reagent (Diagnostica Stago S.A.S, France) on fully automated coagulometer (STA Compact, Diagnostica stago S.A.S, France). Fibrinogen level above 400 mg/dl is considered above the normal range  .
Detection of fibrinogen -455G/A polymorphism (rs1800790)
DNA extraction: Two milliliters of venous blood sample was collected under complete aseptic conditions in vacuum tubes containing EDTA to be stored at -20°C for subsequent DNA extraction. Genomic DNA was extracted using the QIAamp DNA blood mini kit (California, USA) according to the manufacturer's instructions.
Genotyping: Fibrinogen -455G/A polymorphism (rs1800790) was detected using the PCR-RFLP method. As a negative control, PCR mix without DNA sample was used to ensure contamination-free PCR product. The genotyping of fibrinogen -455G/A was detected using the forward, 5′-AGGGTCTTTCTGATGTGT-3′, and the reverse, 5′-AAGTTAGGGCACTCCTCA-3′, primers, as previously described  .
PCR reaction was performed in a 25 μl volume with 5 μl DNA (50 ng), 1 ml forward primer and 1 μl reverse primer (the primer concentration was 20 pmol for each primer), 12.5 μl master mix Thermo Scientific (Dreamtaq Green PCR master mix, Thermo Fisher Scientific Inc, USA), and 5.5 μl nuclease-free water. Amplification was performed on an automated thermal cycler (GeneAmp PCR system 2700 thermal cycler, Applied Biosystems, California, USA). PCR conditions for fibrinogen -455G/A polymorphism were: 3 min for initial denaturation at 94°C followed by 35 cycles at 94°C for 60 s for denaturation, 60 s at 55°C for annealing, and 60 s at 72°C for extension, and then final extension was performed for 10 min at 72°C. This reaction yielded a 336-bp fragment.
Restriction enzyme digestion: The PCR products were digested with HaeIII restriction endonuclease (New England BioLabs, MA, USA) and subjected to electrophoresis on a 2% agarose gel. Bands were visualized with ethidium bromide staining under ultraviolet light. The PCR product of -455A allele is not cleaved by HaeIII generating a 336-bp band, whereas the PCR product of -455G allele is cleaved by the enzyme generating 215 and 121-bp fragments  ([Figure 2]). The G/A heterozygote generates three bands: 336, 215, and 121-bp bands.
|Figure 2 Lane 1: marker ladder 100, 200, 300, 400, 500 bp; lane 3, 4, 6, 7, and 9: GG genotype (215, 121 bp); lane 8: AA genotype (336 bp); lane 2, 5, and 8: G/A geno type (336, 215, 121 bp).|
Click here to view
Data were analyzed using IBM SPSS advanced statistics version 20 (SPSS Inc., Chicago, Illinois, USA). The χ2 -test (Fisher's exact test) was used to examine the relationship between qualitative variables. For quantitative data, comparison between two groups was performed using either the Student t-test or the Mann-Whitney U-test as appropriate. Comparison between three groups was performed using the Kruskal-Wallis test (nonparametric analysis of variance) then multiple comparisons were performed on the basis of the Kruskal-Wallis test. Spearman-rho method was used to test correlation between numerical variables. Odds ratio with its 95% confidence interval was used for risk estimation. A P value less than 0.05 was considered significant.
| Results|| |
A total of 137 participants were included; the number of male patients was significantly higher than female patients. However, female sex predominated in the control group. The mean age was 61 years. Age of STEMI patients was significantly higher compared with the other two groups. All risk factors for CAD were significantly higher in the STEMI and stable angina groups when compared with controls ([Table 1]).
|Table 1 Risk factors detected in the STEMI, stable angina, and control groups|
Click here to view
Baseline plasma fibrinogen levels in different studied groups
Fibrinogen level was measured on admission in the three studied groups revealing a range of 170-757 mg/dl with a mean of 371±154 mg/dl and median of 331 mg/dl in the STEMI group, a range of 196-1000 mg/dl with a mean of 331 ± 98 mg/dl and median of 346 mg/dl in the stable angina group, and a range of 102-627 mg/dl with a mean of 314.5 ± 117.1 mg/dl and median of 323 mg/dl in the control group (P = 0.640) ([Table 2]).
|Table 2 Comparison of fi brinogen levels detected in the different studied groups|
Click here to view
Genotypic distribution of fibrinogen -455G/A polymorphism
Of the STEMI, stable angina, and the control groups, the GG genotype was more prevalent occurring in 66.7% (N = 26), 60.7% (N=37), and 56.8% (N = 21), respectively. The GA genotype was 20.5% (N=8) in the STEMI group, 26.2% (N = 16) in stable angina patients, and 16.2% (N = 6) in the control group. However, the AA genotype was more prevalent among the control group occurring in 27% (N = 10), whereas it was less prevalent in the stable angina group and in the STEMI group occurring in 13.1% (N = 8) and 12.8% (N = 5), respectively ([Table 3]).
|Table 3 Genotypic distribution and allele frequencies of fibrinogen -455G/A polymorphism in different studied groups|
Click here to view
Comparison of genotypic distribution of -455G/A fibrinogen polymorphism in patients with elevated fibrinogen level among the three studied groups are presented in [Table 4].
|Table 4 Comparison of genotypic distribution of -455G/A fibrinogen polymorphism in patients with elevated fibrinogen level among the three studied groups|
Click here to view
| Discussion|| |
There is some evidence from previous studies that increased plasma fibrinogen level may be related to increased CAD risk  . Some studies support the relationship between plasma fibrinogen levels and certain fibrinogen gene polymorphisms ,,,, ; however, the link between these polymorphisms and occurrence of CAD is still lacking and understudied with special reference to Egypt.
In the present study we used PCR-RFLP to evaluate the role of -455G/A fibrinogen polymorphism (rs1800790) in the occurrence of CAD in Egyptian patients referred to one of the main referral centers in Cairo. Patients who either had stable CAD proven by nuclear stress test or presented with STEMI were compared with controls.
As expected, the classic risk factors for CAD were significantly more common in the study group.
Similar to other studied populations, Egyptian patients showed more prevalence of the GG genotype followed by the GA, and the least common was AA genotype  .
There was no significant difference between the patients and controls with respect to the fibrinogen gene polymorphism. This is in agreement with the study by Sun et al.  who did not find any difference between control individuals, stable angina patients, and acute coronary syndrome patients with respect to allele frequency and genotype distribution of -455G/A fibrinogen polymorphism.
However, in our study, AA genotype and allele A frequency were more common among the control group, raising the question about the protective role of the A allele previously presented by Lu et al.  and Rallidis et al.  . In addition, through a study conducted by Eirini et al.  , they found that the fibrinogen gene b variants rs1800787 and rs1800789 (that are highly correlated with rs1800790) might confer protection against CAD.
Interestingly, we did not find a significant difference in fibrinogen level between the three groups. However, fibrinogen level above normal range (400 mg/dl) was more common in ischemic patients when compared with the controls. Subgroup analysis according to the genotype showed a trend toward more common patients with abnormal fibrinogen level in GG genotype. This is in contrast with Chinese study conducted by Sun et al.  who found that fibrinogen -455A allele was associated with the highest plasma fibrinogen level in control individuals and patients with stable angina but not in patients with acute coronary syndromes. This difference could be attributed to small sample size in our study, which hampered to reach a definite conclusion.
| Conclusion|| |
The AA genotype and A allele of fibrinogen -455 gene polymorphism may confer some protection against CAD, whereas GG genotype may be more common among Egyptian CAD patients. However, further studies on larger scale and multicenter are needed.
| Acknowledgements|| |
Conflicts of interest
There are no conflicts of interest.
| References|| |
Goff DC, Lloyd-Jones DM, et al.
2013 ACC/AHA guideline on the assessment of cardiovascular risk. J Am Coll Cardiol 2014; 129:S49-S73.
Lu X-F, Yu H-J , Zhou X-Y, Wang L-Y, Huang J-F, Gu D-F. Influence of fibrinogen b-chain gene variations on risk of myocardial infarction in a Chinese Han population. Chin Med J 2008; 121
Atherosclerosis, Thrombosis, and Vascular Biology Italian Study Group. No evidence of association between prothrombotic gene polymorphisms and the development of acute myocardial infarction at a young age. Circulation 2003; 107
Sevimli S, Karakoyun S, Bakirci EM, Topcu S, Kalkan K, Borekci A, Vançelik S. Impact of -455G/A polymorphism of the b-fibrinogen gene on platelet aggregation in patients with acute coronary syndrome. Clin Appl Thromb Hemost
Sun A, Ma H, Xu D, Wang Y, Liu M, Xu L, et al.
Association between -455G/A and fibrinogen in a Chinese population. Acta Cardiol 2009; 64
Abou El-Magd LM, El-Demerdash OH, El-Rehim Saleh SA, Ali Shaheen KY, El-Kei TMK, Sabet SS. Proinsulin/insulin ratio and plasminogen activator inhibitor type 1 (PAI-1) activity as new risk factors for acute myocardial infarction in both type II diabetic and non diabetic subjects. Egypt Heart J
Henschen A, McDonagh J In: Zwaal RFA, Hemker HC, editors. Fibrinogen, fibrin and factor XIII. Blood coagulation
. Amsterdam: Elsevier Science Pub Co; 1986. 171-241.
Atherosclerosis Risk in Community Study. Operation manual, no. 9: hemost determinations. Version 1.0. Bethesda, MD: National Heart, Lung, and Blood Institute; 1987
Bini A, Kudrik BJ. In: Fibrin and its derivatives in the normal and disease vessel wall. Annals of the New York Academy of Sciences; 1992;667: 112-126.
Constantinides P. Cause of thrombosis in human atherosclerotic arteries. Ann J Cardiol
Smith EL, Thompson WD. Fibrin as cofactor in atherogenesis. Thromb Res 1994; 73
Schwartz CJ, Valente AJ, Kelley JL, Sprague EA, Edwards EH. Thrombosis and development of atherosclerosis: Rokitansky revisited. Semin Thromb Hemost 1988; 14
Di Minno G, Mancini M. Measuring plasma fibrinogen to predict stroke and myocardial infarction. Arteriosclerosis 1990; 10
Cook NS, Ubben D. Fibrinogen as a major risk factor for cardiovascular disease. Trends Pharmacol Sci 1990; 11
Ernst E, Resch KL. Fibrinogen as a cardiovascular risk factor: a meta-analysis and review of the literature. Ann Intern Med 1993; 118:956-963.
Anderson JL, Adams CD, Antman EM, et al.
2012 ACCF/AHA focused update incorporated into the ACCF/AHA 2007 guidelines for the management of patients with unstable angina/non-ST-elevation myocardial infarction: a report of the American College of Cardiology Foundation/American Heart Association Task Force on practice guidelines. J Am Coll Cardiol 2013; 61
Behague I, Poirier O, Nicaud V, Evans A, Arveiler D, Luc G, et al.
Fibrinogen gene polymorphisms are associated with plasma fibrinogen and coronary artery disease in patients with myocardial infarction: the ECTIM Study. Circulation 1996; 93
Carter AM, Mansfield MW, Strickland MH, Grant PJ. Beta-fibrinogen gene -455 G/A polymorphism and fibrinogen levels: risk factors for coronary artery disease in subjects with NIDDM. Diabetes Care 1996; 19
Yu Q, Safavi F, Roberts R, Marian AJ. A variant of beta fibrinogen is a genetic risk factor for coronary artery disease and myocardial infarction. J Invest Med
Zito F, Di Castelnuovo A, Amore C, D'Orazio A, Donati MB, Iacoviello L. Bcl I polymorphism in the fibrinogen b-chain gene is associated with the risk of familial myocardial infarction by increasing plasma fibrinogen levels: a case-control study in a sample of GISSI-2 patients. Arterioscler Thromb Vasc Biol 1997; 17
Leander K, Wiman B, Hallqvist J, Falk G, De Faire U. The G -455A polymorphism of the fibrinogen B beta-gene relates to plasma fibrinogen in male cases, but does not interact with environmental factors in causing myocardial infarction in either men or women. J Intern Med 2002; 252
Yamada Y, Izawa H, Ichihara S, Takatsu F, Ishihara H, Hirayama H, et al.
Prediction of the risk of myocardial infarction from polymorphisms in candidate genes. N Engl J Med 2002; 347
Tobin MD, Braund PS, Burton PR, Thompson JR, Steeds R, Channer K, et al.
Genotypes and haplotypes predisposing to myocardial infarction: a multilocus case-control study. Eur Heart J 2004; 25
Mannila MN, Eriksson P, Lundman P, Samnegard A, Boquist S, Ericsson CG, et al.
Contribution of haplotypes across the fibrinogen gene cluster to variation in risk of myocardial infarction. Thromb Haemost 2005; 93
Maresca G, Blasio A Di, Marchioli R, Minno G Di. Measuring plasma fibrinogen to predict stroke and myocardial infarction: an update. Arterioscler Thromb Vasc Biol 1999; 19
Clauss A. Rapid physiological coagulation method in determination of fibrinogen. Acta Haematol 1957; 17
Samama M, Conrad J, Horellou MH, Lecompte T. Physiologie et exploration de L' hemostase.
Paris: Doin; 1990:153-155.
Danesh J, Collins R, Appleby P, Peto R. Association of fibrinogen, C reactive protein, albumin, or leukocyte count with coronary heart disease: meta-analyses of prospective studies. JAMA 1998; 279
Mannila MN, Eriksson P, Leander K, Wiman B, de Faire U, Hamsten A, Silveira A. The association between fibrinogen haplotypes and myocardial infarction in men is partly mediated through pleiotropic effects on the serum IL-6 concentration. J Intern Med 2007; 261
Carty CL, Cushman M, Jones D, Lange LA, Hindorff LA, Rice K, et al.
Associations between common fibrinogen gene polymorphisms and cardiovascular disease in older adults. The Cardiovascular Health Study. Thromb Haemost 2008; 99
Jacquemin B, Antoniades C, Nyberg F, Plana E, Muller M, Greven S, et al.
Common genetic polymorphisms and haplotypes of fibrinogen alpha, beta, and gamma chains affect fibrinogen levels and the response to proinflammatory stimulation in myocardial infarction survivors: the AIRGENE study. J Am Coll Cardiol 2008; 52
Tybjaerg-Hansen A, Agerholm-Larsen B, Humphries SE, Abildgaard S, Schnohr P, Nordestgaard BG. A common mutation (G -455->A) in the beta-fibrinogen promoter is an independent predictor of plasma fibrinogen, but not of ischemic heart disease. A study of 9127 individuals based on the Copenhagen City Heart Study. J Clin Invest 1997; 99
Van 't Hooft FM, von Bahr SJ, Silveira A, Iliadou A, Eriksson P, Hamsten A. Two common, functional polymorphisms in the promoter region of the beta-fibrinogen gene contribute to regulation of plasma fibrinogen concentration. Arterioscler Thromb Vasc Biol 1999; 19
Rallidis LS, Gialeraki A, Fountoulaki K, Politou M, Sourides V, Travlou A, et al.
G -455A polymorphism of beta-fibrinogen gene and the risk of premature myocardial infarction in Greece. Thromb Res 2010; 125
Eirini VT, Tiit N, Julia S, Vassileios P, Panagiota F, Genovefa K, et al.
Fibrinogen beta variants confer protection against coronary artery disease in a Greek case-control study. BMC Med Genet 2010; 11:28.
[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3], [Table 4]