Association of C677T polymorphism in the methylenetetrahydrofolate reductase gene (MTHFR gene) with ischemic stroke: a metaanalysis Amit Kumar, Pradeep Kumar, Manya Prasad, Ram Sagar, Arun Kumar Yadav, Awadh Kishor Pandit, Vidishaa Prasad Jali, Abhishek Pathak Department of Neurology, All India Institute of Medical Sciences, New Delhi, India Objective: Studies on association between methylenetetrahydrofolate reductase gene (MTHFR) C677T gene polymorphism and ischemic stroke have shown conflicting results. We have conducted a metaanalysis to determine the precise estimation of the role of the C677T polymorphism of MTHFR gene with risk of ischemic stroke. Materials and methods: We searched electronic databases Medline, EMBASE, and Google Scholar (last search dated August 2014). Pooled odds ratios (ORs) with 95% confidence intervals (CIs) from random or fixed-effects models were calculated. The methodological quality of included studies was determined by the quality assessment scale. Results: Thirty eight case–control studies fulfilled our inclusion criteria comprising 6310 patients and 8297 controls. The significant associations between MTHFR C677T genetic polymorphism and risk of ischemic stroke were observed in dominant (OR, 1.09; 95% CI, 1.06–1.12, P-value , 0.001) and recessive (OR, 1.31; 95% CI, 1.19–1.44, P-value , 0.001) inheritance models. In an Asian population, significant association between the MTHFR polymorphism and ischemic stroke was observed (dominant model: OR 1.36, 95% CI 1.23–1.49 and under recessive model OR, 1.29; 95% CI, 1.15–1.45). In the Caucasian population borderline, non-significant association was observed (OR, 1.05; 95% CI, 0.99–1.10) but significant association was observed under the recessive model of inheritance (OR, 1.33; 95% CI, 1.13– 1.58). Conclusion: The present study results suggest that MTHFR C677T genetic polymorphism is a probable risk of ischemic stroke. Keywords: Methylenetetrahydrofolate reductase, Polymorphism, Meta-analysis, Ischemic stroke, Cardiovascular disorder
Introduction Stroke has emerged as the second most common cause of mortality worldwide and is a major public health problem. Stroke accounted for nearly 5.7 million deaths worldwide in 2005.1 More than two-thirds of these deaths occurred in underdeveloped countries.2 Substantial evidence from the twin studies,3,4 family history studies,5,6 and animal stroke model studies have suggested that stroke is partly due to genetic influences.7 Stroke is believed to occur due to consequence of multifactorial and polygenic influences whereby multiple genes exert a small influence or risk on phenotype.7,8 Methylenetetrahydrofolate reductase (MTHFR) catalyses the reduction of 5, 10-methylenetetrahydrofolate Correspondence to: Amit Kumar, Department of Neurology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi, India. Email: [email protected]
ß W. S. Maney & Son Ltd 2014 DOI 10.1179/1743132815Y.0000000008
to 5-methylenetetrahydrofolate, which is the main form of folate in plasma and a carbon donor for the remethylation of homocysteine (Hcy) to methionine.9 A single base pair (677C/T) transition in the MTHFR gene changes the alanine to valine amino acid residue, which influences enzyme thermolability, decreased activity and, in turn the elevated level of Hcy. Several studies have shown that an elevated level of Hcy is a risk factor for stroke.10 A recent study has suggested that hyperhomocysteinemia is associated with small vessel disease stroke (SVD) and large vessel disease stroke (LVD).11 Thus, MTHFR polymorphism is suggested to be a genetic risk factor in cerebrovascular diseases, including ischemic stroke.12 Studies on association between MTHFR polymorphism and risk of ischemic stroke have shown conflicting results.10 Meta-analysis can be a powerful tool that
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can enable pooling data from smaller inconclusive studies, yield it with a greater statistical power, and allow quantifying precise estimation of hidden or unclear association. However, there is concern about the risk of false positives due to publication bias, incomplete searches of the literature, and an insufficient assessment of the methodological quality of the included studies. Therefore, we have taken these concerns and performed the largest meta-analysis of studies that have been published to determine the precise association between 677C/T polymorphism of MTHFR gene and ischemic stroke.
Materials and Methods Literature search Electronic databases (Medline, EMBASE, and Google Scholar) were searched until September 2014 for all case–control studies evaluating MTHFR C677T gene polymorphism and ischemic stroke in humans. All published manuscripts including letters, previous meta-analyses, and abstracts were searched. The retrieved studies were examined thoroughly to assess their appropriateness for inclusion in our study. Search term and text words used for the search were ‘MTHFR polymorphism,’ ‘ischemic stroke,’ and ‘cerebrovascular disorder.’ The search results were limited to human. All languages were searched initially, but only articles in English language were selected. The references of all computer-identified publications were searched for any additional studies, and the MEDLINE option-related articles were used for all the relevant articles.
Inclusion and exclusion criteria Studies were included if: (a) study design was case– control and (b) had confirmed diagnosis of ischemic stroke with magnetic resonance imaging (MRI) or computed tomography (CT). Studies were excluded if (a) the associations between MTHFR polymorphism and stroke in children were investigated, (b) genotype frequencies were not reported, (c) quantitative traits or intermediate phenotypes were investigated, and (d) study design was other than case–control.
Data extraction and quality assessment The primary search generated 108 potentially relevant articles, of which 38 met the inclusion criteria. Data for analysis were extracted independently and entered into separate databases by two reviewers (AK and PK). The results were compared and disagreements were resolved by consensus. A standardized data collection form was used for data extraction; this form mainly included the following content: (i) name of the first author, year of publication, country, and racial descent; (ii) demographics, number of cases and controls, and source of cases and controls; (iii) distribution of genotypes and alleles; (iv) genotyping method; (v) Hardy–Weinberg equilibrium
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(HWE); and (vi) statistical methods used for association assessment. We also evaluated the methodological quality of each study, which is included in our analysis using a quality assessment scale developed for genetic association studies,13 which was modified by us to increase the relevance of our study. This scale took into account both traditional epidemiologic considerations and genetic issues.13 The scores ranged from 0 (bad) to 16 (best). Details of the scale are presented in Table 1. We performed subgroup analyses including ethnicity (Asian and Caucasian) and methodological quality (High and Low) of studies included in the meta-analysis.
Statistical analysis Data were analyzed using software Review Manager, version 5; Cochrane Collaboration, Syracuse, NY, USA. For MTHFR C677T gene variant, a pooled odds ratio (OR) and 95% confidence interval (CI) were calculated. Heterogeneity was examined using Higgins I-squared. Fixed effects model were used if Isquared statistic was ,50%, otherwise random effects Table 1 Scale for methodological quality assessment Criteria Representativeness of cases Selected from any population disease registry or multiple center sites Selected from any cardiology/neurology Not described Source of controls Population or neighbor based Hospital based Healthy volunteers with total description Healthy volunteers without total description Not described Matching of controls Age and sex match Not matched Definition of variables Yes No Ascertainment of IS Adequate confirmation Diagnosis of IS by patient medical record Not described Ascertainment of controls Stroke frees status by using appropriate QVSS or CT/MRI Not described Genotyping under condition Genotyping done under blinded conditions Unblinded or not mentioned Genotyping method DNA sequencing/multiplex PCR/DHPLC PCR-RFLP with sequencing confirmation PCR-RFLP Hardy–Weinberg equilibrium (HWE) Allelic frequency in accordance HWE Not HWE but followed statistics to adjust confounding Not Checked Association assessment Appropriate statistics and examining confounders and effect modifiers Appropriate statistics not used
Score
2 1 0 3 2 1 0.5 0 1 0 1 0 2 1 0 1 0 1 0 3 2 1 2 1 0 1 0
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Figure 1 Study flow diagram.
model was used.
Results Study characteristics Thirty-eight studies, including 6310 ischemic stroke patients, 8297 controls conducted from the period of 1997–2012 were included in our meta-analysis. Age and sex matching was described in 24 studies. There were 18 Studies of Asian population and 20 from Caucasian population. The search results in the flow diagram are summarized in Fig. 1 and the characteristics of included studies are listed in Table 2.
Meta-analysis results The combined results of all studies showed that the MTHFR 677T allele increased the risk of ischemic stroke under the dominant model (TT z CT vs CC: OR, 1.09; 95% CI, 1.06–1.12, P , 0.001) (Fig. 2A) and recessive model (TT vs CC z CT: OR, 1.31; 95% CI, 1.19–1.44, P , 0.001) (Fig. 2B). We stratified the data by studies reported from Caucasian and Asian population. In Asian population, significant association between the MTHFR gene polymorphism and risk of ischemic stroke was observed
under both the dominant model of inheritance (OR, 1.36; 95% CI, 1.23–1.49) (Fig. 3A) and recessive model of inheritance (OR, 1.29; 95% CI, 1.15–1.45) (Fig. 3B). Pooling data of studies reported from the Caucasian population showed that there was borderline association between MTHFR gene polymorphism and risk of ischemic stroke under a dominant model (OR, 1.05; 95% CI, 0.99–1.10) (Fig. 4A), but there was significant association under the recessive mode of inheritance (OR, 1.33; 95% CI, 1.13–1.58) (Fig. 4B). Analysis stratified by quality of studies suggested a higher risk in low-quality studies (OR, 1.34; 95% CI, 1.20–1.50) than high-quality study (OR, 1.07; 95% CI, 0.99–1.16) under the dominant mode of inheritance. Recessive mode of inheritance suggested that there was similar risk of stroke in both low-quality studies (OR, 1.10; 95% CI, 1.06–1.13) and highquality studies (OR, 1.24; 95% CI, 1.03–1.49) with MTHFR gene polymorphism.
Publication bias The shape of the funnel plot seemed symmetrical suggesting that there was no significant publication bias (Fig. 5).
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Table 2 Characteristics of studies included in the meta-analysis on association between methylenetetrahydrofolate reductase (MTHFR) C 677 T polymorphism and ischemic stroke
S. No.
4
Year
Study
1997
H. S. Markus
U.K.
2004
F. Ucar
2001
Sample size
Origin
Hardy– Weinberg equilibrium (HWE)
Matching criteria
345/161
Yes
Turkey
30/242
No
G. Zhang
China
102/100
Yes
–
1998
V. Destefano
Italy
72/198
No
–
2000
J. W. Eikelboom
Australia
219/205
Yes
Age/sex
2003
Zhaohui Li
China
513/1832
No
Age
2001
Y. Wu
Japan
77/229
Yes
–
2001
E. Topic
Croatia
92/124
No
Age/sex
1999
D. L. Harmon
Ireland
174/183
Yes
–
1999
R. D. Press
Portland
136/52
Yes
–
1998
K. Kostulas
Sweden
126/126
Yes
Age
2003
B. O. Choi
China
195/198
Yes
Age/sex
2005
R. V. Alluri
India
75/49
Yes
–
2004
L. Baum
China
241/304
Yes
Age/sex
2011
T. P. They-They
Morocco
91/182
Yes
Age/sex
2010
Ion Bon Han
Korea
264/234
Yes
–
2009
Al-Allawi Nasir
Iraq
70/50
Yes
Age/sex
2006
Sazki Ali
Turkey
92/259
Yes
Age/sex
1997
H. Morita
Japan
256/325
Yes
–
2005
A. Pezzini
Italy
163/158
Yes
Age/sex
2005
X. Gao
China
100/100
Yes
Age/sex
2012
Xiong Lihui
China
89/102
Yes
–
2008
C. Shi
China
95/99
Yes
Age/sex
2002
R. Grossmann
Germany
93/186
No
Age/sex
1998
N. Salooja
U.K.
271/173
Yes
Age/sex
2005
A. J. C. Slooter
Netherland
193/767
Yes
Age/sex
2011
B. I. Somarajan
India
215/188
Yes
Age/sex
2008
Zhang Yan
China
245/282
Yes
Age/sex
2006
M. P. Harmans
Belgium
23/142
Yes
Age/sex
2006
A. Pezzini
Italy
174/155
Yes
Age/sex
2002
A. Pezzini
Italy
31/36
Yes
Age/sex
2006
P. Inusha
India
32/60
Yes
Age/sex
1999
W. Lalouschek
Austria
96/96
No
Age/sex
2007
K. S. Shinjo
Brazil
127/126
No
Age
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Male/female
Age
Quality score
209/136 78/83 19/11 182/60 56/46 70/30 35/37 78/120 140/79 131/64 326/187 1051/781 49/28 120/109 62/30
65.7 z 10.5 12/16 65.4 z 9.5 50 z 12 5/16 42 z 13 65(30–78) 7.5/16 50(22–70) 33.9 7/16 49 66.1 z 12.4 12/16 67.0 z 11.8 61.z 8.5 10/16 59.6 z 8.8 61.4z 6.8 9/16 59.6 z 6.9 64 z 17 3.5/16
82/92 46/137 122/14 30/22 – – 106/89 111/87 65/10
78.8 8/16 72.9 65 z 8 6/16 67 z 9 – 9/16 – 61.4 z 10.9 11/16 60.8 z 12.2 – 6.5/16 – 70.7z 12 12/16 70.8 z 5.7 49 z 5 10/16 46 z 11 59.5 z 11 9/16 62.3 z 11 M 60 12/16 M 62.0 53 z 9 7.5/16 56 z 15 70 z 8 7/16 67.7 z 7.5 35 z 7.5 11/16 34.8 z 6.1 61z 10 14/16 60 z 10 68.6 z 10 9/16 67.5 z 9.9 38.7 z 6.4 11/16 38.6 z 5.9 36(16–49) 9/16 33(18–63) 68(22–94) 12/16 69(23–93) 38.6 12/16 38.7 54 z 15.9 11/16 55 z 10.9 8/16
131/110 137/167 46/45 94/88 124/143 116/118 38/32 26/24 67/53 144/115 122/134 175/150 84/79 85/73 71/29 71/29 64/25 24/28 79/18 80/19 46/47 92/94 141/130 91/82 0/193 0/767 152/55 126/62 146/109 106/176 16/7 82/60 89/85 83/72 20/11 23/13 22/10 58/38 58/38 53/74 72/54 20/21
71.7(9.3) 67.1(11.3) 35(7.3) 34(5.9) 42.3 z 11.2 39.9 z 4.2
7/16 12/16 7/16 7/16
64.4 z 13.1 62.4 z 11.7 63.5 64 35.5 z 6.5
7/16 7/16
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S. No.
C677T polymorphism and ischemic stroke association
Continued
Sample size
Hardy– Weinberg equilibrium (HWE)
Ireland
64/71
No
Age
R. Kawamoto
Japan
97/241
Yes
Age/sex
2009
A. Biswas
India
120/120
Yes
Age/sex
2011
Mejia Mohamed
Malaysia
150/142
Yes
Age/sex
Year
Study
2002
S. P. Mcllroy
2005
Origin
Discussion The present meta-analysis of association between MTHFR C677T polymorphism and risk of ischemic stroke has shown that MTHFR 677C/T polymorphism significantly contributes in increasing the risk of ischemic stroke. Several studies have been conducted to determine the association between MTHFR C677T polymorphism and ischemic stroke; however, the conclusion remains controversial. In order to determine the more precise estimation of this association, we performed a metaanalysis of earlier published studies, which included 38 studies involving 5513 cases and 8297 controls, along with assessing the methodological quality of studies. The finding of present meta-analysis on the association between MTHFR C677T polymorphism and risk of ischemic stroke are in agreement with the previously carried out meta-analysis, which included 19 case–control studies involving 2223 cases and 2936 controls where this polymorphism was found to be potentially associated with the risk of ischemic stroke.14 The strength of association was stronger in the recessive model (OR 1.66) than the dominant model (OR 1.25) representing predominant recessive pattern of association between MTHFR C677T polymorphism and risk of ischemic stroke. The association between MTHFR polymorphism and ischemic stroke under the dominant model of inheritance is still controversial. It is unlikely that any polymorphism to a complex trait act in a simple Mendelian dominant fashion.15 To determine the genetic differences between the two ethnic groups, we did a stratified analysis between Asian and Caucasian population. The present metaanalysis suggests a significant association between MTHFR C677T polymorphism and ischemic stroke in both the populations; however, strength of association was higher in Asian as compared to Caucasian Population. This result was consistent with the study published by Li.14 A recent meta-analysis, which only included published studies from Chinese population, demonstrated a significant association between
Matching criteria
Male/female 37/27 14/57 65/32 152/89 – – 92/58 71/71
Age 73.8 z 8.1 74.3 z 7.6 78 z 8.3 76 z 7.1 – – 60 z 10.1 60.6 z 7.1
Quality score 4.5/16 8/16 8/16 9/16
MTHFR C677T polymorphism and risk of ischemic stroke.16 A study suggested that level of Hcy was higher in low folate intake region like Asia than in an area of folate fortification.17 The findings of our study have clear implication for the policy makers to design programs to enable the increase in the intake of folate to reduce the risk of ischemic stroke in Asian Countries. A meta-analysis suggested that MTHFR A1298C polymorphism increased the susceptibility to ischemic stroke in Asian population but not in Caucasian population.18 Earlier studies on MTHFR polymorphism in ischemic stroke have suggested that strength of association between MTHFR polymorphism and coronary artery disease is weaker in European population than Asian population, which might be partially attributable to difference in folate intake between the two ethnic groups.18 Another study demonstrated the significant association between elevated plasma Hcy levels and TT genotype of MTHFR C677T polymorphism in healthy South Asians.19 Meta-analysis of studies on association between MTHFR C677T polymorphism and hemorrhagic stroke also suggested that MTHFR polymorphism is a risk factor for hemorrhagic stroke.20 A meta-analysis of 14 studies, conducted by SareckaHujar et al. showed that TT genotype of MTHFR gene was more common in pediatric stroke patient than controls (OR 1.57, 95% CI 1.02–2.41).21 Another meta-analysis, which included 19 case–control studies involving 2223 cases and 2936 controls also suggested that MTHFR C677T polymorphism was significantly associated with increased risk of ischemic stroke.14 The mechanism of association between MTHFR polymorphism and ischemic stroke is not clear; however, studies have shown that a single base pair replacement of Cytosine nucleotide with Thymine at 677 nucleotide position of MTHFR gene influences enzyme thermolability, its decreased activity and, in turn, the elevated level of Hcy, an endothelial toxin especially in the presence of low folate levels22 and in effect to elevated risk of stroke.9,12,20 Elevated level of Hcy may lead to endothelial dysfunction, an early stage for the initiation of atherosclerosis.12 Vitamin
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Figure 2 (A) Forest plot and pooled ORs of risk from studies investigating MTHFR C677T polymorphism and ischemic stroke under Dominant model; (B) Forest plot and pooled ORs of risk from studies investigating MTHFR C677T polymorphism and ischemic stroke under Recessive Model.
intervention may bring down the higher level of Hcy.23 A study showed lower Folate levels in patient MI and CAD.23 A meta-analysis of 72 studies showed significant association between higher plasma Hcy level and MTHFR gene polymorphism with stroke, ischemic heart disease, and pulmonary embolism.24 A
meta-analysis, which involved 72 case–control studies reported an OR of 1.42 for 5 mmol/l and increase in Hcy level. A meta-analysis suggested a mean difference in serum Hcy between TT and CC genotype of 2.7 mmol/L.25 One genome wide association study also suggested that there was strong association
Figure 3 (A) Asian population under the Dominant model of inheritance; (B) Asian population under the recessive model of inheritance.
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Figure 4 (A) Caucasian population under the Dominant model of inheritance; (B) Caucasian population under the recessive model of inheritance.
between MTHFR variant and Hcy in specific subgroup of subjects.26 In Europe and North America, no significant association between MTHFR polymorphism and Hcy level was observed in contrast to Middle East and Japan.27 Recent studies have also suggested that an elevated level of Hcy might be a risk factor for ischemic stroke.
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The frequency of polymorphic alleles has been observed to vary among different ethnic groups of the world.28–30 According to Fowler,31 the frequency of homozygous variant of MTHFR gene was present in 5–18% of population. Our meta-analysis suggests that frequency distribution of homozygous TT genotype was present in 23.5% in Asian and 12.8%
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from the hospital in the included study, which may have led to selection bias in the selection of controls.
Conclusion
Figure 5 Funnel plot for assessing publication bias.
in Caucasian population. Heterogeneity is a potential issue in the interpretation of results of metaanalyses.32 Higher value of I2 statistic represents a higher degree of heterogeneity. Our study suggested a moderate degree of heterogeneity, 46% under dominant model and 21% under the recessive model. Moderate heterogeneity in Asian population (I2 49% in dominant model and I2 33% in recessive model) was observed; however, in Caucasian population relatively low heterogeneity (I2 5 22% under dominant model and I2 5 4% under recessive model) was observed. Quality of study determines precise estimation of the association.13 Some studies follow the appropriate methodological guidelines; however, others fail to follow the methodological guidelines, which may lead to conflicting results. Therefore, we decided to stratify the data based on the methodological quality of studies. High-quality studies suggest moderately weak association than poor quality studies.
Limitation of study A meta-analysis based on the quality of study is an ideal method for improving the effect of association between the risk factors and outcome; however, several factors can influence the results of metaanalysis. There are many limitations in this metaanalysis. The result of the present meta-analysis could be influenced by lack of known environment and unstudied genetic factors. In our meta-analysis, there was no haplotype analysis, which plays a crucial role for association studies of complex diseases. The interaction between gene and environment and gene–gene interaction was not studied in this metaanalysis. Some studies included in the meta-analysis had a small sample size, which may have provided inconsistent results due to low statistical power. Stroke risk varies as per specific subtypes of stroke; however, most of the studies included in the metaanalysis have not presented the data as per subtype of stroke, therefore, meta-analysis based on association between MTHFR polymorphism and subtype of stroke has not been done. Controls were included
Evidence from this meta-analysis, which included 38 studies on the association between MTHFR C677T polymorphism and ischemic stroke, sustains the notion of association between polymorphism of MTHFR gene and susceptibility to ischemic stroke mainly in Asian population. This largest metaanalysis has an important significance suggesting that MTHFR genotyping may be helpful for identification of patients, susceptible for risk of stroke, prevention, and management of stroke. The findings of our study have clear implication for the policy makers to design programs to enable the increase in the intake of folate to reduce the risk of ischemic stroke in Asian Countries. Larger prospective studies with correction for multiple comparisons are required to confirm the findings.
Disclaimer Statements Contributors AK has written the manuscript. PK and MP extracted the data. RS, AKP, and AP have helped in writing draft of manuscript. Funding Conflicts of interest None. Ethics approval Not required.
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22 Tokgo¨zog˘lu SL, Alikas¸ifog˘lu M, Unsal I, Atalar E, Aytemir K, Ozer N, et al. Methylene tetrahydrofolate reductase genotype and the risk and extent of coronary artery disease in a population with low plasma folate. Heart. 1999;81:518–22. 23 Cattaneo M. Hyperhomocysteinemia, atherosclerosis and thrombosis. Thromb Haemost. 1999;81:165–76. 24 Faraci FM. Hyperhomocysteinemia: a million ways to lose control. Arterioscler Thromb Vasc Biol. 2003;23:371–3. 25 Wald DS, Law M, Morris JK. The dose-response relation between serum homocysteine and cardiovascular disease: implications for treatment and screening. Eur J Cardiovasc Prev Rehabil. 2004;11:250–3. 26 Lange LA, Croteau-Chonka DC, Marvelle AF, Qin L, Gaulton KJ, Kuzawa CW, et al. Genome-wide association study of homocysteine levels in Filipinos provides evidence for CPS1 in women and a stronger MTHFR effect in young adults. Hum Mol Genet. 2010;19:2050–8. 27 Lewis SJ, Ebrahim S, Davey Smith G. Meta-analysis of MTHFR 677C-.T polymorphism and coronary heart disease: does totality of evidence support causal role for homocysteine and preventive potential of folate? BMJ. 2005;331:1053. 28 Angchaisuksiri P, Pingsuthiwong S, Sura T, Aryuchai K, Busabaratana M, Atichartakarn V. Prevalence of the C677T methylenetetra-hydrofolate reductase mutation in Thai patients with deep vein thrombosis. Acta Haematol. 2000;103:191–6. 29 Arruda VR, Siqueira LH, Gonc¸alves MS, von Zuben PM, Soares MC, Menezes R, et al. Prevalence of the mutation C677 –. T in the methylene tetrahydrofolate reductase gene among distinct ethnic groups in Brazil. Am J Med Genet. 1998;78: 332–5. 30 Rady PL, Tyring SK, Hudnall SD, Vargas T, Kellner LH, Nitowsky H, et al. Methylenetetrahydrofolate reductase (MTHFR): the incidence of mutations C677T and A1298C in the Ashkenazi Jewish population. Am J Med Genet. 1999;86:380–4. 31 Fowler B. Disorders of homocysteine metabolism. J Inherit Metab Dis. 1997;20:270–85. 32 Munafo` MR, Flint J. Meta-analysis of genetic association studies. Trends Genet. 2004;20:439–44.
Introduction Stroke has emerged as the second most common cause of mortality worldwide and is a major public health problem. Stroke accounted for nearly 5.7 million deaths worldwide in 2005.1 More than two-thirds of these deaths occurred in underdeveloped countries.2 Substantial evidence from the twin studies,3,4 family history studies,5,6 and animal stroke model studies have suggested that stroke is partly due to genetic influences.7 Stroke is believed to occur due to consequence of multifactorial and polygenic influences whereby multiple genes exert a small influence or risk on phenotype.7,8 Methylenetetrahydrofolate reductase (MTHFR) catalyses the reduction of 5, 10-methylenetetrahydrofolate Correspondence to: Amit Kumar, Department of Neurology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi, India. Email: [email protected]
ß W. S. Maney & Son Ltd 2014 DOI 10.1179/1743132815Y.0000000008
to 5-methylenetetrahydrofolate, which is the main form of folate in plasma and a carbon donor for the remethylation of homocysteine (Hcy) to methionine.9 A single base pair (677C/T) transition in the MTHFR gene changes the alanine to valine amino acid residue, which influences enzyme thermolability, decreased activity and, in turn the elevated level of Hcy. Several studies have shown that an elevated level of Hcy is a risk factor for stroke.10 A recent study has suggested that hyperhomocysteinemia is associated with small vessel disease stroke (SVD) and large vessel disease stroke (LVD).11 Thus, MTHFR polymorphism is suggested to be a genetic risk factor in cerebrovascular diseases, including ischemic stroke.12 Studies on association between MTHFR polymorphism and risk of ischemic stroke have shown conflicting results.10 Meta-analysis can be a powerful tool that
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can enable pooling data from smaller inconclusive studies, yield it with a greater statistical power, and allow quantifying precise estimation of hidden or unclear association. However, there is concern about the risk of false positives due to publication bias, incomplete searches of the literature, and an insufficient assessment of the methodological quality of the included studies. Therefore, we have taken these concerns and performed the largest meta-analysis of studies that have been published to determine the precise association between 677C/T polymorphism of MTHFR gene and ischemic stroke.
Materials and Methods Literature search Electronic databases (Medline, EMBASE, and Google Scholar) were searched until September 2014 for all case–control studies evaluating MTHFR C677T gene polymorphism and ischemic stroke in humans. All published manuscripts including letters, previous meta-analyses, and abstracts were searched. The retrieved studies were examined thoroughly to assess their appropriateness for inclusion in our study. Search term and text words used for the search were ‘MTHFR polymorphism,’ ‘ischemic stroke,’ and ‘cerebrovascular disorder.’ The search results were limited to human. All languages were searched initially, but only articles in English language were selected. The references of all computer-identified publications were searched for any additional studies, and the MEDLINE option-related articles were used for all the relevant articles.
Inclusion and exclusion criteria Studies were included if: (a) study design was case– control and (b) had confirmed diagnosis of ischemic stroke with magnetic resonance imaging (MRI) or computed tomography (CT). Studies were excluded if (a) the associations between MTHFR polymorphism and stroke in children were investigated, (b) genotype frequencies were not reported, (c) quantitative traits or intermediate phenotypes were investigated, and (d) study design was other than case–control.
Data extraction and quality assessment The primary search generated 108 potentially relevant articles, of which 38 met the inclusion criteria. Data for analysis were extracted independently and entered into separate databases by two reviewers (AK and PK). The results were compared and disagreements were resolved by consensus. A standardized data collection form was used for data extraction; this form mainly included the following content: (i) name of the first author, year of publication, country, and racial descent; (ii) demographics, number of cases and controls, and source of cases and controls; (iii) distribution of genotypes and alleles; (iv) genotyping method; (v) Hardy–Weinberg equilibrium
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(HWE); and (vi) statistical methods used for association assessment. We also evaluated the methodological quality of each study, which is included in our analysis using a quality assessment scale developed for genetic association studies,13 which was modified by us to increase the relevance of our study. This scale took into account both traditional epidemiologic considerations and genetic issues.13 The scores ranged from 0 (bad) to 16 (best). Details of the scale are presented in Table 1. We performed subgroup analyses including ethnicity (Asian and Caucasian) and methodological quality (High and Low) of studies included in the meta-analysis.
Statistical analysis Data were analyzed using software Review Manager, version 5; Cochrane Collaboration, Syracuse, NY, USA. For MTHFR C677T gene variant, a pooled odds ratio (OR) and 95% confidence interval (CI) were calculated. Heterogeneity was examined using Higgins I-squared. Fixed effects model were used if Isquared statistic was ,50%, otherwise random effects Table 1 Scale for methodological quality assessment Criteria Representativeness of cases Selected from any population disease registry or multiple center sites Selected from any cardiology/neurology Not described Source of controls Population or neighbor based Hospital based Healthy volunteers with total description Healthy volunteers without total description Not described Matching of controls Age and sex match Not matched Definition of variables Yes No Ascertainment of IS Adequate confirmation Diagnosis of IS by patient medical record Not described Ascertainment of controls Stroke frees status by using appropriate QVSS or CT/MRI Not described Genotyping under condition Genotyping done under blinded conditions Unblinded or not mentioned Genotyping method DNA sequencing/multiplex PCR/DHPLC PCR-RFLP with sequencing confirmation PCR-RFLP Hardy–Weinberg equilibrium (HWE) Allelic frequency in accordance HWE Not HWE but followed statistics to adjust confounding Not Checked Association assessment Appropriate statistics and examining confounders and effect modifiers Appropriate statistics not used
Score
2 1 0 3 2 1 0.5 0 1 0 1 0 2 1 0 1 0 1 0 3 2 1 2 1 0 1 0
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Figure 1 Study flow diagram.
model was used.
Results Study characteristics Thirty-eight studies, including 6310 ischemic stroke patients, 8297 controls conducted from the period of 1997–2012 were included in our meta-analysis. Age and sex matching was described in 24 studies. There were 18 Studies of Asian population and 20 from Caucasian population. The search results in the flow diagram are summarized in Fig. 1 and the characteristics of included studies are listed in Table 2.
Meta-analysis results The combined results of all studies showed that the MTHFR 677T allele increased the risk of ischemic stroke under the dominant model (TT z CT vs CC: OR, 1.09; 95% CI, 1.06–1.12, P , 0.001) (Fig. 2A) and recessive model (TT vs CC z CT: OR, 1.31; 95% CI, 1.19–1.44, P , 0.001) (Fig. 2B). We stratified the data by studies reported from Caucasian and Asian population. In Asian population, significant association between the MTHFR gene polymorphism and risk of ischemic stroke was observed
under both the dominant model of inheritance (OR, 1.36; 95% CI, 1.23–1.49) (Fig. 3A) and recessive model of inheritance (OR, 1.29; 95% CI, 1.15–1.45) (Fig. 3B). Pooling data of studies reported from the Caucasian population showed that there was borderline association between MTHFR gene polymorphism and risk of ischemic stroke under a dominant model (OR, 1.05; 95% CI, 0.99–1.10) (Fig. 4A), but there was significant association under the recessive mode of inheritance (OR, 1.33; 95% CI, 1.13–1.58) (Fig. 4B). Analysis stratified by quality of studies suggested a higher risk in low-quality studies (OR, 1.34; 95% CI, 1.20–1.50) than high-quality study (OR, 1.07; 95% CI, 0.99–1.16) under the dominant mode of inheritance. Recessive mode of inheritance suggested that there was similar risk of stroke in both low-quality studies (OR, 1.10; 95% CI, 1.06–1.13) and highquality studies (OR, 1.24; 95% CI, 1.03–1.49) with MTHFR gene polymorphism.
Publication bias The shape of the funnel plot seemed symmetrical suggesting that there was no significant publication bias (Fig. 5).
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Table 2 Characteristics of studies included in the meta-analysis on association between methylenetetrahydrofolate reductase (MTHFR) C 677 T polymorphism and ischemic stroke
S. No.
4
Year
Study
1997
H. S. Markus
U.K.
2004
F. Ucar
2001
Sample size
Origin
Hardy– Weinberg equilibrium (HWE)
Matching criteria
345/161
Yes
Turkey
30/242
No
G. Zhang
China
102/100
Yes
–
1998
V. Destefano
Italy
72/198
No
–
2000
J. W. Eikelboom
Australia
219/205
Yes
Age/sex
2003
Zhaohui Li
China
513/1832
No
Age
2001
Y. Wu
Japan
77/229
Yes
–
2001
E. Topic
Croatia
92/124
No
Age/sex
1999
D. L. Harmon
Ireland
174/183
Yes
–
1999
R. D. Press
Portland
136/52
Yes
–
1998
K. Kostulas
Sweden
126/126
Yes
Age
2003
B. O. Choi
China
195/198
Yes
Age/sex
2005
R. V. Alluri
India
75/49
Yes
–
2004
L. Baum
China
241/304
Yes
Age/sex
2011
T. P. They-They
Morocco
91/182
Yes
Age/sex
2010
Ion Bon Han
Korea
264/234
Yes
–
2009
Al-Allawi Nasir
Iraq
70/50
Yes
Age/sex
2006
Sazki Ali
Turkey
92/259
Yes
Age/sex
1997
H. Morita
Japan
256/325
Yes
–
2005
A. Pezzini
Italy
163/158
Yes
Age/sex
2005
X. Gao
China
100/100
Yes
Age/sex
2012
Xiong Lihui
China
89/102
Yes
–
2008
C. Shi
China
95/99
Yes
Age/sex
2002
R. Grossmann
Germany
93/186
No
Age/sex
1998
N. Salooja
U.K.
271/173
Yes
Age/sex
2005
A. J. C. Slooter
Netherland
193/767
Yes
Age/sex
2011
B. I. Somarajan
India
215/188
Yes
Age/sex
2008
Zhang Yan
China
245/282
Yes
Age/sex
2006
M. P. Harmans
Belgium
23/142
Yes
Age/sex
2006
A. Pezzini
Italy
174/155
Yes
Age/sex
2002
A. Pezzini
Italy
31/36
Yes
Age/sex
2006
P. Inusha
India
32/60
Yes
Age/sex
1999
W. Lalouschek
Austria
96/96
No
Age/sex
2007
K. S. Shinjo
Brazil
127/126
No
Age
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History of risk factor
Male/female
Age
Quality score
209/136 78/83 19/11 182/60 56/46 70/30 35/37 78/120 140/79 131/64 326/187 1051/781 49/28 120/109 62/30
65.7 z 10.5 12/16 65.4 z 9.5 50 z 12 5/16 42 z 13 65(30–78) 7.5/16 50(22–70) 33.9 7/16 49 66.1 z 12.4 12/16 67.0 z 11.8 61.z 8.5 10/16 59.6 z 8.8 61.4z 6.8 9/16 59.6 z 6.9 64 z 17 3.5/16
82/92 46/137 122/14 30/22 – – 106/89 111/87 65/10
78.8 8/16 72.9 65 z 8 6/16 67 z 9 – 9/16 – 61.4 z 10.9 11/16 60.8 z 12.2 – 6.5/16 – 70.7z 12 12/16 70.8 z 5.7 49 z 5 10/16 46 z 11 59.5 z 11 9/16 62.3 z 11 M 60 12/16 M 62.0 53 z 9 7.5/16 56 z 15 70 z 8 7/16 67.7 z 7.5 35 z 7.5 11/16 34.8 z 6.1 61z 10 14/16 60 z 10 68.6 z 10 9/16 67.5 z 9.9 38.7 z 6.4 11/16 38.6 z 5.9 36(16–49) 9/16 33(18–63) 68(22–94) 12/16 69(23–93) 38.6 12/16 38.7 54 z 15.9 11/16 55 z 10.9 8/16
131/110 137/167 46/45 94/88 124/143 116/118 38/32 26/24 67/53 144/115 122/134 175/150 84/79 85/73 71/29 71/29 64/25 24/28 79/18 80/19 46/47 92/94 141/130 91/82 0/193 0/767 152/55 126/62 146/109 106/176 16/7 82/60 89/85 83/72 20/11 23/13 22/10 58/38 58/38 53/74 72/54 20/21
71.7(9.3) 67.1(11.3) 35(7.3) 34(5.9) 42.3 z 11.2 39.9 z 4.2
7/16 12/16 7/16 7/16
64.4 z 13.1 62.4 z 11.7 63.5 64 35.5 z 6.5
7/16 7/16
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S. No.
C677T polymorphism and ischemic stroke association
Continued
Sample size
Hardy– Weinberg equilibrium (HWE)
Ireland
64/71
No
Age
R. Kawamoto
Japan
97/241
Yes
Age/sex
2009
A. Biswas
India
120/120
Yes
Age/sex
2011
Mejia Mohamed
Malaysia
150/142
Yes
Age/sex
Year
Study
2002
S. P. Mcllroy
2005
Origin
Discussion The present meta-analysis of association between MTHFR C677T polymorphism and risk of ischemic stroke has shown that MTHFR 677C/T polymorphism significantly contributes in increasing the risk of ischemic stroke. Several studies have been conducted to determine the association between MTHFR C677T polymorphism and ischemic stroke; however, the conclusion remains controversial. In order to determine the more precise estimation of this association, we performed a metaanalysis of earlier published studies, which included 38 studies involving 5513 cases and 8297 controls, along with assessing the methodological quality of studies. The finding of present meta-analysis on the association between MTHFR C677T polymorphism and risk of ischemic stroke are in agreement with the previously carried out meta-analysis, which included 19 case–control studies involving 2223 cases and 2936 controls where this polymorphism was found to be potentially associated with the risk of ischemic stroke.14 The strength of association was stronger in the recessive model (OR 1.66) than the dominant model (OR 1.25) representing predominant recessive pattern of association between MTHFR C677T polymorphism and risk of ischemic stroke. The association between MTHFR polymorphism and ischemic stroke under the dominant model of inheritance is still controversial. It is unlikely that any polymorphism to a complex trait act in a simple Mendelian dominant fashion.15 To determine the genetic differences between the two ethnic groups, we did a stratified analysis between Asian and Caucasian population. The present metaanalysis suggests a significant association between MTHFR C677T polymorphism and ischemic stroke in both the populations; however, strength of association was higher in Asian as compared to Caucasian Population. This result was consistent with the study published by Li.14 A recent meta-analysis, which only included published studies from Chinese population, demonstrated a significant association between
Matching criteria
Male/female 37/27 14/57 65/32 152/89 – – 92/58 71/71
Age 73.8 z 8.1 74.3 z 7.6 78 z 8.3 76 z 7.1 – – 60 z 10.1 60.6 z 7.1
Quality score 4.5/16 8/16 8/16 9/16
MTHFR C677T polymorphism and risk of ischemic stroke.16 A study suggested that level of Hcy was higher in low folate intake region like Asia than in an area of folate fortification.17 The findings of our study have clear implication for the policy makers to design programs to enable the increase in the intake of folate to reduce the risk of ischemic stroke in Asian Countries. A meta-analysis suggested that MTHFR A1298C polymorphism increased the susceptibility to ischemic stroke in Asian population but not in Caucasian population.18 Earlier studies on MTHFR polymorphism in ischemic stroke have suggested that strength of association between MTHFR polymorphism and coronary artery disease is weaker in European population than Asian population, which might be partially attributable to difference in folate intake between the two ethnic groups.18 Another study demonstrated the significant association between elevated plasma Hcy levels and TT genotype of MTHFR C677T polymorphism in healthy South Asians.19 Meta-analysis of studies on association between MTHFR C677T polymorphism and hemorrhagic stroke also suggested that MTHFR polymorphism is a risk factor for hemorrhagic stroke.20 A meta-analysis of 14 studies, conducted by SareckaHujar et al. showed that TT genotype of MTHFR gene was more common in pediatric stroke patient than controls (OR 1.57, 95% CI 1.02–2.41).21 Another meta-analysis, which included 19 case–control studies involving 2223 cases and 2936 controls also suggested that MTHFR C677T polymorphism was significantly associated with increased risk of ischemic stroke.14 The mechanism of association between MTHFR polymorphism and ischemic stroke is not clear; however, studies have shown that a single base pair replacement of Cytosine nucleotide with Thymine at 677 nucleotide position of MTHFR gene influences enzyme thermolability, its decreased activity and, in turn, the elevated level of Hcy, an endothelial toxin especially in the presence of low folate levels22 and in effect to elevated risk of stroke.9,12,20 Elevated level of Hcy may lead to endothelial dysfunction, an early stage for the initiation of atherosclerosis.12 Vitamin
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Figure 2 (A) Forest plot and pooled ORs of risk from studies investigating MTHFR C677T polymorphism and ischemic stroke under Dominant model; (B) Forest plot and pooled ORs of risk from studies investigating MTHFR C677T polymorphism and ischemic stroke under Recessive Model.
intervention may bring down the higher level of Hcy.23 A study showed lower Folate levels in patient MI and CAD.23 A meta-analysis of 72 studies showed significant association between higher plasma Hcy level and MTHFR gene polymorphism with stroke, ischemic heart disease, and pulmonary embolism.24 A
meta-analysis, which involved 72 case–control studies reported an OR of 1.42 for 5 mmol/l and increase in Hcy level. A meta-analysis suggested a mean difference in serum Hcy between TT and CC genotype of 2.7 mmol/L.25 One genome wide association study also suggested that there was strong association
Figure 3 (A) Asian population under the Dominant model of inheritance; (B) Asian population under the recessive model of inheritance.
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Figure 4 (A) Caucasian population under the Dominant model of inheritance; (B) Caucasian population under the recessive model of inheritance.
between MTHFR variant and Hcy in specific subgroup of subjects.26 In Europe and North America, no significant association between MTHFR polymorphism and Hcy level was observed in contrast to Middle East and Japan.27 Recent studies have also suggested that an elevated level of Hcy might be a risk factor for ischemic stroke.
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The frequency of polymorphic alleles has been observed to vary among different ethnic groups of the world.28–30 According to Fowler,31 the frequency of homozygous variant of MTHFR gene was present in 5–18% of population. Our meta-analysis suggests that frequency distribution of homozygous TT genotype was present in 23.5% in Asian and 12.8%
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from the hospital in the included study, which may have led to selection bias in the selection of controls.
Conclusion
Figure 5 Funnel plot for assessing publication bias.
in Caucasian population. Heterogeneity is a potential issue in the interpretation of results of metaanalyses.32 Higher value of I2 statistic represents a higher degree of heterogeneity. Our study suggested a moderate degree of heterogeneity, 46% under dominant model and 21% under the recessive model. Moderate heterogeneity in Asian population (I2 49% in dominant model and I2 33% in recessive model) was observed; however, in Caucasian population relatively low heterogeneity (I2 5 22% under dominant model and I2 5 4% under recessive model) was observed. Quality of study determines precise estimation of the association.13 Some studies follow the appropriate methodological guidelines; however, others fail to follow the methodological guidelines, which may lead to conflicting results. Therefore, we decided to stratify the data based on the methodological quality of studies. High-quality studies suggest moderately weak association than poor quality studies.
Limitation of study A meta-analysis based on the quality of study is an ideal method for improving the effect of association between the risk factors and outcome; however, several factors can influence the results of metaanalysis. There are many limitations in this metaanalysis. The result of the present meta-analysis could be influenced by lack of known environment and unstudied genetic factors. In our meta-analysis, there was no haplotype analysis, which plays a crucial role for association studies of complex diseases. The interaction between gene and environment and gene–gene interaction was not studied in this metaanalysis. Some studies included in the meta-analysis had a small sample size, which may have provided inconsistent results due to low statistical power. Stroke risk varies as per specific subtypes of stroke; however, most of the studies included in the metaanalysis have not presented the data as per subtype of stroke, therefore, meta-analysis based on association between MTHFR polymorphism and subtype of stroke has not been done. Controls were included
Evidence from this meta-analysis, which included 38 studies on the association between MTHFR C677T polymorphism and ischemic stroke, sustains the notion of association between polymorphism of MTHFR gene and susceptibility to ischemic stroke mainly in Asian population. This largest metaanalysis has an important significance suggesting that MTHFR genotyping may be helpful for identification of patients, susceptible for risk of stroke, prevention, and management of stroke. The findings of our study have clear implication for the policy makers to design programs to enable the increase in the intake of folate to reduce the risk of ischemic stroke in Asian Countries. Larger prospective studies with correction for multiple comparisons are required to confirm the findings.
Disclaimer Statements Contributors AK has written the manuscript. PK and MP extracted the data. RS, AKP, and AP have helped in writing draft of manuscript. Funding Conflicts of interest None. Ethics approval Not required.
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