INVESTIGATE MUTATION GENE OF BETA CHAIN FIBRINOGEN (FGB) IN A THROMBOPHILIA PATIENT WITH ACUTE MYOCARDIAL INFARCTION

Trang Thi Nguyen,¹ Phan Duc Tran¹, Huong Thanh Truong ¹, Anh Thi Lan Luong¹,

Binh Thanh Le², LyThi Minh Nguyen¹, An Hong Le³

1- Hanoi Medical University

2- Vietnam Heart Institute, Bachmai Hospital

3-Chu Van An high school

Hanoi Medical University, Department Biomedicine and genetics, 1, Ton That Tung, Dong Da, Ha Noi, Viet Nam. Tel: 04.38523798; Fax:04.38525115. trangtrang1182@yahoo.com.

ABSTRACT

Objectives: To investigate mutation on promoter region of fibrinogen beta chain gene in thrombophilia patient with acute myocardial infarction (AMI). Subjects: A 50 year - old male patient, history of left leg deep vein thrombosis; was diagnosed of AMI. Coronary angiogram: thrombosis caused total occlusion of the middle region of right coronary artery (RCA), middle region of left circumflex coronary artery (LCx), middle segment of left coronary artery (LAD) and partial thrombotic occlusive the distal end of the left main coronary artery and the proximal segment of LAD. Methods: clinical examination, lab tests, echocardiography, emergency angiogram, sequencing beta chain fibrinogen binding gene, using reference and control. Results: find out point mutation C148T at promoter region of FGB gene. Conclusion: following a case of AMI thrombophilia patient with C148T mutation on FGB gene, we aim to develop a research in a wider population of familial myocardial infarction patients in order to find out a relationship between genetic polymorphism of the fibrinogen gene, the corresponding plasma level of fibrinogen and the risk for clinical event (AMI).

Key words: fibrinogen, acute myocardial infarction (AMI), FGB gene, promoter.

INTRODUCTION

Myocardial infarction (MI), one of the leading causes of death worldwide, is a complex disorder influenced by multiple genetic and environmental factors. Thrombosis is generally accepted as the most common pathogenetic pathway of acute MI. As the last target of coagulation, fibrinogen plays a crucial role in the

process of thrombus formation and evolution, and elevated plasma fibrinogen levels are strongly associated with the risk for MI¹. Fibrinogen is a glycoprotein consisting of two subunits of three distinct polypeptide chains (Aα, Bβ, and γ). Each of the chains is encoded by a different gene situated on the long arm of chromosome 4 at 4q23-32. It has been demonstrated that the synthesis of the Bβ chain is the rate limiting factor in the production of the mature fibrinogen protein³. Thus, most studies focus on the association of polymorphisms in the fibrinogen β-chain (FGB) gene with MI. Several polymorphisms in the FGB gene were found to be associated with increased plasma fibrinogen levels^4-6. But the role of the FGB polymorphisms as a risk factor of MI has been debated⁷.

In view of the conflicting findings, we performed an association study to assess the effect of common genetic variants in FGB gene on the risk of MI in a Viet Nam population. We hereby report a case of acute myocardial infarction; the location was easy to form blood clots that can detect changes in the promoter region of the FGB gene.

METHODS

Subjects

A 50 year old male patient admitted to hospital because of chest pain at the 10^th hour of onset. The patient has a history of heavy smoking (1 packet per day for 30 years). He has also suffered from deep vein thrombosis of the left leg since Sept 2010 and is on treatment with Sintrom (Acenocoumarol) with 1mg per day.

On admission, the patient was examined, conducted functional diagnostic testings, finally came with a definitive diagnosis of acute myocardial infarction (MI). He also was taken blood for genetic tests. The patient’s blood sample was stored in a test tube with EDTA as an anticoagulant and obtained for genetic studies of FGB gene at the Department of Biology and Medical Genetics, Hanoi Medical University, Vietnam. A control blood sample for comparison was taken from a healthy man of that age with the patient with no history of myocardial infarction or angina pectoris.

Diagnostic Imagings and Pre-clinical tests

Patient was checked by doppler echocardiography, emergency coronary angiography and 64 - slide computed tomography of the chest. Pre-clinical tests were done in his bood, including coagulation basic, protein C, protein S, antithrombin III, lipid profile, blood glucose, liver function, kidney, etc.

Genotyping

Genomic DNA was extracted from peripheral blood by phenol - chloroform method and stored at -20°C until analysis. The DNA was purified and checked by the amount of spectrophotometer nano-drop. FGB gene was replicated by standard polymerase chain reaction (PCR) in the promoter region of a specific nucleotide position which is determined by international bank sequences (GenBank, M 64983,1). Specific primers used for the promoter region of the FGB, forward: 5’-AATAACTTCCCATCATTTTGTCCAATTCC-3’, reverse: 5’-AGTCGTTGACACCTTGGGACTTAACTTG-3’ (primer pairs FGBf3m- FGBr4m). For PCR amplification, the standard program was used as follows: an initial denaturation step at 95°C for 5 min was followed by 40denaturation cycles of 30 s at 95°C, 30 s of annealing at 55°C, and 30 s of extension at 72°C, and at last followed by a final elongation cycle at 72°C for 5 min. PCR reactions were carried out in a final volume of 50 µl containing genomic DNA template, primers and the reaction components for PCR (Taq DNA polymerase, dNTPs, MgCl2, dH2O). PCR products were purified by PureLinkTM kit (Invitrogen).

Sequencing FGB gene was performed on a Genetic Analyzer ABI PRISM 3100. The quality parameters and the peak were collected, tested by the ABI Data Collection software v2.0 and Sequencing Analysis v5.3 Software. The sequence of the promoter region of the FGB gene case and control in Vietnam were compared with reference sequences, which is published in GenBank by using analysis software Chromas Lite v2.1.1 and Seaview to identify point mutations.

RESULTS

Clinical characteristics of patients with myocardial infarction

On admission, the patient still had chest pain, heart rate 120 bpm and blood pressure 90/60 mmHg (both arms), fine crackles at the base of both lungs, mild, tender hepatomegaly (congestion).

Electrocardiography (ECG): sinus tachycardia, ST segment elevation in leads DII, DIII, aVF, and from V3 to V6.

Emergency Echocardiography: LVDd 52mm, LVDs 41mm, EF Simpson (4 chamber view) 53%, (2 chamber view) 14%, hypokinetic movement of different left ventricle (LV) wall regions, with more profoundly in the inferio - posterior region of the LV wall. There was mild pericardial effusion.

The patient was treated with drugs for acute MI. Emergency coronary angiography was indicated for him.

Pre-clinical characteristics of patients

Analyses determined the state of the heart muscle necrosis due to myocardial infarction: troponin T, CK and CK MB increased up to 8 ng / mL, 9600 IU / mL, 1100 UI / L, respectively. ProBNP increased with time up to nearly 1700 pmol / l (expressed condition with severe heart failure), GOT and GPT rose to 823 and 127 IU / l.

The coagulation tests of the patient were closely monitored over time to see the changes in blood clotting, namely: PT% reduction in speed slightly less than 20% (17.2%; INR increased to 3.35 (patient receives anticoagulant vitamin K antagonists for the treatment of the left leg deep vein thrombosis); APTT was abnormal with a value ranging from 174ms - 79.5 ms - 42 ms - 28ms makes APTT disease outcome / evidence also ranged from 5.5 to 0.99 (the highest stage additional factors often using continuous infusion of heparin after fibrinolytic therapy. Protein C 47,4% (↓), protein S 22,7% (↓), Antithrombin III: 96,6% (normal) (Blood samples were taken to test the condition of patients still using anticoagulant vitamin K antagonist). Fibrinogen 4.7 g / l (up slightly). Other preclinical studies: platelet count was reduced to 103.000 cell /ml; Bilan lipids, blood glucose, renal function were within normal ranges.

Emergency coronary angiography images: complete occlusion of the 2^nd segment of the right coronary artery, the circumflex coronary artery and the left anterior descending coronary artery. Angiography revealed a thrombotic sub-total occlusion of left main coronary artery and the 1^st segment of the left descending coronary artery.

Figure 1. Coronary angiography image of the patient

With the above results of the coronary angiography, team of interventionists decided to stop the procedure. The patient was transfered back to the cardiac care unit (CCU), prepared for receiving thrombolytic therapy. Alteplase was the drug of choice. The protocol was as followed: bolus 15mg, then 0.75 mg/kg within 30 minutes, and then 0.5 mg over 60 minutes. Continuous intravenous heparin infusion was given at the same time at the rate of 1000UI/h, and also dobutamine infusion at the dose of 5 mcg/kg/min. the patient’s hemodynamics was stable during fibrinolytic therapy.

After fibrinolysis, the patient’s chest pain was improved. The following days, the patient was being medically treated intensively for thrombotic disease, coronary artery disease and heart failure. The anticoagulant and anti-platelet aggregation drugs used include: aspegic 200mg/day, clopidogrel 75 mg/day, anticoagulant- antagonist vitamin K: sintrom 1mg/day.

The patient was reevaluated with Doppler echocardiography after 3 days in hospital: LVDd 55mm, LVDs 44mm, EF simpson (2B) 32%, moderate pulmonary artery hypertension; akinesis of the LV wall region, supplied by left anterior descending and right coronary artery; mild pericardial effusion. Doppler echocardiography of the lower limbs revealed old thrombosis of the left iliofemoral vein which was partially revascularized.

In the 64 slide MSCT revealed no pulmonary artery infarction, there was pleural effusion both sides mildly.

The patient was discharged after 2 weeks in better physical condition: heart failure improved, no sign of chest pain. Patients continued medical therapy 2 weeks, improved heart failure, chest pain disappeared.

The patient readmitted after 2 weeks: full stability, no chest pain, myocardial tests for cell injury was in normal ranges.

Mutation C148T of FGB gene in a patient with MI

PCR amplifying FGB promoter regions

The extracted genomic DNA was used for the matrix in the replication FGB promoter. To analyze the changes in the promoter region of the FGB up to 728 bp, we used primers FGBf3m-FGBr4m. The amplified products were detected on 1.5% agarose gel, along with a 100-bp DNA ladder. The result of electrophoresis of product PCR (Figure 2) showed the specificity and quality of the isolated DNA.

1 - Control sample

2 - Case sample

3 - Ladder

Figure 2. Electrophoresis of products PCR patients and control samples.

Ladder: molecular weight marker 100 bp

Promoter sequences

PCR products were sequenced after purification in both directions (Forward - F, reverse - R), compared to the control sequences in the GenBank, we found at nucleotide position -148 promoter region of a patient with FGB gene point mutations such as nucleotide Cytozine (C) is replaced by thymine (T), designated -148C à T or C148T (Fig. 3).

Figure 3. Comparison of nucleotide sequence of the promoter region of the FGB in control and case samples

On the forward and reverse chain in FGB in case (Patient-F and Patient-R) found a point mutation that C àT, TT genotype. Meanwhile, CC genotype was confirmed in the control.

Figure 4. Peaks of nucleotides in the promoter region of the FGB

A: Case - sample with mutation -140CàT. B: mutation is not found in Control

DISCUSSION

Patients aged 50 years with a history of smoking and thrombosis of deep veins of the left leg, continuing anticoagulant therapy – a vitamin K antagonist (INR = 2.1). He was hospitalized with acute MI. Coronary angiography revealed three trunks thrombosis coronary artery. Clinical assays showed a decrease in the platelet count, coagulation outer (PT%, INR) in the limit of a vitamin K antagonist therapy, fibrinogen levels in normal (2-4 g/l), a decrease ratio of protein C, S (but the patient still taking vitamin K antagonists when had blood withdrawal for analysis). In situations where the patient has arterial and venous thrombosis, wherein the clinical coagulation assays showed changes are unknown, we doubt that may be part of the development of the disease the patient is affected by other factors including genetic factors? On the basis of the scientific literature in the world, we decided to take a patient's blood for genetic diagnostics, focusing on genes encoding fibrinogen, especially because the FGB has quite a lot of research on the world's stock is the relationship between polymorphisms of these genes and cardiovascular risk factors in general, the risk of coronary heart disease in particular.

As a result of the genetic studies of polymorphic markers promoter region of the FGB in patients compared with sequences control samples was detected C148T mutation in the promoter region of the FGB. At the Center of stroke prevention in Australia, a study demonstrates the first evidence of a significant association between the T/T148 genotype at the b-fibrinogen gene and carotid atherosclerosis⁸. Ewa Wypasek et al (2012) conducted analysis FGB 243 patients with myocardial infarction, coronary bypass surgery (coronary artery bypass grafting - CABG), found 101 patients with mutation C148T, that has genotype CT or TT. They found that CT+TT genotype is an independent predictor of high postoperative CRP levels in CABG patients. Carrying of the -148T allele has also been associated with increased postoperative IL-6 levels in CABG patients⁹.

Looking at the results of our investigation in the case with mutation C148T, the patient has the TT genotype. This patient is treated with sintrom daily, although fibrinogen levels on admission in the normal range, then increased. However, a history of venous thrombosis and were acute myocardial infarction with thrombosis of three trunks of the coronary arteries. This may indicate that the C148T mutation may lead to a breach of the synthesis reaction of fibrinogen that influence inflammation and blood clotting.

Detection of C148T mutation in a patient with acute MI with being easy formation of blood clots suggests that we need to expand the study of association of polymorphisms of FGB with MI in Vietnam to determine the relationship between genotype and phenotype or pathological manifestations. In fact, until now, modification of the gene considered polymorphism FGB and scientists explore these polymorphisms with respect to the pathological manifestations. Behague I. et al evaluated the association between genetic polymorphisms in the FGB gene and the severity of coronary artery disease (CAD) in patients with familial acute MI show that among 11 variants of the β fibrinogen gene that were investigated in the ECTIM study, 8 were mutually very tightly or completely associated to the concentration of fibrinogen in the plasma, especially in smokers and patients with MI⁷.

Some studies have shown strong correlations between polymorphism genotype of the gene for the beta chain synthesis and plasma fibrinogen concentrations.

For example, polymorphism of the gene beta fibrinogen was detected at 3 ' position by restriction enzyme BclI (polymorphism BclI) as polymorphism allele pairs with low frequency of B2, only 0.18 in the general population. B2B2 genotype is rare, but was associated with increase levels of fibrinogen in blood serum 15% - 20% compared with the genotype B1B1¹¹. In addition, studies of Van’t Hooft F., Sekar K. and his colleagues showed polymorphisms G455A and G854A gene beta fibrinogen significantly influenced by the concentration of serum fibrinogen. G455A mutation of a gene beta fibrinogen is most associate with increase levels of fibrinogen, found in both sexes in society as a whole. This study has also shown a link between the C148T and G455A mutations, in pathological cases, usually appears one of the two mutations^7,11-12.

Although the relationship polymorphisms FGB with the risk of cardiovascular diseases in general and coronary heart disease risk in particular still has differences but research FGB gene has continue with time and detecting each patient explaining on pathological mutations or polymorphisms.

In conclusion, new areas of research that we would like to mention the case of the C148T mutation FGB gene in a patient with MI being easy for the formation of pathological thrombosis opens new research direction in the most general population of patients with myocardial infarction, family, nature, understanding the relationship between genotype polymorphism on regulation synthesis fibrinogen, the response of serum fibrinogen levels, as well as a risk of clinical events (acute myocardial infarction). These links will serve as a basis for studying the etiologic role of fibrinogen in acute MI, as well as the basis for evaluation of interventions that can be made for incident heart attack in patients with familial mutation arising properties and genetic counseling for carriers.

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