PhD, Mesut DEMIR, 1. MD, Mustafa DEMIRTAS, Murat ÇAYLI, 1 MD, Cumhur ALHAN, 1 MD, and Fikri BASLAMISLI,

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1 Experimental Studies Prothrombin 20210GA and Factor V Leiden Mutations in Patients Less Than 55 Years Old With Myocardial Infarction Yurdaer DÖNMEZ, 1 MD, Mehmet KANADASI, 1 MD, Kahraman TANRIVERDI, 2,3 PhD, Mesut DEMIR, 1 MD, Mustafa DEMIRTAS, 1 MD, Murat ÇAYLI, 1 MD, Cumhur ALHAN, 1 MD, and Fikri BASLAMISLI, 2 MD SUMMARY Several studies claim that prothrombin 20210GA and factor V Leiden mutations are related to arterial thrombosis. We investigated the frequencies of these mutations and their significance in the development of early atherosclerosis in acute myocardial infarction (AMI) patients younger than 55 years of age. We investigated 96 patients with AMI and 77 control subjects. The diagnosis of AMI was established by typical chest pain and ST elevations on the presentation electrocardiogram and characteristic cardiac enzyme elevations. None of the control subjects had evidence of cardiovascular disease. DNA samples were isolated from all subjects and prothrombin 20210GA and factor V Leiden mutations were determined by the RealTime PCR technique with the aid of a Light Cycler device. The prevalence of factor V Leiden mutation was 6.3% and 5.2% in the patient and control groups, respectively (OR 0.6 [95% CI ], P = 0.6), whereas the prevalence of prothrombin G20210A mutation was 4.2% and 2.6% in the patient and control groups, respectively (OR 2.8 [95% CI ], P = 0.4). None of the patients had both mutations. Prothrombin 20210GA and factor V Leiden mutations are not significant risk factors for the development of myocardial infarction in patients less than 55 years old in Southern Turkey. (Jpn Heart J 2004; 45: ) Key words: infarction Prothrombin 20210GA mutation, Factor V Leiden mutation, Myocardial ACUTE myocardial infarction develops as a result of thrombosis in the coronary arteries. 1) In recent years, there has been growing interest in the genetic etiology of thrombosis. With the advent of improved techniques for DNA analysis, several different mutants of coagulation factors have been determined. Prothrombin 20210GA mutation was discovered in 1996 by Poort, et al. 2) There is a replacement of guanine by adenine at nucleotide 20210GA in the 3'- untranslated From the 1 Department of Cardiology, 2 Department of Hematology, Çukurova University School of Medicine, Adana, Turkey, 3 Whitaker Cardiovascular Institute, Boston University Medical Center, Boston, USA. Address for correspondence: Yurdaer Dönmez, MD, Department of Cardiology, Çukurova Üniversitesi Tip Fakültesi Kardiyoloji Anabilim Dali Balcali, Adana, Turkey. Received for publication August 21, Revised and accepted November 27,

2 506 DÖNMEZ, ET AL Jpn Heart J May 2004 region of the prothrombin gene. This mutation results in increased synthesis and secretion of prothrombin by the liver. It has been shown that there is a definite link between venous thrombosis and this mutation in all age groups. 3) Presently, the prevalence of this mutation is reported to be 1 to 5% in Caucasians. 4) Three independent laboratories reported the discovery of factor V Leiden in Factor V Leiden is defined as a mutant factor V which is resistant to activated protein C. There is a transition of guanine to adenine at nucleotide 1691 of the gene coding for factor V, which results in replacement of the amino acid Arg by Gln at the 506th position. 5) Factor V Leiden mutation is responsible for the great majority of cases (more than 90%) with hereditary resistance for activated protein C. 6) Factor V Leiden is inactivated 10 times slower than the normal factor V, 7-10) which leads to an increased risk for venous thrombosis. 11,12) The prevalence of factor V Leiden frequency is reported to be 3 to 12% in Caucasians ) Several recently published studies claim that there is a relation between both mutations and the risk for myocardial infarction. 16,17) However, there is no consensus yet on this subject. This study was planned to determine the prevalence of prothrombin 20210GA and factor V Leiden mutations, and their impacts on the development of myocardial infarction in patients younger than 55 years of age in Southern Turkey. METHODS Subjects: We recruited 96 patients consecutively (group I) who were admitted with acute myocardial infarction to the coronary care unit and 77 healthy subjects (group II) with no previous history or clinical evidence of cardiovascular disease. We excluded diabetic patients from the study since diabetes mellitus is considered to be a main risk factor for the development of atherosclerosis according to the results of the US National Cholesterol Education Program. 18) All patients were recruited consecutively, and all patients had had their first myocardial infarction. The diagnosis of acute myocardial infarction was established by the presence of typical chest pain and ST elevations on the presentation electrocardiogram and an increase in plasma cardiac enzyme levels. Control subjects were randomly selected from the same population. All conventional coronary risk factors such as age, gender, hypertension, diabetes mellitus, and family history were recorded for both groups. After fasting for 12 hours, venous blood samples were collected from all subjects and plasma lipid levels (LDL, HDL, total cholesterol, and triglycerides) were determined by the enzymatic colorimetric method. According to the Adult Treatment Panel III guidelines in the National Cholesterol Education Program, LDL-cholesterol levels 130 mg/dl were considered to be

3 Vol 45 No 3 PROTHROMBIN 20210GA AND FACTOR V LEIDEN MUTATIONS 507 hyperlipidemia. 18) Blood samples for DNA analysis were drawn within the first hours of hospital admission. DNA isolation: K3EDTA-anticoagulated venous blood samples (2 ml) were collected from all subjects for DNA analysis. DNA samples were isolated from whole blood with the aid of a MagNa Pure LC DNA Isolation Kit I using a MagNA Pure LC Automated DNA isolation instrument (Roche Molecular Biochemicals). DNA samples were stored at -20 C until the mutations were investigated. Real-Time PCR: Factor V Leiden and prothrombin 20210GA mutations were detected with the aid of LightCycler-Factor V Leiden and LightCycler-Prothrombin 20210GA Mutation Detection Kits (Roche Molecular Biochemicals), respectively. All mutation-related gene regions were amplified in 20 µl PCR capillary tubes. After preparation of the master mixture, 18 µl of the reaction mixture and 2 µl (approximately 40 ng) of genomic DNA or control template were added to each LightCycler capillary tube. For negative control, PCR grade water was added instead of template. The capillary tubes were sealed and briefly centrifuged in a microcentrifuge and then placed into the LightCycler carousel. The PCR products were detected using 3'-fluorescein (FLU) labeled and 5'-Red 640 labeled probes. When both probes hybridize in close proximity, fluorescence resonance energy transfer (FRET) occurs, producing a specific fluorescence emission of LC-Red as a result of FLU excitation. The fluorescence intensity depends on the amount of specific PCR products. Amplification per cycle can be monitored with the LightCycler instrument. At the end of the amplification process, the LightCycler instrument increases the temperature and the fluorescence obtained is plotted against the temperature. The mutations are then identified by their characteristic curves. Total assay time is approximately 40 minutes. Statistical analysis: SPSS 9.0 for Windows was used for statistical analysis. The data are presented as the mean ± standard deviation. The significance of the classical risk factors and the presence of prothrombin 20210GA and factor V Leiden mutations were determined with the χ 2 test. The relationships between all the risk factors and acute myocardial infarction were investigated by multiple logistic regression analysis. The suitability of the distribution of the frequency of mutations was tested according to the Hardy-Weinberg equation. A P value < 0.05 was considered statistically significant. RESULTS The mean age and gender distributions were similar in both groups. The prevalence of hypertension, a positive family history of coronary artery disease, and mean plasma LDL-cholesterol and HDL-cholesterol levels were significantly

4 508 DÖNMEZ, ET AL Jpn Heart J May 2004 higher in group I. Smoking, mean plasma triglycerides, and total cholesterol levels were similar in both groups. The clinical characteristics of the subjects are shown in Table I. The prevalence of factor V Leiden mutation was 6.3% and 5.2% in group I and group II, respectively (OR 0.6 [95% CI ], P = 0.6), whereas the prevalence of Prothrombin 20210GA was 4.2% and 2.6% in group I and group II, respectively (OR 2.8 [95% CI ], P = 0.4) (Table II). None of the patients had both mutations. Multiple logistic regression analysis was performed to determine the effects of conventional coronary risk factors, and prothrombin 20210GA and factor V Leiden mutations on myocardial infarction. Prothrombin 20210GA and factor V Leiden mutations did not increase the risk of myocardial infarction. We found that hypertension and a positive family history of coronary artery disease increased the risk of acute myocardial infarction (Table III). Table I. Clinical and Laboratory Characteristics of the Patients and Controls Age Gender (M/F) Hypertension Diabetes mellitus Smoking Family history of CAD LDL-cholesterol (mg/dl) HDL-cholesterol (mg/dl) Triglycerides (mg/dl) Total cholesterol (mg/dl) * P < 0.05 Patients 43.8 ± /28 31* * ± 40.4* 41.8 ± 9.3* ± ± 47.7 Controls 42.4 ± / ± ± ± ± 34.7 Table II. Mutation Distribution of Subjects Patients Control P N % N % Factor V Leiden Prothrombin

5 Vol 45 No 3 PROTHROMBIN 20210GA AND FACTOR V LEIDEN MUTATIONS 509 Table III. Multiple Logistic Regression Analysis of Conventional Coronary Risk Factors, Prothrombin 20210, and Factor V Leiden Mutations on Myocardial Infarction Odds ratio CI (95%) P Age Gender (male/female) Hypertension Smoking Family history of CAD Hyperlipidemia Prothrombin Factor V Leiden DISCUSSION Myocardial infarction usually develops as a result of thrombosis originating from a ruptured atherosclerotic plaque in one of the coronary arteries. 1) But, sometimes, even individuals with anatomically normal coronary arteries can suffer from a myocardial infarction. 19) There is growing interest in the defects of the coagulation system in order to explain the occurrence of acute myocardial infarction in the absence of obvious coronary artery disease and/or risk factors for coronary artery disease. The relation between venous thrombosis and both genetic coagulation defects has been clearly documented in the literature. 3) However, there is some controversy in the literature about the relation between these mutations and the development of acute myocardial infarction. 16,17,20) Van de Water, et al investigated the prevalence of prothrombin 20210GA and factor V Leiden mutations in 271 patients with myocardial infarction. Sixty of these patients (41 patients aged < 50 years and 19 patients aged > 50 years) had normal or near normal coronary arteries. The remaining 211 control subjects (114 patients aged < 50 years and 97 patients aged > 50 years) had angiographically proven significant coronary artery stenosis. When patients aged <50 years were investigated, the prevalence of factor V Leiden mutation was determined to be 14.6% and 3.6% in the patient and control groups, respectively (OR 4.7 [95% CI ], P = 0.04). Similarly, the prevalence of prothrombin 20210GA mutation was determined to be 7.3% and 1.8% in the patient and control groups, respectively (OR 4.9 [95% CI ], P = 0.04). Seventeen percent of the patients were female and the prevalence of a positive family history for ischemic heart disease was similar in both groups. However, when the age distribution was not taken into account, the prevalence of prothrombin 20210GA mutation was 6.7%

6 510 DÖNMEZ, ET AL Jpn Heart J May 2004 (4/60) and 1.4% (3/211) in the patient and control groups, respectively (OR 4.9 [95% CI ], P = 0.04). 16) When the population characteristics of this study are compared with the present study, the mean age is essentially similar, although the female gender ratio and the prevalence of a positive family history were higher in our subjects. We are not able to comment on the coronary status of our patients, as we have not performed routine angiograms following acute MI in these patients. On the other hand, both patients and control subjects included in the study of Van de Water, et al had had a myocardial infarction, while their patients were selected by excluding significant coronary disease on coronary angiography. Their method of selecting patients may therefore have had a significant effect on their results as it is very likely that the prevalence of risk factors for hypercoagulability, such as prothrombin 20210GA and factor V Leiden mutations, were higher in their patient group. In a study involving 560 myocardial infarction patients and 646 healthy control subjects, Doggen, et al reported that the presence of prothrombin 20210GA mutation increased the risk for myocardial infarction (OR 1.5 [95% CI ]). The presence of factor V Leiden mutation, on the other hand, increased the risk for myocardial infarction by as much as 40% (OR 1.4 [95% CI ]). The mean age in this study was 56.2 and 57.3 years for the patient and control groups, respectively. In the subgroup of 314 individuals consisting of both patients and control subjects aged < 50 years (mean ages, 44.4 and 42.5 years, respectively), however, the risk for myocardial infarction was not significantly increased by the presence of prothrombin 20210GA mutation (OR 0.9 [95% CI ]), while the relative risk for myocardial infarction in the presence of factor V Leiden mutation was calculated as 1.8 (OR 1.8 [95% CI ]). 17) It is noted that the mean age values for this subgroup are essentially similar to that of our study groups. On the other hand, Ridker, et al investigated a subgroup of 2607 patients with a prothrombin 20210GA mutation in a prospective trial consisting of 14,916 men, with a follow-up period of ten years. During this period, 833 patients (mean age, 59.9 ± 9.0 years) developed myocardial infarction, stroke, or venous thrombosis while the remainder (1774 patients, mean age, 59.1 ± 8.6 years) remained free of a thrombotic event. Comparing the actual and expected risk for a major thrombotic event for these two groups, the authors reported that a prothrombin 20210GA mutation is not associated with increased risk for myocardial infarction or stroke (OR 1.05 [95% CI ], P = 0.8). 20) The reasons behind these conflicting results are not clear. The prevalence of prothrombin 20210GA and factor V Leiden mutations significantly vary in different populations. The prevalence of a factor V Leiden mutation is reported to vary

7 Vol 45 No 3 PROTHROMBIN 20210GA AND FACTOR V LEIDEN MUTATIONS 511 between 3 and 12 percent, while the prevalence of a prothrombin 20210GA mutation is reported to be between 1 and 5 percent in different studies involving Caucasians. 4,13-15) The prevalence of a factor V Leiden mutation in American and European populations is significantly higher than its prevalence in African and Asian populations. 20,21) The prevalence of factor V Leiden mutation is 7 to 8 percent according to studies performed in Turkey. 22,23) The results of several studies published so far suggest that both of these mutations can have a significant impact on the risk for myocardial infarction, especially in the presence of other conventional risk factors such as smoking, obesity, hypertension, diabetes mellitus, and hypercholesterolemia. 24,25) The risk for myocardial infarction increased with the presence of hypertension and a positive family history for myocardial infarction in our patient group. There are two limitations to this study, the main one of which is the small size of the patient and control groups. Another significant limitation we would like to acknowledge is the lack of coronary angiographic evaluation of the subjects included in our study. Considering the significant variation in carriership frequencies in different populations, we suggest that further studies are needed for determination of the age group at risk. Conclusion: Factor V Leiden and prothrombin 20210GA mutations do not seem to represent increased risk for early myocardial infarction development in inhabitants of Southern Turkey. ACKNOWLEDGEMENTS The authors are grateful to Ms. Tülay Özgören for her assistance in the preparation and editing of this manuscript. REFERENCES 1. Fallon JT. Pathology of myocardial infarction and reperfusion. In: Fuster V, Ross R and Topol EJ, editors. A therosclerosis and Coronary Artery Disease. Philadelphia: Lippincott-Raven, 1996; Poort SR, Rosendaal FR, Reitsma PH, Bertina RM. A common genetic variation 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: Rosendaal FR, Vos HL, Poort SL, Bertina RM. Prothrombin 20210A variant and age at thrombosis. Thromb Haemost 1998; 79: Rosendaal FR, Doggen CJ, Zivelin A, et al. Geographic distribution of the G to A prothrombin variant. Thromb Haemost 1998; 79: Greengard JS, Sun X, Xu X, Fernandez JA, Griffin JH, Evatt B. Activated protein C resistance caused by Arg506Gln mutation in factor Va. Lancet 1994; 343: Zivelin A, Griffin JH, Xu X, et al. A single genetic origin for a common Caucasian risk factor for venous thrombosis. Blood 1997; 89:

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