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C677T Mutation in the Methylene Tetrahydrofolate Reductase Gene as a Risk Factor for Venous Thrombotic Disease in Austrian Patients
Thrombosis Research 100 (2000) 405 ± 407
MINI-REPORT
C677T Mutation in the Methylene Tetrahydrofolate Reductase Gene as a Risk Factor for Venous Thrombotic Disease in Austrian Patients Gerald Seinost, Wilfried Renner, Marianne Brodmann, Monika Winkler, Herwig KoÈppel and Ernst Pilger Division of Angiology, Department of Medicine, Karl-Franzens University Graz, Graz, Austria (Received 6 June 2000 by Editor W. Muntean; revised/accepted 7 August 2000)
Key Words: Thrombosis; Homocysteine; C677T gene polymorphism; MTHFR
M
oderately raised concentrations of total homocysteine have been associated with an increased risk of arterial and venous thrombosis [1,2,3]. A point mutation, C to T substitution at the nucleotide 677, in the coding sequence of the gene for methylene tetrahydrofolate reductase (MTHFR) is the most common enzyme defect associated with moderately raised homocysteine concentrations, particularly in the presence of a suboptimal folate intake [4]. Genetic analyses studying the prevalence of the 677 C to T mutation in the MTHFR gene have recently shown divergent results in patients with venous thrombotic disease from different geographic regions. Two recent studies on Italian patients with venous thrombosis failed to demonstrate an association of the homozygous MTHFR 677 TT genotype with an increased risk of venous thrombotic disease [5,6]. On the other hand Arruda et al. found the prevalence of the MTHFR 677 C to T mutation to be significantly increased in 127 Brazilian patients with venous thrombosis [7]. In addition, Salomon et al. found the homocysteine MTHFR C677T mutation to be an independent
Corresponding author: Gerald Seinost, Division of Angiology, Department of Medicine, University Hospital Graz, Auenbruggerplatz 15 A-8036 Graz, Austria. Tel: +43 (316) 385 2911; Fax: +43 (316) 385 3788; E-mail:.
risk factor for venous thromboembolism in patients from Israel [8]. As there is no existing data for the Austrian population, we aimed to study the frequency of this polymorphism.
1. Materials and Methods In order to establish the frequency of the C677T mutation in the MTHFR gene and its association with venous thrombotic disease occurrence in the Austrian population, we investigated 155 unrelated patients with deep venous thrombosis admitted to the Division of Angiology, Department of Medicine, University Hospital Graz, between 1997 and 1999, and 298 individuals without venous thrombosis. The patient group included 61 males (39.4%) and 94 females (60.6%). The control group included 113 males (37.9%) and 185 females (62.1%). The mean age ( SD) was 54.1 ( 19.2) in the patient group and 54.1 ( 19.3) in the control group. In the patient group 16 patients (10.3%) suffered from recurrent venous thrombosis, two patients (1.3%) had systemic disease (lupus erythematodes). Subjects recruited from staff and inpatients were eligible as controls if they were without previous or current venous disease (deep venous thrombosis, pulmonary embolism, primary varicosis). The study was performed according to the Austrian Gene Technology Act and to the guidelines of the local Ethics Committee. Written
0049-3848/00/$ ± see front matter D 2000 Elsevier Science Ltd. All rights reserved. PII S0049-3848(00)00341-8
406
G. Seinost et al./Thrombosis Research 100 (2000) 405±407
Table 1. Distribution of the methylenetet ahydrofolate reductase (MTHFR) genotypes of venous thrombotic disease cases and controls Genotype Wild type (C/C) Heterozygous (C/T) Homozygous (T/T) T-allele frequency
Patients (%) 67 (43.2) 73 (47.1) 15 (9.7) 0.332
Controls (%) 122 (40.9) 141 (47.3) 35 (11.7) 0.354
B12, which are needed to break down excess homocysteine, as well as methionine overabundance from dietary protein, may play a critical role in homocysteine metabolism in different socio-cultural environments.
References informed consent was obtained from all participating subjects. All investigated individuals were of Austrian ancestry. DNA was extracted by conventional methods and genetic analysis of the C677T MTHFR polymorphism was performed by allele-specific restriction enzyme digestion of PCR products [9]. Statistical analysis was done using SPSS 8.0 for Windows. Proportions of two groups were compared by c2 test. Odds ratio (OR) and 95% confidence interval (CI) were calculated by multiple logistic regression analysis. The criterion for statistical significance was p < 0.05.
2. Results The frequency of the mutated MTHFR allele was not significantly different between the patient group (33.2%) and the control group (35.4%, c2). In addition, the prevalence of the homozygous genotype of the MTHFR mutation was not significantly different between the two groups (Table 1). Accordingly, the odds ratio for having deep venous thrombosis associated with the homozygous MTHFR 677TT genotype was not significantly increased (0.8, 95% confidence interval 0.42±1.52).
3. Conclusion In our study we found that the MTHFR C677T genotype was not associated with an increased risk of deep venous thrombosis. Further studies are needed to elucidate the fact that genetic defects causing moderate to severe hyperhomocysteinemia differently influence the risk of initial and recurrent venous thrombosis in various geographic regions. Dietary imbalances such as an inadequate intake of folate and vitamin
1. Fermo I, D'Angelo CV, Paroni R, Mazzola G, Calori G, D'Angelo A. Prevalence of moderate hyperhomocysteinemia in patients with earlyonset venous and arterial disease. Ann Intern Med 1995;123:747±53. 2. Clarke R, Daly L, Robinson K, Naughten E, Cahalane S, Fowler B, Graham I. Hyperhomocysteinemia: an independent risk factor for vascular disease. N Engl J Med 1991;324: 1149±55. 3. Ray JG. Meta-Analysis of hyperhomocysteinemia as a risk factor for venous thromboembolic disease. Arch Intern Med 1998;158: 2101±16. 4. Jacques PF, Bostom AG, Williams RR, Ellison RC, Eckfeldt JH, Rosenberg ICH, Selhub J, Rozen R. Relation between folate status, a common mutation in methylenetetrahydrofolate reductase, and plasma homocysteine concentrations. Circulation 1996;93: 7±9. 5. De Stefano V, Chiusolo P, Paciaroni K, Serra FG, Voso MT, Casorelli I, Rossi E, Leone G. Prevalence of the 677C to T mutation in the methylenetetrahydrofolate reductase gene in Italian patients with venous thrombotic disease. Thromb Haemost 1998;79: 686±7. 6. Tosetto A, Missiaglia E, Frezzato M, Rodeghiero F. The VITA project: C677T mutation in the methylene-tetrahydrofolate reductase (MTHFR) gene and risk of venous thromboembolism. Br J Haematol 1997;97:804±6. 7. Arruda VR, von Zuben PM, Chiaparini LC, Annichino-Bizzacchi JM, Costa FF. The mutation Ala677 ! Val in the methylene-tetrahydrofolate reductase gene: a risk factor for arterial disease and venous thrombosis. Thromb Haemost 1997;77:818±21.
G. Seinost et al./Thrombosis Research 100 (2000) 405±407
8. Salomon O, Steinberg DM, Zivelin A, Gitel S, Dardik R, Rosenberg N, Berliner S, Inbal A, Many A, Lubetsky A, Varon D, Martinowitz U, Seligsohn U. Single and combined prothrombotic factors in patients with idiopathic venous thromboembolism. Arterioscler Thromb Vasc Biol 1999;19:511±8.
407
9. Frosst P, Blom HJ, Milos R, Goyette P, Sheppard CA, Matthews RG, Boers GJ, den Heijer M, Kluijtmans LA, van den Heuvel LP, Rozen R. A candidate genetic risk factor for vascular disease: a common mutation in methylenetetrahydrofolate reductase. Nature Genet. 1995;10:111±3.
MINI-REPORT
C677T Mutation in the Methylene Tetrahydrofolate Reductase Gene as a Risk Factor for Venous Thrombotic Disease in Austrian Patients Gerald Seinost, Wilfried Renner, Marianne Brodmann, Monika Winkler, Herwig KoÈppel and Ernst Pilger Division of Angiology, Department of Medicine, Karl-Franzens University Graz, Graz, Austria (Received 6 June 2000 by Editor W. Muntean; revised/accepted 7 August 2000)
Key Words: Thrombosis; Homocysteine; C677T gene polymorphism; MTHFR
M
oderately raised concentrations of total homocysteine have been associated with an increased risk of arterial and venous thrombosis [1,2,3]. A point mutation, C to T substitution at the nucleotide 677, in the coding sequence of the gene for methylene tetrahydrofolate reductase (MTHFR) is the most common enzyme defect associated with moderately raised homocysteine concentrations, particularly in the presence of a suboptimal folate intake [4]. Genetic analyses studying the prevalence of the 677 C to T mutation in the MTHFR gene have recently shown divergent results in patients with venous thrombotic disease from different geographic regions. Two recent studies on Italian patients with venous thrombosis failed to demonstrate an association of the homozygous MTHFR 677 TT genotype with an increased risk of venous thrombotic disease [5,6]. On the other hand Arruda et al. found the prevalence of the MTHFR 677 C to T mutation to be significantly increased in 127 Brazilian patients with venous thrombosis [7]. In addition, Salomon et al. found the homocysteine MTHFR C677T mutation to be an independent
Corresponding author: Gerald Seinost, Division of Angiology, Department of Medicine, University Hospital Graz, Auenbruggerplatz 15 A-8036 Graz, Austria. Tel: +43 (316) 385 2911; Fax: +43 (316) 385 3788; E-mail:
risk factor for venous thromboembolism in patients from Israel [8]. As there is no existing data for the Austrian population, we aimed to study the frequency of this polymorphism.
1. Materials and Methods In order to establish the frequency of the C677T mutation in the MTHFR gene and its association with venous thrombotic disease occurrence in the Austrian population, we investigated 155 unrelated patients with deep venous thrombosis admitted to the Division of Angiology, Department of Medicine, University Hospital Graz, between 1997 and 1999, and 298 individuals without venous thrombosis. The patient group included 61 males (39.4%) and 94 females (60.6%). The control group included 113 males (37.9%) and 185 females (62.1%). The mean age ( SD) was 54.1 ( 19.2) in the patient group and 54.1 ( 19.3) in the control group. In the patient group 16 patients (10.3%) suffered from recurrent venous thrombosis, two patients (1.3%) had systemic disease (lupus erythematodes). Subjects recruited from staff and inpatients were eligible as controls if they were without previous or current venous disease (deep venous thrombosis, pulmonary embolism, primary varicosis). The study was performed according to the Austrian Gene Technology Act and to the guidelines of the local Ethics Committee. Written
0049-3848/00/$ ± see front matter D 2000 Elsevier Science Ltd. All rights reserved. PII S0049-3848(00)00341-8
406
G. Seinost et al./Thrombosis Research 100 (2000) 405±407
Table 1. Distribution of the methylenetet ahydrofolate reductase (MTHFR) genotypes of venous thrombotic disease cases and controls Genotype Wild type (C/C) Heterozygous (C/T) Homozygous (T/T) T-allele frequency
Patients (%) 67 (43.2) 73 (47.1) 15 (9.7) 0.332
Controls (%) 122 (40.9) 141 (47.3) 35 (11.7) 0.354
B12, which are needed to break down excess homocysteine, as well as methionine overabundance from dietary protein, may play a critical role in homocysteine metabolism in different socio-cultural environments.
References informed consent was obtained from all participating subjects. All investigated individuals were of Austrian ancestry. DNA was extracted by conventional methods and genetic analysis of the C677T MTHFR polymorphism was performed by allele-specific restriction enzyme digestion of PCR products [9]. Statistical analysis was done using SPSS 8.0 for Windows. Proportions of two groups were compared by c2 test. Odds ratio (OR) and 95% confidence interval (CI) were calculated by multiple logistic regression analysis. The criterion for statistical significance was p < 0.05.
2. Results The frequency of the mutated MTHFR allele was not significantly different between the patient group (33.2%) and the control group (35.4%, c2). In addition, the prevalence of the homozygous genotype of the MTHFR mutation was not significantly different between the two groups (Table 1). Accordingly, the odds ratio for having deep venous thrombosis associated with the homozygous MTHFR 677TT genotype was not significantly increased (0.8, 95% confidence interval 0.42±1.52).
3. Conclusion In our study we found that the MTHFR C677T genotype was not associated with an increased risk of deep venous thrombosis. Further studies are needed to elucidate the fact that genetic defects causing moderate to severe hyperhomocysteinemia differently influence the risk of initial and recurrent venous thrombosis in various geographic regions. Dietary imbalances such as an inadequate intake of folate and vitamin
1. Fermo I, D'Angelo CV, Paroni R, Mazzola G, Calori G, D'Angelo A. Prevalence of moderate hyperhomocysteinemia in patients with earlyonset venous and arterial disease. Ann Intern Med 1995;123:747±53. 2. Clarke R, Daly L, Robinson K, Naughten E, Cahalane S, Fowler B, Graham I. Hyperhomocysteinemia: an independent risk factor for vascular disease. N Engl J Med 1991;324: 1149±55. 3. Ray JG. Meta-Analysis of hyperhomocysteinemia as a risk factor for venous thromboembolic disease. Arch Intern Med 1998;158: 2101±16. 4. Jacques PF, Bostom AG, Williams RR, Ellison RC, Eckfeldt JH, Rosenberg ICH, Selhub J, Rozen R. Relation between folate status, a common mutation in methylenetetrahydrofolate reductase, and plasma homocysteine concentrations. Circulation 1996;93: 7±9. 5. De Stefano V, Chiusolo P, Paciaroni K, Serra FG, Voso MT, Casorelli I, Rossi E, Leone G. Prevalence of the 677C to T mutation in the methylenetetrahydrofolate reductase gene in Italian patients with venous thrombotic disease. Thromb Haemost 1998;79: 686±7. 6. Tosetto A, Missiaglia E, Frezzato M, Rodeghiero F. The VITA project: C677T mutation in the methylene-tetrahydrofolate reductase (MTHFR) gene and risk of venous thromboembolism. Br J Haematol 1997;97:804±6. 7. Arruda VR, von Zuben PM, Chiaparini LC, Annichino-Bizzacchi JM, Costa FF. The mutation Ala677 ! Val in the methylene-tetrahydrofolate reductase gene: a risk factor for arterial disease and venous thrombosis. Thromb Haemost 1997;77:818±21.
G. Seinost et al./Thrombosis Research 100 (2000) 405±407
8. Salomon O, Steinberg DM, Zivelin A, Gitel S, Dardik R, Rosenberg N, Berliner S, Inbal A, Many A, Lubetsky A, Varon D, Martinowitz U, Seligsohn U. Single and combined prothrombotic factors in patients with idiopathic venous thromboembolism. Arterioscler Thromb Vasc Biol 1999;19:511±8.
407
9. Frosst P, Blom HJ, Milos R, Goyette P, Sheppard CA, Matthews RG, Boers GJ, den Heijer M, Kluijtmans LA, van den Heuvel LP, Rozen R. A candidate genetic risk factor for vascular disease: a common mutation in methylenetetrahydrofolate reductase. Nature Genet. 1995;10:111±3.