Original article 869
Association study of methylenetetrahydrofolate reductase C677T mutation with cerebral venous thrombosis in an Iranian population Habib Ghaznavia, Zahra Soheilib, Shahram Samieic and Mohammad S. Soltanpourd There are limited data on the role of methylenetetrahydrofolate reductase C677T polymorphism and hyperhomocysteinemia as risk factors for cerebral venous thrombosis in Iranian population. We examined a possible association between fasting plasma homocysteine levels, methylenetetrahydrofolate reductase C677T polymorphism, and cerebral venous thrombosis in 50 patients with a diagnosis of cerebral venous thrombosis (20–63 years old) and 75 healthy controls (18–65 years old). Genotyping of the methylenetetrahydrofolate reductase C677T gene polymorphism was performed by PCR–restriction fragment length polymorphism analysis, and homocysteine levels were measured by enzyme immunoassay. Fasting plasma homocysteine levels were significantly higher in cerebral venous thrombosis patients than in controls (P U 0.015). Moreover, plasma homocysteine levels were significantly higher in methylenetetrahydrofolate reductase 677TT genotype compared to 677CT and 677CC genotypes in both cerebral venous thrombosis patients (P U 0.01) and controls (P U 0.03). Neither 677CT heterozygote genotype [odds ratio (OR) 1.35, 95% confidence interval (CI) 0.64–2.84, P U 0.556] nor 677TT homozygote genotype (OR 1.73, 95% CI 0.32–9.21, P U 0.833) was significantly associated with cerebral venous thrombosis. Additionally, no significant differences in the frequency of 677T allele between cerebral
Introduction Cerebral venous thrombosis (CVT) is a rare form of cerebrovascular disease with an associated mortality rate ranging from 10 to 13% [1,2]. It accounts for 0.5% of all strokes, with an annual incidence of three to four cases per million populations and often affects younger age groups [1,2]. Cerebral venous thrombosis is slightly more common in women, particularly in the age group of 20–35 years, due to pregnancy, puerperium, and oral contraceptive use [3]. CVT is typically multifactorial, and its predisposing factors can be identified in up to 80% of the patients. Hyperhomocysteinemia, a prothrombotic state, causes vascular endothelial damage, resulting in venous thromboembolism and ischemic strokes [4]. A large number of studies have demonstrated that mild to moderate hyperhomocysteinemia is an independent risk factor for CVT [5–7]. Martinelli et al. [8] reported that patients with 0957-5235 Copyright ß 2015 Wolters Kluwer Health, Inc. All rights reserved.
venous thrombosis patients and controls were identified (OR 1.31, 95% CI 0.69–2.50, P U 0.512). In conclusion, our study demonstrated that elevated plasma homocysteine levels are significant risk factors for cerebral venous thrombosis. Also, methylenetetrahydrofolate reductase 677TT genotype is not linked with cerebral venous thrombosis, but is a determinant of elevated plasma homocysteine levels. Blood Coagul Fibrinolysis 26:869–873 Copyright ß 2015 Wolters Kluwer Health, Inc. All rights reserved.
Blood Coagulation and Fibrinolysis 2015, 26:869–873 Keywords: cerebral venous thrombosis, homocysteine, methylenetetrahydrofolate reductase, PCR, polymorphism, restriction fragment length polymorphism a Cellular and Molecular Research Centre, Zahedan University of Medical Sciences, Zahedan, bDepartment of Basic Sciences, National Institute of Genetic Engineering and Biotechnology, cIranian Blood Transfusion Organization, Tehran and dDepartment of Laboratory Sciences, School of Paramedical Sciences, Zanjan University of Medical Sciences, Zanjan, Iran
Correspondence to Mohammad S. Soltanpour, PhD, Department of Laboratory Sciences, School of Paramedical Sciences, Zanjan University of Medical Sciences, Zanjan, Iran Tel: +98 24 3377 2092; fax: +98 24 3377 3153; mob: þ98 914 322 9473; e-mail: [email protected]/[email protected] Received 31 August 2014 Revised 27 January 2015 Accepted 12 February 2015
hyperhomocysteinemia had a four-fold increased risk to develop CVT. A high plasma level of total homocysteine (tHcy) may result from acquired factors such as an inadequate folate intake in diet, smoking, renal failure, and from inherited gene polymorphisms in enzymes involved in homocysteine (Hcy) metabolism such as methylenetetrahydrofolate reductase (MTHFR) and cystathionine b synthase [4,9]. Methylenetetrahydrofolate reductase is one of the main regulatory enzymes of Hcy metabolism, catalyzing the conversion of 5,10-methylenetetrohydrofolate into 5-methyltetrahydrofolate, which is a carbon donor for re-methylation of Hcy to methionine [10]. The common C677T missense mutation in the MTHFR gene results in a thermolabile variant associated with decreased specific enzymatic activity, decreased DOI:10.1097/MBC.0000000000000292
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870 Blood Coagulation and Fibrinolysis 2015, Vol 26 No 8
concentration of folate in serum, and moderately increased plasma tHcy levels [10,11].
was aspirated and transferred to plastic tubes and stored at 208C until analysis for tHcy. Plasma tHcy levels were determined by microplate enzyme immunoassay using a commercial kit (Axis Homocysteine EIA KIT) according to manufacturer’s instruction. The assay’s detection range was 2.5–50.0 mmol/l.
Several studies have examined MTHFR C677T polymorphism as a risk factor for CVT, but conflicting results have been reported [8,12–14]. Recently, in a metaanalysis by Marjot et al. [15], a significant association was found between this polymorphism and CVT.
MTHFR C677T genotype analysis
Since the association of MTHFR C677T polymorphism with CVT is controversial and there is little information on the prevalence of this polymorphism in Iranian CVT patients, we aimed to perform a case–control study to assess the frequency of MTHFR C677T genotypes in CVT patients and healthy controls, in order to establish whether this hereditary mutation may have a causative role in CVT.
DNA was extracted from blood leukocyte according to the method described by Lahiri and Nurnberger [16]. MTHFR C677T mutation was analyzed by PCR– restriction fragment length polymorphism (PCR-RFLP) method as previously described by Frosst et al. [10] with slight modification. PCR was carried out on 40 ml reaction mixture containing 200 ng genomic DNA in 1 PCR buffer, 2.2 mmol/l MgCl2, 200 mmol/l dNTPs, 0.5 mmol/l primers, and 1.5 U AmpliTaq DNA polymerase (Applied Biosystems, Life Technologies, Foster City, California, USA). The sequences of the primers were: 50 -TGA AGG AGG TGT CTG CGG GA-30 and 50 -AGG ACG GTG CGG TGA GAG TG-30 . The PCR conditions included an initial denaturation at 948C for 2 min, followed by 35 cycles of denaturation at 948C for 30 s, annealing at 628C for 30 s, extension at 728C for 30 s, and a final extension at 728C for 7 min. The amplified products were digested with 5 units of HinfI (Promega, Madison, Wisconsin, USA) at 378C for 12 h.
Materials and methods Patients and controls
Fifty-five unrelated patients with a diagnosis of CVT were referred to the coagulation center of Iranian blood transfusion organization for thrombophilia screening. The clinical records and objective documentation of cerebral vein thrombosis were reviewed by a neurologist to confirm the diagnosis. Five patients were excluded from the analysis because their diagnosis was uncertain. Therefore, 50 patients (17 men and 33 women; mean age 38 years, ranging from 20 to 63 years) with objectively confirmed diagnosis of CVT were included in the study.
The digested products were separated by electrophoresis on a 3% agarose gel and visualized using ethidium bromide. Individuals with the 677CC (wild-type) genotype showed a single band of 198 bp, those with the 677CT (heterozygote) genotype showed bands of 198 and 175 bp, and those with the 677TT (homozygote) genotype showed a single band of 175 bp (Fig. 1).
The controls consisted of 75 healthy individuals (25 men and 50 women; mean age 36 years; ranging from 18 to 65 years) matched for age and sex with the patients, and without any history of thrombosis and/or use of vitamin B supplements and folate. Informed consent was obtained from all participants. Plasma total homocysteine assay
Statistical analysis
Blood from fasting patients was drawn in EDTA-containing vacutainer tubes for analysis of tHcy in plasma. Samples were centrifuged at 48C, and plasma fraction
Allele frequencies were calculated by gene counting in CVT patients and control individuals. Differences in mean age and tHcy levels between CVT patients and
Fig. 1
198
1
2
3
4
5
6
7
8
9
10
11
12
175
Polymorphism analysis of MTHFR C677T. The PCR products were digested by restriction enzyme Hinf I: 1 (ladder 100 bp); lanes 2, 5, 6, 8, 9, and 10 – wild-type (677CC) genotypes (198 bp); lanes 4, 7, and 12 – heterozygote (677CT) genotypes (198 and 175 bp); lanes 3 and 11 – homozygote (677TT) genotypes (175 bp). MTHFR, methylenetetrahydrofolate reductase.
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MTHFR gene polymorphism and cerebral vein thrombosis Ghaznavi et al. 871
controls were tested by Student’s t test. Analysis of variance (ANOVA) was used to compare Hcy levels among the genotypes. Chi-square test was used to assess the association of MTHFR C677T polymorphism with venous thrombosis, and odds ratio (OR) and 95% confidence interval (CI) were calculated. Chi-square test was also used to analyze the distribution of MTHFR C677T genotypes between men and women in CVT patients and controls, and to test sex differences between CVT patients and controls. Statistical analysis was performed by SPSS 15 software (SPSS Inc., Chicago, Illinois, USA) with a statistical significance level of P less than 0.05.
Moreover, no significant differences were observed in the frequency of 677T allele (27 vs. 22%) between CVT patients and controls (OR 1.31, 95% CI 0.69–2.50, P ¼ 0.512). Additionally, the ANOVA test results showed significant association between MTHFR C677T genotypes and Hcy levels in both CVT patients (P ¼ 0.01) and controls (P ¼ 0.03), as shown in Fig. 2. According to Table 3, no significant differences were observed in the genotype distribution of MTHFR C677T polymorphism between men and women in both CVT patients (P ¼ 0.88) and controls (P ¼ 0.93).
Discussion Results The population under study consisted of 50 patients with CVT and 75 apparently healthy individuals matched for age and sex as controls. As shown in Table 1, the differences in the sex distribution (P ¼ 0.85) and mean age (P ¼ 0.5) were not significant between CVT patients and controls. Fasting plasma tHcy levels were significantly higher in the CVT patients than in controls (P ¼ 0.015). Moreover, mean normal values (8.9 mmol/l) of the tHcy obtained from Hcy assay kit were used as a cut-off point in order to determine abnormal tHcy values in both control individuals and CVT patients. In control individuals, 33 out of 75 (44%) had abnormal tHcy values, whereas in the CVT patients, 38 out of 50 (76%) had abnormal tHcy values. Statistical analysis using chisquare test showed that the frequency of abnormal tHcy values was significantly higher in CVT patients than in control individuals (P < 0.01). The highest tHcy levels in CVT patients and control individuals were 26.80 and 22.20 mmol/l, respectively. Moreover, the number of cases with high plasma tHcy values (tHcy > 14 mmol/l) were significantly higher in CVT patients than in the control individuals (25 vs.15; P < 0.01). The distribution of MTHFR C677T genotypes in CVT patients and controls is shown in Table 2. Among the CVT patients, the distribution of different MTHFR C677T genotypes was 6.0% for 677TT, 42% for 677CT, and 52% for 677CC genotypes. Among controls, these values were 4% for 677TT, 36% for 677CT, and 60% for 677CC genotypes. Neither 677CT heterozygote genotype (OR 1.35, 95% CI 0.64–2.84, P ¼ 0.556) nor 677TT homozygote genotype (OR 1.73, 95% CI 0.32– 9.21, P ¼ 0.833) was significantly associated with CVT. Homocysteine levels and other characteristics in cerebral venous thrombosis patients and control individuals Table 1
Study population Age (years) Sex (male/female) Plasma tHcy (mmol/l)
CVT patients (n ¼ 50)
Controls (n ¼ 75)
P value
38 28.2 17/33 13.7 4.5
36 30.6 25/50 9.9 3.6
0.5 0.85 0.015
Values are mean SD. Chi-square test was used to compare sex between the two groups, and mean values of age and homocysteine levels were analyzed by Student’s t test. CVT, cerebral venous thrombosis; tHcy, total homocysteine.
The main findings of this study were that elevated plasma tHcy levels were significantly associated with CVT development; the MTHFR C677T polymorphism, though not associated with a risk of CVT, contributed to significantly higher plasma tHcy levels in CVT cases and controls in the Iranian population. Elevated plasma tHcy levels have been defined as a risk factor for CVT development in several studies [5–8,17,18]. Ventura et al. [19] reported that elevated plasma tHcy levels increased the risk of idiopathic CVT by 6.8-fold. In a case report by Bharatkumar et al. [20], hyperhomocysteinemia was reported to be associated with sinovenous thrombosis of the brain in a vegetarian man. In accordance with these reports, our study implied a positive association between hyperhomocysteinemia and CVT (P ¼ 0.015). Elevated plasma tHcy levels are thought to accelerate vascular disease in a number of ways, possibly by inhibition of protein C activation, inducing procoagulant activity of monocytes, promoting endothelial tissue factor expression, and suppressing heparin sulfate expression on endothelial cells [4]. The C677T polymorphism in the MTHFR gene, a contributing factor for elevated plasma tHcy levels, has been suggested as a candidate risk factor for CVT, although the results are controversial [12,13,18–26]. Our study is in agreement with those studies that showed no association between MTHFR C677T polymorphism and CVT [14,22,27–29]. However, our results were inconsistent with those studies that identified MTHFR C677T polymorphism as a risk factor for CVT [15,21, 23–25]. The possible reasons for inconsistency of association studies are numerous, and many factors such as population heterogeneity, ethnic stratification, confounding sampling bias, variation in study design, and sample size may contribute to variable association results [30]. In a recent meta-analysis regarding the association of MTHFR C677T polymorphism and CVT, Marjot et al. [15] identified seven case–control studies included 233 CVT cases and 1323 controls, and their results
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872 Blood Coagulation and Fibrinolysis 2015, Vol 26 No 8
Table 2 Methylenetetrahydrofolate reductase C677T genotypes and allelic frequencies of cerebral venous thrombosis patients and control individuals MTHFR C677T gene polymorphism 677T allele 677 CC genotype 677 CT genotype 677 TT genotype
Patients [n (%)] 27 26 21 3
(27) (52) (42) (6)
Controls [n (%)] 33 45 27 3
(22) (60) (36) (4)
OR (95% CI)
P value
1.31 (0.69–2.50) 1 1.35 (0.64–2.84) 1.73 (0.32–9.21)
0.512 – 0.556 0.833
677CC, wild type; 677CT, heterozygote; 677TT, homozygote. CI, confidence interval; CVT, cerebral venous thrombosis; MTHFR, methylenetetrahydrofolate reductase; OR, odds ratio.
found a slightly increased risk of CVT for MTHFR 677TT carriers (OR 2.30, 95% CI 1.20–4.42, P ¼ 0.02) in this pooled analysis. Our study is inconsistent with this meta-analysis. Possible explanation for this controversy may be related to the likelihood of publication bias in metaanalysis studies which may result in overestimation of the risk due to under-representation of studies that found no association [31]. Also, small sample size of our study population and the presence of other confounding factors may contribute to this inconsistency. In the most recent study, Bharatkumar et al. [20] identified hyperhomocysteinemia, but not MTHFR 677TT genotype, as a significant risk marker for CVT, and also showed that MTHFR 677TT genotype is a determinant of elevated plasma tHcy levels. Our results were in complete agreement with this recent study. The frequency of the MTHFR 677T allele in our study was 22%, which is lower than the frequency reported in the Chinese (55.2%) [32] and the Turkish
(33.6%) [33] population, and higher than the frequency reported in the Canadian (6%) [34] and the West African (9%) [35] population. The MTHFR 677T allele frequency in our population was also comparable with the MTHFR 677T allele frequency in the Brazilian (24%) [36] and the Taiwanese Chinese (24.4%) [37] population. Despite the limitation of the small sample size, results of the present study indicated an association of mild hyperhomocysteinemia with CVT, but did not prove a causal relationship between MTHFR C677T polymorphism and CVT. Since hyperhomocysteinemia can be safely and inexpensively corrected by vitamin supplementation, Hcy should be routinely determined in CVT patients, and even slightly higher plasma tHcy levels should be treated in the hope that the risks associated with CVT will be reduced [38]. In conclusion, on the basis of this study, we suggest that hyperhomocysteinemia is a significant risk factor for CVT development. However, MTHFR 677TT genotype is not linked with CVT, but is a determinant of elevated plasma tHcy levels.
Fig. 2
26 24 22 20 18 16 14 12 10 8 6 4 2 0
Homocysteine (µmol/l )
(b) 28
26 24 22 20 18 16 14 12 10 8 6 4 2 0
Homocysteine (µmol/l )
(a) 28
CC (n = 26)
CT (n = 21)
P value= 0.01
TT (n = 3)
CC (n = 45)
CT (n = 27)
TT(n = 3)
P value= 0.03
Effect of different MTHFR C677T genotypes on total Hcy levels in CVT patients (a) and controls (b). Analysis of variance (ANOVA) was used to compare Hcy levels among the genotypes. CVT, cerebral venous thrombosis; Hcy, homocysteine; MTHFR, methylenetetrahydrofolate reductase.
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MTHFR gene polymorphism and cerebral vein thrombosis Ghaznavi et al. 873
The genotype distribution of methylenetetrahydrofolate reductase C677T polymorphism according to sex in cerebral venous thrombosis patients and control individuals
17
MTHFR C677T gene polymorphism
18
Table 3
Men [n (%)]
CVT patients 677CC genotype 677CT genotype 677TT genotype P value Control individuals 677CC genotype 677CT genotype 677TT genotype P value
17 9 7 1
(100) (53.0) (41.1) (5.9)
25 14 10 1
(100) (56.0) (40.0) (4.0)
Women [n (%)] 33 (100) 17 (51.5) 14 (42.4) 2 (6.1) 0.88 50 (100) 31 (62.0) 17 (34.0) 2 (4.0) 0.93
Total [N (%)] 50 26 21 3
(100) (52) (42) (6.0)
75 45 27 3
(100) (60.0) (36.0) (4.0)
677CC, wild type; 677CT, heterozygote; 677TT, homozygote. CVT, cerebral venous thrombosis; MTHFR, methylenetetrahydrofolate reductase.
19
20
21 22
23
Acknowledgements Conflicts of interest
24
There are no conflicts of interest. 25
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Association study of methylenetetrahydrofolate reductase C677T mutation with cerebral venous thrombosis in an Iranian population Habib Ghaznavia, Zahra Soheilib, Shahram Samieic and Mohammad S. Soltanpourd There are limited data on the role of methylenetetrahydrofolate reductase C677T polymorphism and hyperhomocysteinemia as risk factors for cerebral venous thrombosis in Iranian population. We examined a possible association between fasting plasma homocysteine levels, methylenetetrahydrofolate reductase C677T polymorphism, and cerebral venous thrombosis in 50 patients with a diagnosis of cerebral venous thrombosis (20–63 years old) and 75 healthy controls (18–65 years old). Genotyping of the methylenetetrahydrofolate reductase C677T gene polymorphism was performed by PCR–restriction fragment length polymorphism analysis, and homocysteine levels were measured by enzyme immunoassay. Fasting plasma homocysteine levels were significantly higher in cerebral venous thrombosis patients than in controls (P U 0.015). Moreover, plasma homocysteine levels were significantly higher in methylenetetrahydrofolate reductase 677TT genotype compared to 677CT and 677CC genotypes in both cerebral venous thrombosis patients (P U 0.01) and controls (P U 0.03). Neither 677CT heterozygote genotype [odds ratio (OR) 1.35, 95% confidence interval (CI) 0.64–2.84, P U 0.556] nor 677TT homozygote genotype (OR 1.73, 95% CI 0.32–9.21, P U 0.833) was significantly associated with cerebral venous thrombosis. Additionally, no significant differences in the frequency of 677T allele between cerebral
Introduction Cerebral venous thrombosis (CVT) is a rare form of cerebrovascular disease with an associated mortality rate ranging from 10 to 13% [1,2]. It accounts for 0.5% of all strokes, with an annual incidence of three to four cases per million populations and often affects younger age groups [1,2]. Cerebral venous thrombosis is slightly more common in women, particularly in the age group of 20–35 years, due to pregnancy, puerperium, and oral contraceptive use [3]. CVT is typically multifactorial, and its predisposing factors can be identified in up to 80% of the patients. Hyperhomocysteinemia, a prothrombotic state, causes vascular endothelial damage, resulting in venous thromboembolism and ischemic strokes [4]. A large number of studies have demonstrated that mild to moderate hyperhomocysteinemia is an independent risk factor for CVT [5–7]. Martinelli et al. [8] reported that patients with 0957-5235 Copyright ß 2015 Wolters Kluwer Health, Inc. All rights reserved.
venous thrombosis patients and controls were identified (OR 1.31, 95% CI 0.69–2.50, P U 0.512). In conclusion, our study demonstrated that elevated plasma homocysteine levels are significant risk factors for cerebral venous thrombosis. Also, methylenetetrahydrofolate reductase 677TT genotype is not linked with cerebral venous thrombosis, but is a determinant of elevated plasma homocysteine levels. Blood Coagul Fibrinolysis 26:869–873 Copyright ß 2015 Wolters Kluwer Health, Inc. All rights reserved.
Blood Coagulation and Fibrinolysis 2015, 26:869–873 Keywords: cerebral venous thrombosis, homocysteine, methylenetetrahydrofolate reductase, PCR, polymorphism, restriction fragment length polymorphism a Cellular and Molecular Research Centre, Zahedan University of Medical Sciences, Zahedan, bDepartment of Basic Sciences, National Institute of Genetic Engineering and Biotechnology, cIranian Blood Transfusion Organization, Tehran and dDepartment of Laboratory Sciences, School of Paramedical Sciences, Zanjan University of Medical Sciences, Zanjan, Iran
Correspondence to Mohammad S. Soltanpour, PhD, Department of Laboratory Sciences, School of Paramedical Sciences, Zanjan University of Medical Sciences, Zanjan, Iran Tel: +98 24 3377 2092; fax: +98 24 3377 3153; mob: þ98 914 322 9473; e-mail: [email protected]/[email protected] Received 31 August 2014 Revised 27 January 2015 Accepted 12 February 2015
hyperhomocysteinemia had a four-fold increased risk to develop CVT. A high plasma level of total homocysteine (tHcy) may result from acquired factors such as an inadequate folate intake in diet, smoking, renal failure, and from inherited gene polymorphisms in enzymes involved in homocysteine (Hcy) metabolism such as methylenetetrahydrofolate reductase (MTHFR) and cystathionine b synthase [4,9]. Methylenetetrahydrofolate reductase is one of the main regulatory enzymes of Hcy metabolism, catalyzing the conversion of 5,10-methylenetetrohydrofolate into 5-methyltetrahydrofolate, which is a carbon donor for re-methylation of Hcy to methionine [10]. The common C677T missense mutation in the MTHFR gene results in a thermolabile variant associated with decreased specific enzymatic activity, decreased DOI:10.1097/MBC.0000000000000292
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870 Blood Coagulation and Fibrinolysis 2015, Vol 26 No 8
concentration of folate in serum, and moderately increased plasma tHcy levels [10,11].
was aspirated and transferred to plastic tubes and stored at 208C until analysis for tHcy. Plasma tHcy levels were determined by microplate enzyme immunoassay using a commercial kit (Axis Homocysteine EIA KIT) according to manufacturer’s instruction. The assay’s detection range was 2.5–50.0 mmol/l.
Several studies have examined MTHFR C677T polymorphism as a risk factor for CVT, but conflicting results have been reported [8,12–14]. Recently, in a metaanalysis by Marjot et al. [15], a significant association was found between this polymorphism and CVT.
MTHFR C677T genotype analysis
Since the association of MTHFR C677T polymorphism with CVT is controversial and there is little information on the prevalence of this polymorphism in Iranian CVT patients, we aimed to perform a case–control study to assess the frequency of MTHFR C677T genotypes in CVT patients and healthy controls, in order to establish whether this hereditary mutation may have a causative role in CVT.
DNA was extracted from blood leukocyte according to the method described by Lahiri and Nurnberger [16]. MTHFR C677T mutation was analyzed by PCR– restriction fragment length polymorphism (PCR-RFLP) method as previously described by Frosst et al. [10] with slight modification. PCR was carried out on 40 ml reaction mixture containing 200 ng genomic DNA in 1 PCR buffer, 2.2 mmol/l MgCl2, 200 mmol/l dNTPs, 0.5 mmol/l primers, and 1.5 U AmpliTaq DNA polymerase (Applied Biosystems, Life Technologies, Foster City, California, USA). The sequences of the primers were: 50 -TGA AGG AGG TGT CTG CGG GA-30 and 50 -AGG ACG GTG CGG TGA GAG TG-30 . The PCR conditions included an initial denaturation at 948C for 2 min, followed by 35 cycles of denaturation at 948C for 30 s, annealing at 628C for 30 s, extension at 728C for 30 s, and a final extension at 728C for 7 min. The amplified products were digested with 5 units of HinfI (Promega, Madison, Wisconsin, USA) at 378C for 12 h.
Materials and methods Patients and controls
Fifty-five unrelated patients with a diagnosis of CVT were referred to the coagulation center of Iranian blood transfusion organization for thrombophilia screening. The clinical records and objective documentation of cerebral vein thrombosis were reviewed by a neurologist to confirm the diagnosis. Five patients were excluded from the analysis because their diagnosis was uncertain. Therefore, 50 patients (17 men and 33 women; mean age 38 years, ranging from 20 to 63 years) with objectively confirmed diagnosis of CVT were included in the study.
The digested products were separated by electrophoresis on a 3% agarose gel and visualized using ethidium bromide. Individuals with the 677CC (wild-type) genotype showed a single band of 198 bp, those with the 677CT (heterozygote) genotype showed bands of 198 and 175 bp, and those with the 677TT (homozygote) genotype showed a single band of 175 bp (Fig. 1).
The controls consisted of 75 healthy individuals (25 men and 50 women; mean age 36 years; ranging from 18 to 65 years) matched for age and sex with the patients, and without any history of thrombosis and/or use of vitamin B supplements and folate. Informed consent was obtained from all participants. Plasma total homocysteine assay
Statistical analysis
Blood from fasting patients was drawn in EDTA-containing vacutainer tubes for analysis of tHcy in plasma. Samples were centrifuged at 48C, and plasma fraction
Allele frequencies were calculated by gene counting in CVT patients and control individuals. Differences in mean age and tHcy levels between CVT patients and
Fig. 1
198
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11
12
175
Polymorphism analysis of MTHFR C677T. The PCR products were digested by restriction enzyme Hinf I: 1 (ladder 100 bp); lanes 2, 5, 6, 8, 9, and 10 – wild-type (677CC) genotypes (198 bp); lanes 4, 7, and 12 – heterozygote (677CT) genotypes (198 and 175 bp); lanes 3 and 11 – homozygote (677TT) genotypes (175 bp). MTHFR, methylenetetrahydrofolate reductase.
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MTHFR gene polymorphism and cerebral vein thrombosis Ghaznavi et al. 871
controls were tested by Student’s t test. Analysis of variance (ANOVA) was used to compare Hcy levels among the genotypes. Chi-square test was used to assess the association of MTHFR C677T polymorphism with venous thrombosis, and odds ratio (OR) and 95% confidence interval (CI) were calculated. Chi-square test was also used to analyze the distribution of MTHFR C677T genotypes between men and women in CVT patients and controls, and to test sex differences between CVT patients and controls. Statistical analysis was performed by SPSS 15 software (SPSS Inc., Chicago, Illinois, USA) with a statistical significance level of P less than 0.05.
Moreover, no significant differences were observed in the frequency of 677T allele (27 vs. 22%) between CVT patients and controls (OR 1.31, 95% CI 0.69–2.50, P ¼ 0.512). Additionally, the ANOVA test results showed significant association between MTHFR C677T genotypes and Hcy levels in both CVT patients (P ¼ 0.01) and controls (P ¼ 0.03), as shown in Fig. 2. According to Table 3, no significant differences were observed in the genotype distribution of MTHFR C677T polymorphism between men and women in both CVT patients (P ¼ 0.88) and controls (P ¼ 0.93).
Discussion Results The population under study consisted of 50 patients with CVT and 75 apparently healthy individuals matched for age and sex as controls. As shown in Table 1, the differences in the sex distribution (P ¼ 0.85) and mean age (P ¼ 0.5) were not significant between CVT patients and controls. Fasting plasma tHcy levels were significantly higher in the CVT patients than in controls (P ¼ 0.015). Moreover, mean normal values (8.9 mmol/l) of the tHcy obtained from Hcy assay kit were used as a cut-off point in order to determine abnormal tHcy values in both control individuals and CVT patients. In control individuals, 33 out of 75 (44%) had abnormal tHcy values, whereas in the CVT patients, 38 out of 50 (76%) had abnormal tHcy values. Statistical analysis using chisquare test showed that the frequency of abnormal tHcy values was significantly higher in CVT patients than in control individuals (P < 0.01). The highest tHcy levels in CVT patients and control individuals were 26.80 and 22.20 mmol/l, respectively. Moreover, the number of cases with high plasma tHcy values (tHcy > 14 mmol/l) were significantly higher in CVT patients than in the control individuals (25 vs.15; P < 0.01). The distribution of MTHFR C677T genotypes in CVT patients and controls is shown in Table 2. Among the CVT patients, the distribution of different MTHFR C677T genotypes was 6.0% for 677TT, 42% for 677CT, and 52% for 677CC genotypes. Among controls, these values were 4% for 677TT, 36% for 677CT, and 60% for 677CC genotypes. Neither 677CT heterozygote genotype (OR 1.35, 95% CI 0.64–2.84, P ¼ 0.556) nor 677TT homozygote genotype (OR 1.73, 95% CI 0.32– 9.21, P ¼ 0.833) was significantly associated with CVT. Homocysteine levels and other characteristics in cerebral venous thrombosis patients and control individuals Table 1
Study population Age (years) Sex (male/female) Plasma tHcy (mmol/l)
CVT patients (n ¼ 50)
Controls (n ¼ 75)
P value
38 28.2 17/33 13.7 4.5
36 30.6 25/50 9.9 3.6
0.5 0.85 0.015
Values are mean SD. Chi-square test was used to compare sex between the two groups, and mean values of age and homocysteine levels were analyzed by Student’s t test. CVT, cerebral venous thrombosis; tHcy, total homocysteine.
The main findings of this study were that elevated plasma tHcy levels were significantly associated with CVT development; the MTHFR C677T polymorphism, though not associated with a risk of CVT, contributed to significantly higher plasma tHcy levels in CVT cases and controls in the Iranian population. Elevated plasma tHcy levels have been defined as a risk factor for CVT development in several studies [5–8,17,18]. Ventura et al. [19] reported that elevated plasma tHcy levels increased the risk of idiopathic CVT by 6.8-fold. In a case report by Bharatkumar et al. [20], hyperhomocysteinemia was reported to be associated with sinovenous thrombosis of the brain in a vegetarian man. In accordance with these reports, our study implied a positive association between hyperhomocysteinemia and CVT (P ¼ 0.015). Elevated plasma tHcy levels are thought to accelerate vascular disease in a number of ways, possibly by inhibition of protein C activation, inducing procoagulant activity of monocytes, promoting endothelial tissue factor expression, and suppressing heparin sulfate expression on endothelial cells [4]. The C677T polymorphism in the MTHFR gene, a contributing factor for elevated plasma tHcy levels, has been suggested as a candidate risk factor for CVT, although the results are controversial [12,13,18–26]. Our study is in agreement with those studies that showed no association between MTHFR C677T polymorphism and CVT [14,22,27–29]. However, our results were inconsistent with those studies that identified MTHFR C677T polymorphism as a risk factor for CVT [15,21, 23–25]. The possible reasons for inconsistency of association studies are numerous, and many factors such as population heterogeneity, ethnic stratification, confounding sampling bias, variation in study design, and sample size may contribute to variable association results [30]. In a recent meta-analysis regarding the association of MTHFR C677T polymorphism and CVT, Marjot et al. [15] identified seven case–control studies included 233 CVT cases and 1323 controls, and their results
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872 Blood Coagulation and Fibrinolysis 2015, Vol 26 No 8
Table 2 Methylenetetrahydrofolate reductase C677T genotypes and allelic frequencies of cerebral venous thrombosis patients and control individuals MTHFR C677T gene polymorphism 677T allele 677 CC genotype 677 CT genotype 677 TT genotype
Patients [n (%)] 27 26 21 3
(27) (52) (42) (6)
Controls [n (%)] 33 45 27 3
(22) (60) (36) (4)
OR (95% CI)
P value
1.31 (0.69–2.50) 1 1.35 (0.64–2.84) 1.73 (0.32–9.21)
0.512 – 0.556 0.833
677CC, wild type; 677CT, heterozygote; 677TT, homozygote. CI, confidence interval; CVT, cerebral venous thrombosis; MTHFR, methylenetetrahydrofolate reductase; OR, odds ratio.
found a slightly increased risk of CVT for MTHFR 677TT carriers (OR 2.30, 95% CI 1.20–4.42, P ¼ 0.02) in this pooled analysis. Our study is inconsistent with this meta-analysis. Possible explanation for this controversy may be related to the likelihood of publication bias in metaanalysis studies which may result in overestimation of the risk due to under-representation of studies that found no association [31]. Also, small sample size of our study population and the presence of other confounding factors may contribute to this inconsistency. In the most recent study, Bharatkumar et al. [20] identified hyperhomocysteinemia, but not MTHFR 677TT genotype, as a significant risk marker for CVT, and also showed that MTHFR 677TT genotype is a determinant of elevated plasma tHcy levels. Our results were in complete agreement with this recent study. The frequency of the MTHFR 677T allele in our study was 22%, which is lower than the frequency reported in the Chinese (55.2%) [32] and the Turkish
(33.6%) [33] population, and higher than the frequency reported in the Canadian (6%) [34] and the West African (9%) [35] population. The MTHFR 677T allele frequency in our population was also comparable with the MTHFR 677T allele frequency in the Brazilian (24%) [36] and the Taiwanese Chinese (24.4%) [37] population. Despite the limitation of the small sample size, results of the present study indicated an association of mild hyperhomocysteinemia with CVT, but did not prove a causal relationship between MTHFR C677T polymorphism and CVT. Since hyperhomocysteinemia can be safely and inexpensively corrected by vitamin supplementation, Hcy should be routinely determined in CVT patients, and even slightly higher plasma tHcy levels should be treated in the hope that the risks associated with CVT will be reduced [38]. In conclusion, on the basis of this study, we suggest that hyperhomocysteinemia is a significant risk factor for CVT development. However, MTHFR 677TT genotype is not linked with CVT, but is a determinant of elevated plasma tHcy levels.
Fig. 2
26 24 22 20 18 16 14 12 10 8 6 4 2 0
Homocysteine (µmol/l )
(b) 28
26 24 22 20 18 16 14 12 10 8 6 4 2 0
Homocysteine (µmol/l )
(a) 28
CC (n = 26)
CT (n = 21)
P value= 0.01
TT (n = 3)
CC (n = 45)
CT (n = 27)
TT(n = 3)
P value= 0.03
Effect of different MTHFR C677T genotypes on total Hcy levels in CVT patients (a) and controls (b). Analysis of variance (ANOVA) was used to compare Hcy levels among the genotypes. CVT, cerebral venous thrombosis; Hcy, homocysteine; MTHFR, methylenetetrahydrofolate reductase.
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MTHFR gene polymorphism and cerebral vein thrombosis Ghaznavi et al. 873
The genotype distribution of methylenetetrahydrofolate reductase C677T polymorphism according to sex in cerebral venous thrombosis patients and control individuals
17
MTHFR C677T gene polymorphism
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Table 3
Men [n (%)]
CVT patients 677CC genotype 677CT genotype 677TT genotype P value Control individuals 677CC genotype 677CT genotype 677TT genotype P value
17 9 7 1
(100) (53.0) (41.1) (5.9)
25 14 10 1
(100) (56.0) (40.0) (4.0)
Women [n (%)] 33 (100) 17 (51.5) 14 (42.4) 2 (6.1) 0.88 50 (100) 31 (62.0) 17 (34.0) 2 (4.0) 0.93
Total [N (%)] 50 26 21 3
(100) (52) (42) (6.0)
75 45 27 3
(100) (60.0) (36.0) (4.0)
677CC, wild type; 677CT, heterozygote; 677TT, homozygote. CVT, cerebral venous thrombosis; MTHFR, methylenetetrahydrofolate reductase.
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Acknowledgements Conflicts of interest
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There are no conflicts of interest. 25
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