Cardiovascular Research

Cardiovascular Research 1998 37(1):42-45; doi:10.1016/S0008-6363(97)00211-3
© 1998 by European Society of Cardiology

This Article Abstract FREE Full Text (PDF) E-letters: Submit a response Alert me when this article is cited Alert me when E-letters are posted Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to My Personal Archive Download to citation manager Request Permissions Disclaimer Google Scholar Articles by Arruda, V. R Articles by Annichino-Bizzacchi, J. M Search for Related Content PubMed PubMed Citation Articles by Arruda, V. R Articles by Annichino-Bizzacchi, J. M Social Bookmarking          What's this?

Copyright © 1998, European Society of Cardiology

Prevalence of the prothrombin gene variant 20210 GA among patients with myocardial infarction

Valder R Arruda^a,*, Lucia H Siquiera^a, Luiz C Chiaparini^a, Otavio R Coelho^a, Antonio P Mansur^b, Antonio Ramires^b and Joyce M Annichino-Bizzacchi^a

^aHematology–Hemotherapy Center, State University of Campinas, C.P. 6198, C.E.P. 13081-970, Campinas, SP Brazil
^bHeart Institute, University of São Paulo, São Paulo, Brazil

^* Corresponding author. Fax: +55-19-788-8750/+55-192-393511; E-mail: vrarruda@hotmail.com

Received 26 May 1997; accepted 25 July 1997

	Abstract

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
Objective: The aim of this study was to determine the prevalence of the prothrombin variant allele 20210A among survivors of myocardial infarction Background: The prothrombin gene variant has been identified as a novel genetic risk factor for venous thrombosis. However, the risk of developing arterial thrombosis as a result of the presence of this mutated allele is unknown. Methods: The GA transition at position 20210 of the 3'-untranslated region was determined in 220 survivors of myocardial infarction and in 295 individuals from the general population. Results: The prevalence of heterozygotes for the prothrombin mutated allele was 3% among patients with myocardial infarction and 0.7% in the general population (P = 0.03). No age-related difference in the prevalence of the mutated allele was observed. However, for individuals over 45 years old the prevalence among females was higher than among males (5% vs. 0%). Conclusion: These data suggest that being heterozygote for the allele variant 20210A of the prothrombin gene could be a genetic risk factor for developing myocardial infarction.

KEYWORDS Myocardial infarction; Risk factors; Prothrombin; DNA polymorphism

	1 Introduction

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
Thrombin plays a central role in the control of blood coagulation, and has procoagulant as well as anticoagulant properties [1]. Low levels of thrombin markedly increase the amount of activated natural anticoagulant protein C, whereas high levels of thrombin induce a procoagulant effect as a result of fibrinogen to fibrin conversion and the generation of factor Va and VIIIa [2–4]. The control of thrombin generation has attracted considerable interest because of its additional properties, including direct effects on platelet aggregation, regulation of endothelial cell proliferation, and fibroblast mitogenesis, all of which may be important in the pathogenesis of arteriosclerosis [5]. Thrombin is formed from a zymogen, prothrombin, by a series of reactions between serine proteases and cofactors which are assembled as a prothrombinase complex on a phospholipid surface, such as the activated platelet [1]. Recently, Poort et al. [6]described an inherited variant of the prothrombin (factor II) gene caused by GA transition at position 20210 in the 3'-untranslated region of the prothrombin gene. This prothrombin variant 20210A was associated with higher prothrombin levels and a nearly threefold greater risk of venous thrombosis. The mechanism of the prothrombin mutation causing venous thrombosis is unknown. The fact that this mutated allele was also identified in 2.3% of the controls suggests that it could be a common risk factor for vascular disease. It is not known whether the prothrombin gene variant is associated with arterial thrombosis. Several factors involved in coagulation and fibrinolysis have been correlated with an increased risk of myocardial infarction including fibrinogen, factor VII, and plasminogen-activator inhibitor [7–10]. In this study, we have determined the prevalence of the prothrombin gene variant 20210A among survivors of myocardial infarction and compared this with the prevalence of the allele in the general population.

	2 Methods

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
2.1 Selection of the patients and controls
A cross-sectional study was carried out at the University Hospital of the State University of Campinas and at the Heart Institute of The State University of São Paulo. Survivors of myocardial infarction during the period from January, 1994 to December, 1995 which had been attended at the State University of Campinas and from July, 1996 to December, 1996 at the Heart Institute were invited to participate in this investigation of the inherited risk factors involved in coronary artery disease. This protocol was approved by the Ethical Committee of the Hospital.

2.2 Patients
Myocardial infarction was documented in 220 individuals based on clinical, laboratory, and electrocardiography data. Coronary arteriography was carried out in almost all patients. The patients were classified into two groups, the first of which consisted of 63 young adults (42 male and 21 female) with a median age of 38 years (range:17 to 45 years), and was termed the premature disease group. The second group, known as the non-premature disease group, comprised 157 adults (82 male and 75 female), with a median age of 59.9 (range: 46 to 82 years) (Table 1). In 31 out of the 220 patients, the diagnosis of myocardial infarction was carried out in the absence of a systemic disease or the following risk factors for arterial disease, based on the Brazilian consensus on dyslipidemias [11]: hyperlipoproteinemia (total cholesterol>6.18 mmol per liter and/or total triglycerides>2.39 mmol per liter), hypertension (diastolic blood pressure higher than 90 mmHg), and diabetes mellitus (fasting plasma glucose levels higher than 6.7 mmol per liter) [12].

View this table: [in this window] [in a new window]   Table 1 Clinical and laboratorial data of the patients with myocardial infarction

 
2.3 General population
This group consisted of 295 consecutive neonates from the same University Hospital in order to provide a representative group of the general population that seeks medical assistance in this region, which includes Campinas and São Paulo.

2.4 Diagnosis of the transition 202021GA in the prothrombin gene
Genomic DNA was obtained from peripheral blood cells for the adult patients and from umbilical vein neonates following extraction by a standard method [13]. A fragment of the 3'-untranslated region of the prothrombin gene was amplified by the polymerase chain reaction (PCR) [14]in a mixture of 54 mM Tris-HCl, pH 8.8, 5.4 mM MgCl₂, 5.4 µM EDTA, 0.8 mM of each nucleoside triphosphate, 400 ng of each primer described by Poort et al. [6]sense (5'-TCTAGAAACAGTTGCCTGGC-3') and mutagenic antisense (5'-ATAGCACTGGGAGCATTGAAGC-3'), genomic DNA and 2 U of Taq polymerase. The reaction involved 35 cycles of incubation at 94°C (1 min), 58°C (1 min) and 72°C (1 min) and resulted in a 345 bp fragment. The overnight digestion of 10–15 µl of the PCR product with 2.5 U of endonuclease HindIII yielded a 322 bp fragment observed when the mutated allele 20210A was present based on electrophoresis in a 2% agarose gel. When the normal allele 20210G was present, there was no cleavage site for HindIII and the 345 bp fragment remained intact (Fig. 1).

View larger version (83K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 Ethidium bromide-stained 2% agarose gel showing PCR products corresponding to a fragment of 3'-untranslated region of the prothrombin gene after digestion with HindIII. When at nucleotide 20210 a substitution GA was present, it created a restriction site to HindIII, resulting in fragments of 322 bp or remained 345 bp when the normal allele was present. Line 1 is the DNA size marker Ladder 100. In the lines 3–4 are shown the results from two patients heterozygous for the mutated allele (G/A), in the line 2 is a homozygous for the normal allele (G/G).

 
2.5 Statistical analysis
The statistical significance of the differences between groups was calculated by Fisher's exact test using the Epi Info program [15].

	3 Results

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
In the general population, the mutated allele 20210A of the prothrombin gene was found in two out of 295 neonates (0.7%, confidence interval: 0.1–2.7%) giving an allele frequency of 0.33%. In the myocardial infarction group, as a whole, the mutated allele was observed in seven out of 220 patients (3.2%, confidence interval: 1.4–6.7%) and was thus significantly (P = 0.03) more common than in the general population (Table 2). These heterozygotes for the prothrombin variant included 3 out of 63 patients (4.7%) in the premature disease group and 4 out of 157 patients (2.5%) in the non-premature group. The clinical and laboratory investigation of these seven cases (two male and five female) revealed that both of the heterozygous males were younger than 45 years old, and that four of the five females were older than 45 years old (median age of 57.5 years). There was a prevalence of 5.3% for the mutated allele among old females (4 out of 75 patients) which contrasts with the absence of this mutation among males older than 45 years old. There was no significant sex-related difference in the prevalence of the mutant allele among young patients (P = 0.34). In 6 out of 7 patients, risk factors for arterial disease such as diabetes mellitus, hypertension, hyperlipidemia, and heterozygous for factor V Leiden mutation (one case), diabetes mellitus and hyperlipidemia (one case), hypertension and hyperlipidemia (one case), hypertension (two cases), hyperlipidemia (one case). The remaining case with no risk factors was a smoking, 41 year old man.

View this table: [in this window] [in a new window]   Table 2 Prevalence of the transition 20210GA in the prothrombin gene among patients with myocardial infarction and in the general population

 

	4 Discussion

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
Vascular disease is a common and serious public health problem in the industrialized world, and coronary artery disease is an important cause of death in the adult Brazilian population [16, 17]. Blood clotting at the site of vascular injury is an important step in the development of an acute coronary event such as myocardial infarction [5]. Hypercoagulability resulting from an inherited thrombophilia involving a deficiency in natural anticoagulants such as protein C, protein S, or antithrombin III is rarely the cause of arterial disease [18]. The factor V Leiden mutation, a point mutation in the clotting factor V gene, causes a 5–10 fold increase in the risk of deep venous thrombosis [19, 20]. However, the risk of arterial disease with factor V Leiden, remains controversial [21–26]. In their study of the genetic risks for vascular disease, Poort et al. [6]described a variant form of prothrombin resulting from a GA transition in the 3' untranslated region of the gene 20210A while they showed it to be a moderate risk factor for venous thrombosis. It is interesting to note that an association of the mutated allele 20210A with elevated plasma prothrombin levels may increase the generation of thrombin and consequently enhance the risk of developing occlusive vascular disease. However, there are no current data on the prevalence or role of this prothrombin gene variant among patients with arterial disease. In this study, we examined 220 patients with myocardial infarction and found that seven (3.4%) were heterozygous for the mutated allele, the prevalence of which was similar among young and old patients alike. However, when the group was analyzed by gender, we observed a higher prevalence of the prothrombin variant among elderly women (4 out of 75 patients; 5%) than among elderly men (none out of 82 patients). Among young patients this sex related difference was not apparent. In 6 out of 7 patients with the mutated allele a major risk factor for arterial disease was identified. Together, these data suggest a possible role for the prothrombin gene variant as a genetic risk factor for arterial disease especially inter-reacting with environmental factors. However, since we studied survivors of an acute ischemic event, the prevalence of the mutated allele could be different if all incomers with a first episode of myocardial infarction were considered. Since the prevalence of the prothrombin gene variant in the general population is not as high as that for other risk factors associated with vascular disease (i.e. 0.7% among Brazilians and 2.3% among Dutch controls), a prospective follow-up study will be necessary in order to delineate the role of this prothrombin gene variant in developing myocardial infarction.

Time for primary review 22 days.

	References

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 

1. Davie E.W., Fujikawa K., Kisiel W. The coagulation cascade: initiation, maintenance, and regulation. Biochemistry (1991) 30:10363–10370.[CrossRef][Web of Science][Medline]
2. Esmon C.T. The protein C anticoagulant pathway. Arterioscler Thromb Vasc Biol (1992) 12:135–145.[Free Full Text]
3. Gruber A., Harker L.A., Hanson S.R., Kelly A.B., Griffin J.H. Antithrombin effects of combining activated protein C and urokinase in nonhuman primates. Circulation (1991) 84:2454–2462.[Abstract/Free Full Text]
4. Gruber A., Pál A., Kiss R.G., Sas G., Griffin J.H. Generation of activated protein C during thrombolysis. Lancet (1993) 342:1275–1276.[CrossRef][Web of Science][Medline]
5. Ross R. Pathogenesis of atherosclerosis. In: Colman RW, Hirsh J, Marder VJ, Salzman EW, editors. Hemostasis and Thrombosis: Basic Principles and Clinical Practice. Philadelphia, PA: JB Lippincott Company, 1994:861–869.
6. Poort S.R., Rosendaal F.R., Reitsma P.H., Bertina R.M. A common genetic variant in the 3'-untranslated region of the prothrombin gene is associated with elevated plasma prothrombin levels and an increase in venous thrombosis. Blood (1996) 88:3698–3703.[Abstract/Free Full Text]
7. Wilhelmensen L., Svärdusudd K., Korsan-Bengtsen K., Larsson B., Welin L., Tibblin G. Fibrinogen as a risk factor for stroke and myocardial infarction. N Engl J Med (1984) 311:501–505.[Abstract]
8. Meade T.W., Mellows S., Brozovic M., Miller G.J., Chakrabarti R.R., North W.R.S., Haines A.P., Stirling Y., Imerson J.D., Thompson S.G. Haemostatic function and ischaemic heart disease: principal results of the Northwick Park Heart Study. Lancet (1986) 2:533–537.[Web of Science][Medline]
9. Hamsten A., Blömback M., Wiman B., Svensson J., Szamosi A., de Faire U., Mettinger L. Haemostatic function in myocardial infarction. Br Heart J (1986) 55:58–66.[Abstract/Free Full Text]
10. Hamsten A., Wiman B., de Faire U., Blömback M. Increased plasma levels of a rapid inhibitor of tissue plasminogen activator inhibitor in young survivors of myocardial infarction. N Engl J Med (1985) 313:1557–1563.[Abstract]
11. Sociedade Brasileira de Cardiologia. Brazilian consensus on dyslipidemias: detection, evaluation, and treatment. Arq Bras Cardiol (1994) 63:1–13.[Medline]
12. Stein EA. Lipids, Lipoproteins, and Apolipoprotrein. In: Tieftz NW editor. Fundamentals of Clinical Chemistry. Philadelphia, PA: W.B. Saunders, 1987:449–481.
13. Millar S.A., Dykes D.D., Polesky H.F. A simple salting out procedure for extracting DNA from nucleated cells. Nucleic Acids Res (1988) 16:1215.[Free Full Text]
14. Saiki R.K., Gelfand D.H., Stoffel S., Scharf S.J., Higuchi R., Horn G.T., Mullis K.B., Erlich H.A. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science (1988) 239:487–491.[Abstract/Free Full Text]
15. Dean AG, Dean JA, Coulombier D, Brendel KA, Smith DC, Burton AH, Dicker RC, Sullivan KM, Fagan RF, Arner TG, Epi Info, version 6: a word processing, database, and statistics program for epidemiology on microcomputers. Atlanta: Center for Disease Control and Prevention, 1994.
16. WHO Technical Report Series, No 841: Cardiovascular disease risk factor: new areas for research. Report of a WHO Scientific Group, 1994.
17. Duncan B.B., Schmidt M.I., Polanczyk C.A., Mengue S.S. High mortality rates in Brazilian adult populations — an international comparison. Rev Ass Med Brasil (1992) 38:138–144.
18. Simioni P., Zanardi S., Saracino A., Girolami A. Occurrence of arterial thrombosis in a cohort of patients with hereditary deficiency of clotting inhibitors. J Med (1992) 23:61–74.[Web of Science][Medline]
19. Dahlbäck B., Carlsson M., Svensson P.J. Familial thrombophilia due to a previously unrecognized mechanism characterized by poor anticoagulant response to activated protein C: prediction of a cofactor to activated protein C. Proc Natl Acad Sci USA (1993) 90:1004–1008.[Abstract/Free Full Text]
20. Bertina R.M., Koeleman B.P.C., Koster T., Rosendaal F.R., Dirven R.J., Ronde H., van der Velden P.A., Reitsma P.H. Mutation in blood coagulation factor V associated with resistance to activated protein C. Nature (1994) 369:64–67.[CrossRef][Medline]
21. Ridker P.M., Hennekens C.H., Lindpaintner K., Stampeer M.J., Eisenberg P.R., Miletich J.P. Mutation in the gene coding for coagulation factor V and the risk of myocardial infarction, stroke, and venous thrombosis in apparently healthy men. N Engl J Med (1995) 332:912–917.[Abstract/Free Full Text]
22. Samani N., Lodwick D., Martin D., Kimber P. Resistance to activated protein C and risk of premature myocardial infarction. Lancet (1994) 344:1709–1710.[Web of Science][Medline]
23. van Bockxmeer F.M., Baker R.I., Taylor R.R. Premature ischaemic heart disease and the gene for coagulation factor V. Nature Med (1995) 1:185.[CrossRef][Web of Science][Medline]
24. Kontula K., Ylikorkala A., Miettinen H., Vuorio A., Kauppinen-Mäkelin R., Hämäläinen L., Palomäki H., Kaste M. Arg506Gln factor V mutation (factor V Leiden) in patients with ischemic cerebrovascular disease and survivors of myocardial infarction. Thromb Haemost (1995) 73:558–601.[Web of Science][Medline]
25. Holm J., Zöller B., Berntorp E., Erhardt L., Dahlbäck B. Prevalence of factor V gene mutation amongst myocardial infarction patients and healthy controls in Sweden than in other countries. J Intern Med (1996) 239:221–226.[CrossRef][Web of Science][Medline]
26. Rosendaal F.R., Siscovick D.S., Schwartz S.M., Beverly R.K., Psaty B.M., Longstreth W.T., Raghunathan T.E., Koepsell T.D., Reitsma P.H. Factor V Leiden (resistance to activated protein C) increases the risk of myocardial infarction in young women. Blood (1997) 89:2817–2821.[Abstract/Free Full Text]

CiteULike    Connotea    Del.icio.us    What's this?

This article has been cited by other articles:

				F Burzotta, K Paciaroni, V De Stefano, F Crea, A Maseri, G Leone, and F Andreotti G20210A Prothrombin gene polymorphism and coronary ischaemic syndromes: a phenotype-specific meta-analysis of 12 034 subjects Heart, January 1, 2004; 90(1): 82 - 86. [Abstract] [Full Text] [PDF]

				P. M. Mannucci, D. Ardissino, P. A. Merlini, F. Peyvandi, Y.-G. Xie, C. Butt, and E. Randell Vagaries of genetic association studies in myocardial infarction Blood, August 15, 2003; 102(4): 1558 - 1560. [Full Text] [PDF]

				C. Butt, H. Zheng, E. Randell, D. Robb, P. Parfrey, and Y.-G. Xie Combined carrier status of prothrombin 20210A and factor XIII-A Leu34 alleles as a strong risk factor for myocardial infarction: evidence of a gene-gene interaction Blood, April 15, 2003; 101(8): 3037 - 3041. [Abstract] [Full Text] [PDF]

				F. Burzotta, K. Paciaroni, V. De Stefano, P. Chiusolo, A. Manzoli, I. Casorelli, A.M. Leone, E. Rossi, G. Leone, A. Maseri, et al. Increased prevalence of the G20210A prothrombin gene variant in acute coronary syndromes without metabolic or acquired risk factors or with limited extent of disease Eur. Heart J., January 1, 2002; 23(1): 26 - 30. [Abstract] [Full Text] [PDF]

				S. M. Boekholdt, N. R. Bijsterveld, A. H.M. Moons, M. Levi, H. R. Buller, and R. J.G. Peters Genetic Variation in Coagulation and Fibrinolytic Proteins and Their Relation With Acute Myocardial Infarction: A Systematic Review Circulation, December 18, 2001; 104(25): 3063 - 3068. [Abstract] [Full Text] [PDF]

				D. M. Herrington and K. P. Klein Genome and Hormones: Gender Differences in Physiology: Invited Review: Pharmacogenetics of estrogen replacement therapy J Appl Physiol, December 1, 2001; 91(6): 2776 - 2784. [Abstract] [Full Text] [PDF]

				Y. Mira, J. Todoli, R. Alonso, M. L. Mico, A. Vaya, F. Ferrando, A. Estelles, P. Villa, and J. Aznar Factor V Leiden and Prothrombin G20210A in Relation to Arterial and/or Vein Rethrombosis: Two Cases Clinical and Applied Thrombosis/Hemostasis, July 1, 2001; 7(3): 234 - 237. [Abstract] [PDF]

				C. Russo, D. Girelli, O. Olivieri, P. Guarini, F. Manzato, F. Pizzolo, B. Zaia, A. Mazzucco, and R. Corrocher G20210A Prothrombin Gene Polymorphism and Prothrombin Activity in Subjects With or Without Angiographically Documented Coronary Artery Disease Circulation, May 22, 2001; 103(20): 2436 - 2440. [Abstract] [Full Text] [PDF]

				B. M. Psaty, N. L. Smith, R. N. Lemaitre, H. L. Vos, S. R. Heckbert, A. Z. LaCroix, and F. R. Rosendaal Hormone Replacement Therapy, Prothrombotic Mutations, and the Risk of Incident Nonfatal Myocardial Infarction in Postmenopausal Women JAMA, February 21, 2001; 285(7): 906 - 913. [Abstract] [Full Text] [PDF]

				A. Vaya, R. Trenor, S. Molla, A. Estelles, Y. Mira, P. Villa, and J. Aznar Acute Myocardial Infarction Associated With the Prothrombin G20210A Mutation Clinical and Applied Thrombosis/Hemostasis, April 1, 2000; 6(2): 111 - 112. [PDF]

				R. D. Rosenberg and W. C. Aird Vascular-Bed-Specific Hemostasis and Hypercoagulable States N. Engl. J. Med., May 20, 1999; 340(20): 1555 - 1564. [Full Text] [PDF]

				M. Redondo, H. H. Watzke, B. Stucki, I. Sulzer, F. D. Biasiutti, B. R. Binder, M. Furlan, B. Lammle, and W. A. Wuillemin Coagulation Factors II, V, VII, and X, Prothrombin Gene 20210G->A Transition, and Factor V Leiden in Coronary Artery Disease : High Factor V Clotting Activity Is an Independent Risk Factor for Myocardial Infarction Arterioscler Thromb Vasc Biol, April 1, 1999; 19(4): 1020 - 1025. [Abstract] [Full Text] [PDF]

This Article Abstract FREE Full Text (PDF) E-letters: Submit a response Alert me when this article is cited Alert me when E-letters are posted Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to My Personal Archive Download to citation manager Request Permissions Disclaimer Google Scholar Articles by Arruda, V. R Articles by Annichino-Bizzacchi, J. M Search for Related Content PubMed PubMed Citation Articles by Arruda, V. R Articles by Annichino-Bizzacchi, J. M Social Bookmarking          What's this?

Online ISSN 1755-3245 - Print ISSN 0008-6363

Copyright © 2009 European Society of Cardiology

Oxford Journals Oxford University Press

• Site Map
• Privacy Policy
• Frequently Asked Questions

Other Oxford University Press sites: Oxford University Press Oxford Journals China Oxford Journals Japan American National Biography Booksellers' Information Service Children's Fiction and Poetry Children's Reference Corporate & Special Sales Dictionaries Dictionary of National Biography Digital Reference English Language Teaching Higher Education Textbooks Humanities International Education Unit Law Medicine Music Online Products Oxford English Dictionary Reference Rights and Permissions Science School Books Social Sciences Very Short Introductions World's Classics