Maternal Methylenetetrahydrofolate Reductase C677T Polymorphism and Down Syndrome Risk: A Meta-Analysis from 34 Studies

Maternal Methylenetetrahydrofolate Reductase C677T Polymorphism and Down Syndrome Risk: A MetaAnalysis from 34 Studies Vandana Rai1*, Upendra Yadav1, Pradeep Kumar1, Sushil Kumar Yadav1, Om Prakesh Mishra2 1 Human Molecular Genetics Laboratory, Department of Biotechnology, VBS Purvanchal University, Jaunpur, India, 2 Department of Pediatrics, Institute of Medical Sciences, Banaras Hindu University, Varanasi, India Abstract Background: Methylenetetrahydrofolate reductase (MTHFR) is a key enzyme of folate metabolic pathway which catalyzes the irreversible conversion of 5, 10-methylenetetrahydrofolate to 5-methyltetrahydrofolate. 5-methyltetrahydrofolate donates methyl group for the methylation of homocysteine to methionine. Several studies have investigated maternal MTHFR C677T polymorphism as a risk factor for DS, but the results were controversial and inconclusive. To come into a conclusive estimate, authors performed a meta-analysis. Aim: A meta-analysis of published case control studies was performed to investigate the association between maternal MTHFR C677T polymorphism and Down syndrome. Methods: PubMed, Google Scholar, Elsevier, Springer Link databases were searched to select the eligible case control studies using appropriate keywords. The pooled odds ratio (OR) with 95%confidence interval were calculated for risk assessment. Results: Thirty four studies with 3,098 DS case mothers and 4,852 control mothers were included in the present metaanalysis. The pooled OR was estimated under five genetic models and significant association was found between maternal MTHFR 677C.T polymorphism and Down syndrome under four genetic models except recessive model (for T vs. C, OR = 1.26, 95% CI = 1.09–1.46, p = 0.001; for TT vs. CC, OR = 1.49, 95% CI = 1.13–1.97, p = 0.008; for CT vs. CC, OR = 1.29, 95% CI = 1.10–1.51, p = 0.001; for TT+CT vs. CC, OR = 1.35, 95% CI = 1.13–1.60, p = 0.0008; for TT vs. CT+CC, OR = 0.76, 95% CI = 0.60–0.94, p = 0.01). Conclusion: The results of the present meta-analysis support that maternal MTHFR C677T polymorphism is a risk factor for DS- affected pregnancy. Citation: Rai V, Yadav U, Kumar P, Yadav SK, Mishra OP (2014) Maternal Methylenetetrahydrofolate Reductase C677T Polymorphism and Down Syndrome Risk: A Meta-Analysis from 34 Studies. PLoS ONE 9(9): e108552. doi:10.1371/journal.pone.0108552 Editor: Balraj Mittal, Sanjay Gandhi Medical Institute, India Received April 26, 2014; Accepted August 28, 2014; Published September 29, 2014 Copyright: ß 2014 Rai et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The authors have no support or funding to report. Competing Interests: The authors have declared that no competing interests exist. * Email: raivandana@rediffmail.com reductase (MTRR) in Asian [1,2,4,5] and Caucasian [6–8] populations. Folate deficiency and dysfunctional MTHFR causes abnormal DNA methylation [9,10] and chromosomal segregation [11,12]. Hypomethylation of the centromeric DNA has been suggested as the causative mechanism of meiotic non-disjunction. Abnormal DNA methylation of centromere lead to aberrant kinetochore formation that results into abnormal segregation of chromosomes during meiosis [3,13]. MTHFR is a key enzyme in folate metabolism, which catalyzes the reduction of 5, 10-methylenetetrahydrofolate to the predominant circulating form of folate i.e. 5-methyltetrahydrofolate (5THF). 5-THF donates methyl group for the conversion of homocysteine to methionine, which is further converted into Sadenosylmethionine (SAM). SAM is the main methyl group donor for all cellular methylation reactions. Folate deficiency and/or dysfunctional MTHFR reduces the conversion of 5, 10-methylene THF to 5-methyl THF, and elevates plasma homocysteine Introduction Down syndrome (DS) is the most common chromosomal disorder with the prevalence of 1/700–1000 live birth. It is characterized by the trisomy 21, which results from maternal meiotic nondisjunction in majority (90%) of cases. The established risk factor for DS is advanced (.35 years) maternal age at the time of conception. However, a fairly high number of DS children born to younger mothers suggest that risk factors other than advanced maternal age might be involved in predisposing younger mothers to DS-affected pregnancy [1,2]. The molecular and biochemical mechanism of maternal meiotic non-disjunction is still not known. James et al. [3] reported that methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism might be a risk factor for maternal meiotic non-disjunction. Since then several studies have investigated the risk of DS to variants of folate pathway genes like MTHFR, Methionine synthase (MTR) and Methionine synthase PLOS ONE | www.plosone.org 1 September 2014 | Volume 9 | Issue 9 | e108552 MTHFR C677T Polymorphism as Risk Factor for Down Syndrome Figure 1. Flow Diagram of Study Searching and Selection Process. doi:10.1371/journal.pone.0108552.g001 conflicting and inconclusive. In light of the above facts, we conducted a meta-analysis of published case control studies relating the C677T polymorphism of the maternal MTHFR gene to the risk of having DS offspring. concentration. Both folate and MTHFR are involved in many complex biochemical reactions like DNA synthesis, repair and methylation. There are more than 40 polymorphisms reported in MTHFR gene and among them C677T variant is the most studied and clinically important. The C677T variant (rs 1801133; Ala 222 Val) has been associated with a decreased activity of MTHFR, and increased homocysteine level [14–16]. Mutant homozygous (TT) individuals have a decreased enzymatic activity , 70% and the heterozygote by 40%. A dysfunctional MTHFR leads to lower levels of SAM resulting into DNA hypomethylation. DNA hypomethylation increases the risk of many diseases and disorders like- neural tube defects [17], cleft lip and palate [18], Alzheimer disease [19], cardiovascular diseases [14], diabetes [20] and psychiatric disorders [21] etc. Several epidemiological studies have investigated the associations of the maternal MTHFR C677T polymorphism with Down syndrome. However, the results were PLOS ONE | www.plosone.org Materials and Methods Selection of studies Electronic searches were conducted using PubMed, Google Scholar, Elsevier and Springer link and all published manuscripts up to January, 2014 were considered in present meta-analysis. The following index terms were used for search ‘MTHFR’ ‘Methylenetetrahydrofolate reductase’, and ‘C677T polymorphism’, ‘maternal risk’ and ‘Down syndrome’. In addition, bibliographies of all articles and reviews were hand searched for additional suitable studies. 2 September 2014 | Volume 9 | Issue 9 | e108552 PLOS ONE | www.plosone.org Table 1. Characteristics of the eligible studies included in the meta-analysis. Study Year Country Case Control Quality Score Reference James et al. 1999 Canada 50 57 7 Am J Clin Nutr 70:495-50 Hobbs et al. 2000 America 157 140 7 Am J Hum Genet 67:623–630 Chadeaux-Vekemans et al. 2002 France 85 70 5 Pediatr Res 51:766–767 O’Leary et al. 2002 Ireland 41 192 5 Am J Med Genet A 107:151–155 Stuppia et al. 2002 Italy 64 112 7 Eur J Hum Genet 10:388–390 Boduroglu et al. 2004 Turkey 158 91 5 Am J Med Genet 127A: 5–10 Acacio et al. 2005 Brazil 70 88 8 Prenat Diagn 25:1196–1199 Da Silva et al. 2005 Brazil 154 158 7 Am J Med Genet Part A 135A: 263–267 Coppede et al. 2006 Italey 79 111 7 Am J Med Genet A 140(10): 1083–1091 Liang et al. 2006 China 30 70 7 China J Modern Medicine 20:011 Rai et al. 2006 India 149 165 6 J Hum Genet 51:278–283 Scala et al. 2006 Italy 94 256 8 Genet Med 8:409–416 Wang et al. 2007 China 100 100 8 Zhonghua Yi Xue Yi Chuan Xue Za Zhi 24:533–537 Biselli et al. 2008 Brazil 82 134 8 Genet Mol Res 7:33–42 3 Kohli et al. 2008 India 103 109 6 Downs Syndr Res Pract 12:133–137 Martinez-Frias et al. 2008 Spain 146 188 4 Am J Med Genet A 146A(11): 1477–1482 Meguid et al. 2008 Egypt 42 48 7 Dis Markers 24:19–26 Santos-Reboucas et al. 2008 Brazil 103 108 7 Dis Markers 25:149–157 2008 China 64 70 8 J Zhejiang Univ Sci B 9(2): 93–99 Brandalize et al. 2009 Brazil 239 197 6 Am J Med Genet 149A (10): 2080–2087 Coppede et al. 2009 Italy 94 113 8 Neurosci Lett 449:15–19 September 2014 | Volume 9 | Issue 9 | e108552 Cyril et al. 2009 India 36 60 6 Indian J Hum Genet 15:60–64 Kokotas et al. 2009 Denmark 177 984 6 Dis Markers 27:279–285 Pozzi et al. 2009 Italy 74 184 8 Am J Obstet Gynecol 63: e1–e6 Coppede et al. 2010 Italy 29 32 5 BMC Med Genomics 3:42 Liao et al. 2010 China 60 68 7 Yi Chuan 32(5): 461–466 Vranekoviz et al. 2010 Croatia 111 141 7 Dis Markers 28:293–298 Bozovic et al. 2011 Croatia 112 221 7 Pediatr Int 53(4): 546–550 Sadiq et al. 2012 Jordan 53 29 6 Genet Test Mol Biomarker 15:1–7 Tayeb 2012 Saudi Arabia 30 40 5 Egyptian J Med Hum Genet 13(3): 263–268 Zampieri et al. 2012 Brazil 105 185 8 Dis Markers 32(2): 73–81 Kaur and Kaur 2013 India 110 111 6 Indian J Hum Genet 19(4): 412–414 Pandey et al. 2013 India 81 99 6 Int J Pharm Bio Sci; 4(2):(B)249–256 Elsayed et al. 2014 Egypt 26 61 9 The Egyptian J Med Hum Genet 15(1): 39–44 doi:10.1371/journal.pone.0108552.t001 MTHFR C677T Polymorphism as Risk Factor for Down Syndrome Wang et al. PLOS ONE | www.plosone.org Table 2. Distributions of MTHFR C677T genotypes and allele frequencies in DS case mothers and control mothers reported in different included studies. CC CT TT C T Study Country Case Control Case Control Case Control Case Control Case Control James et al., 1999 Canada 24 15 22 34 4 8 70 64 30 50 Hobbs et al., 2000 America 51 67 84 59 22 14 186 193 128 87 Chadeaux-Vekemans et al., 2002 France 36 29 42 30 7 11 114 88 56 52 O’Leary et al., 2002 Ireland 18 90 21 84 2 18 57 264 25 120 Stuppia et al., 2002 Italy 20 27 32 62 12 23 72 116 56 108 Boduroglu et al., 2004 Turkey 86 58 55 30 17 3 227 146 89 36 Acacio et al., 2005 Brazil 35 54 30 25 5 9 100 133 40 43 Da Silva et al., 2005 Brazil 67 84 72 67 15 7 206 235 102 81 Coppede et al., 2006 Italey 20 39 43 54 16 18 83 132 75 90 Liang et al., 2006 China 7 16 20 34 3 20 34 66 26 74 Rai et al., 2006 India 97 124 40 39 12 2 234 287 64 43 Scala et al., 2006 Italy 31 74 39 125 24 57 101 273 87 239 Wang et al., 2007 China 28 48 52 42 20 10 108 138 92 62 Biselli et al., 2008 Brazil 29 100 35 77 8 17 93 229 71 39 4 Kohli et al., 2008 India 74 71 29 32 0 6 177 174 29 44 Martinez-Frias et al., 2008 Spain 61 76 61 85 24 27 183 237 109 139 Egypt 20 33 17 12 5 3 57 78 27 18 Santos-Reboucas et al., 2008 Brazil 51 49 43 47 9 12 145 145 61 71 Wang et al., 2008 China 14 36 32 29 18 5 60 101 68 39 Brandalize et al., 2009 Brazil 94 86 113 93 32 18 301 265 177 129 Coppede et al., 2009 Italy 25 40 52 55 17 18 102 135 86 91 September 2014 | Volume 9 | Issue 9 | e108552 Cyril et al., 2009 India 33 60 3 0 0 0 69 120 3 0 Kokotas et al., 2009 Denmark 92 445 72 449 13 90 256 1339 98 629 Pozzi et al., 2009 Italy 28 62 30 93 16 29 86 217 62 151 Coppede et al., 2010 Italy 5 11 19 17 5 4 29 39 29 25 Liao et al., 2010 China 12 23 26 33 22 12 50 79 70 57 Vranekoviz et al., 2010 Croatia 49 66 49 64 13 11 147 196 75 86 Bozovic et al., 2011 Croatia 46 101 55 97 11 23 147 299 77 143 Sadiq et al., 2011 Jordan 23 23 27 5 3 1 73 51 33 7 Tayeb, 2012 Saudi Arabia 16 22 10 14 4 4 42 58 18 22 Zampieri et al., 2012 Brazil 40 94 55 73 10 18 135 261 75 109 Kaur & Kaur, 2013 India 86 89 22 22 2 0 194 200 26 22 Pandey et al., 2013 India 67 87 12 9 2 3 146 183 16 15 Elsayed et al., 2014 Egypt 11 30 12 24 3 7 34 84 18 38 doi:10.1371/journal.pone.0108552.t002 MTHFR C677T Polymorphism as Risk Factor for Down Syndrome Meguid et al., 2008 MTHFR C677T Polymorphism as Risk Factor for Down Syndrome Figure 2. Forest plots (Random effect) showed significant association between MTHFR C677T polymorphism and risk of Down syndrome using allele contrast model (C versus T). Results of individual and summary OR estimates and 95% CI of each study were shown. Horizontal lines represented 95% CI, and dotted vertical lines represent the value of the summary OR. doi:10.1371/journal.pone.0108552.g002 analyses were performed using the computer program MIX version 1.7 [24]. The control genotypes were tested for HardyWeinberg equilibrium (HWE) using the Goodness of fit Chisquare test. The quality of the included studies was measured according to the scoring system for randomized controlled association studies proposed by Clark and Baudouin [25]. Case control studies scoring ,5 were defined as low quality study and those $5 were defined as high quality study. Inclusion criteria Included studies had to meet the following criteria: (1) article should be published; (2) article should have sufficient data to calculate the odds ratio with 95% CI; (3) article should be case control association study; and (4) author should describe the genotyping protocols. Data extraction The following data were extracted from each study: first author’s name, publication year, journal name, country name, genotyping method, and different MTHFR genotype numbers. Publication bias Funnel plots of precision by log (OR) and standard error by log (OR) were plotted to determine publication bias and asymmetrical funnel plots represent publication bias. Begg and Mazumdar rank correlation [26] and Egger’s regression intercept [27] tests were adopted to assess the publication bias. Meta-analysis Statistical analysis of maternal MTHFR C677T polymorphism and DS risk was estimated by Odds ratio (ORs) with 95% confidence intervals (CIs). The heterogeneity was tested by the Qstatistics with p-values ,0.05. Subgroup analysis was done to know the source of heterogeneity. If higher heterogeneity (I2. 50%) would be observed, the random effect model [22] would be applied. Otherwise, fixed-effect model [23] was applied to obtain the summary OR and 95% CI. All p values were two-sided and a p value of less than 0.05 was considered statistically significant. All PLOS ONE | www.plosone.org Results Eligible Studies With our original search criterion, 85 articles were found. After reviewing each original article, 50 publications were excluded including reviews, case studies, editorials etc. (Figure 1). Following these exclusions, 34 individual case-control studies with a total of 5 September 2014 | Volume 9 | Issue 9 | e108552 PLOS ONE | www.plosone.org Table 3. Summary estimates for the odds ratio (OR) of MTHFR C677T in various allele/genotype contrasts, the significance level (p value) of heterogeneity test (Q test), the I2 metric and publication bias p-value (Egger Test) in total studies, Asian, American and European studies. Genetic Contrast All Asian American 6 European Heterogeneity p-value (Q test) Publication Bias (p of Egger’s test) Fixed effect OR (95% CI), p Random effect OR (95% CI), p Allele Contrast (T vs. C) 1.22 (1.1321.31), ,0.0001 1.26 (1.0921.46), 0.001 ,0.0001 69.42 0.14 Co-dominant (CT vs. CC) 1.23 (1.1121.36), ,0.0001 1.29 (1.1021.51), 0.001 0.0002 52.49 0.02 Homozygote (TT vs. CC) 1.44 (1.2221.69), ,0.0001 1.49 (1.1321.97), 0.008 ,0.0001 57.3 0.56 Dominant (TT+CT vs. CC) 1.28 (1.1621.41), ,0.0001 1.35 (1.1321.60), 0.0008 ,0.0001 63.56 0.05 Recessive (CT+CC vs. TT) 0.76 (0.6520.88), 0.0004 0.76 (0.6020.94), 0.01 0.0044 43.68 0.926 Allele Contrast (T vs. C) 1.53 (1.2921.82), ,0.0001 1.52 (1.0922.1), 0.01 0.0003 69.43 0.82 Co-dominant (CT vs. CC) 1.52 (1.2121.91), 0.0003 1.57 (1.1422.14), 0.005 0.09 38.05 0.11 Homozygote (TT vs. CC) 2.41 (1.6223.59), ,0.0001 2.21 (1.0324.74), 0.0411 0.0074 60.04 0.204 I2 (%) 1.64 (1.3222.0), ,0.0001 1.70 (1.1822.4), 0.004 0.01 56.67 0.30 0.54 (0.3720.78), ,0.0001 0.58 (0.2921.16), 0.12 0.0094 58.77 0.334 Allele Contrast (T vs. C) 1.23 (1.0721.39), 0.003 1.19 (0.9921.44), 0.06 0.06 47.69 0.11 Co-dominant (CT vs. CC) 1.42 (1.1721.71), 0.0002 1.42 (0.9722.06), 0.066 0.0005 73.15 0.908 Homozygote (TT vs. CC) 1.68 (1.2422.28), 0.0008 1.58 (0.8422.95), 0.148 0.0007 72.07 0.667 Dominant (TT+CT vs. CC) 1.48 (1.2421.76), ,0.0001 1.44 (0.9522.19), 0.078 ,0.0001 80.11 0.782 Recessive (CT+CC vs. TT) 0.69 (0.5120.92), 0.0136 0.72 (0.4421.18), 0.203 0.0159 59.42 0.753 Allele Contrast (T vs. C) 1.03 (0.9321.15), 0.482 1.04 (0.9321.16), 0.451 0.3576 8.81 0.084 Co-dominant (CT vs. CC) 0.99 (0.8521.16), 0.956 1.00 (0.8521.17), 0.992 0.3774 6.87 0.050 Homozygote (TT vs. CC) 1.09 (0.8721.37), 0.422 1.09 (0.8521.40), 0.455 0.3715 7.45 0.329 Dominant (TT+CT vs. CC) 1.02 (0.8821.17), 0.787 1.03 (0.8721.21), 0.704 0.308 13.58 0.041 Recessive (CT+CC vs. TT) 0.90 (0.7321.10), 0.322 0.90 (0.7221.11), 0.339 0.570 0 0.948 September 2014 | Volume 9 | Issue 9 | e108552 doi:10.1371/journal.pone.0108552.t003 MTHFR C677T Polymorphism as Risk Factor for Down Syndrome Dominant (TT+CT vs. CC) Recessive (CT+CC vs. TT) MTHFR C677T Polymorphism as Risk Factor for Down Syndrome Figure 3. Forest plots (Random effect) showed significant association between MTHFR C677T polymorphism and risk of Down syndrome. Results of individual and summary OR estimates and 95% CI of each study were shown using homozygote model (TT versus CC). doi:10.1371/journal.pone.0108552.g003 12.14% respectively. Frequencies of CC and CT genotypes were highest in both cases and controls (Table 2). In cases and controls, the allele C was the most common. All five genetic models; -allele contrast (T vs C) homozygote (TT vs CC), codominant (CT vs CC), dominant (TT+CT vs CC) and recessive (TT vs CT+CC) models were used to evaluate C677T polymorphism as DS risk. 3,098 cases and 4,852 controls were found to be suitable for inclusion into meta-analysis and listed in Table 1 (Figure 1). These studies were published between 1999 and 2013. All these thirty four studies were performed in different countries- Brazil [28–33], China [4,34–36], Croatia [8,37], Egypt [38,39], France [40], India [1,5,41–43], Ireland [44], Italy [7,13,45–48], Jordan [49], Netherlands [50], Saudi Arabia [2], Spain [51], Turkey [52] and USA [3,6] (Table 1). Meta-analysis Meta-analysis with allele contrast showed significant association between maternal 677T allele and DS with both fixed effect (ORTvsC = 1.22; 95% CI = 1.13–1.31; p = ,0.0001) and random effect models (ORTvsC = 1.26; 95% CI = 1.09–1.45; p = 0.001) (Figure 2) (Table 3). In cumulative meta-analysis using random effect model, the association of maternal T allele with DS turned statistically significant with the addition of study of Wang et al. (2008) and remained significant thereafter. Table 3 summarizes the ORs with corresponding 95% CIs for association between maternal C677T polymorphism and risk of DS in dominant, recessive, homozygote and co-dominant models. With our primary analysis, there was an increased risk of DS among mutant homozygote variants (TT), with both fixed (ORTTvs.CC = 1.44; 95% CI = 1.2221.69, p = ,0.0001) and ran- Characteristics of included studies In thirty four studies included in the present meta-analysis, the smallest case sample size was 26 [39] and highest sample size was 239 [32]. ORs for more than one were reported in twenty four articles [1,2,4–6,8,13,28–30,32,33,35–39,42,43,46–49,51,52]. Except two studies [28,43], control populations of all articles were in Hardy-Weinberg equilibrium. In all thirty four studies, total cases were 3,098 with CC (1,396), CT (1,326) and TT (376), and controls were 4,852 with CC (2,329), CT (2,015), and TT (508) genotypes. In controls genotypes, percentage of CC, CT and TT were 48.00%, 41.53%, and 10.47% respectively. In total cases, genotype percentage of CC, CT, and TT was 45.06%, 42.8% and PLOS ONE | www.plosone.org 7 September 2014 | Volume 9 | Issue 9 | e108552 MTHFR C677T Polymorphism as Risk Factor for Down Syndrome Figure 4. Forest plots (Random effect) showed significant association between MTHFR C677T polymorphism and risk of Down syndrome using dominant model (TT+CT versus CC). Results of individual and summary OR estimates and 95% CI of each study were shown. doi:10.1371/journal.pone.0108552.g004 p = 0.003; I2 = 47.69%; Pheterogeneity = 0.06; PPb = 0.11) (Figure 6) no significant association was found in American and European population (for T vs. C: OR = 1.03; 95% CI = 0.9321.15; p = 0.482; I2 = 8.81%; Pheterogeneity = 0.357; PPb = 0.084) (Figures 7; Table 3). dom (ORTTvs.CC = 1.49; 95% CI = 1.1321.97, p = 0.008) effect models with moderate statistical heterogeneity between-study (Figure 3). Association of mutant heterozygous genotype (CT vs. CC) was observed significant with fixed (ORCTvs.CC = 1.23; 95% CI = 1.1121.36; p = ,0.0001) and random (ORCTvs.CC = 1.29; 95% CI = 1.1021.51; p = 0.001) effect models. Similarly combined mutant genotypes (TT+CT vs. CC) showed significant association with DS using both fixed (ORTT+CTvs.CC = 1.28; 95% CI = 1.162 1.41; p = ,0.0001) and random (ORTT+CTvs.CC = 1.35; 95% CI = 1.1321.60; p = 0.0008) effect models (Figure 4). Heterogeneity and Sensitive analysis A true heterogeneity existed between studies for allele (PheterQ = 107.92, df = 33, I2 = 69.42%, t2 = 0.12) and mutant genotypes (Pheterogeneity = ,0.0001, Q = 74.90, df = 32, I2 = 57.3%, t2 = 0.10) comparisons. The ‘I2’ value of more than 50% for between studies comparison in both allele and genotype analysis shows high level of true heterogeneity. In Asian (Pheterogeneity = 0.0003, I2 = 67.43%) and American (Pheterogene2 ity = ,0.0001, I = 83.25%) allele contrast meta-analysis significant high heterogeneity was observed, in European sub-group metaanalysis low heterogeneity was observed (Pheterogeneity = 0.357, I2 = 8.81) in allele contrast model. In allele contrast meta-analysis, sensitivity analysis performed by exclusion of the studies in which control population was not in Hardy Weinberg equilibrium, studies with small sample size and studies with high p values. Control population of only two studies ogeneity = ,0.0001, Stratified analysis We also performed sub-group analysis which is based on geographic distribution of population. Out of 34 studies included in present meta-analysis, 11 studies were from Asia, 13 from Europe, 8 from America and 2 from Africa. The subgroup analysis by geographical regions revealed that the significant association between the maternal MTHFR C677T polymorphism and DS existed in Asian population (for T vs. C: OR = 1.51; 95% CI = 1.0922.10; p = 0.01; I2 = 69.43%; Pheterogeneity = 0.0003; PPb = 0.82) (Figure 5; Table 3). Except allele contrast model of American population (T vs. C: OR = 1.23; 95% CI = 1.0721.39; PLOS ONE | www.plosone.org 8 September 2014 | Volume 9 | Issue 9 | e108552 MTHFR C677T Polymorphism as Risk Factor for Down Syndrome Figure 5. Forest plots (Random effect) showed significant association between MTHFR C677T polymorphism and risk of Down syndrome in Asian studies using allele contrast model (T versus C). Results of individual and summary OR estimates and 95% CI of each study were shown. doi:10.1371/journal.pone.0108552.g005 [28,43] were not in HW equilibrium and heterogeneity did not decreased after exclusion of these studies (p = ,0.0001, I2 = 70.00%). Exclusion of seven studies with small sample size, less than 50 (O’Leary et al. [44], n = 41; Liang et al. [34], n = 30; Mequid et al [38], n = 42; Cyril et al. [42], n = 36; Coppede et al. [48], n = 29; Tayeb [2], n = 30; Elsayed et al. [39], n = 26), also did not decreased heterogeneity (Pheterogeneity = ,0.0001, I2 = 72.98%). Similarly exclusion of eleven studies with very high p value (O’Leary et al. [44], p = 0.87; Acacio et al. [28], p = 0.40; Scala et al. [7], p = 0.91; Martinez-Frias et al. [51], p = 0.90; Pozzi et al. [13], p = 0.84;Vranekoviz et al. [37], p = 0.43; Bozovic et al. [8], p = 0.58; Tayeb [2], p = 0.74; Elsayed et al. [39], p = 0.65; Kaur and Kaur [5], p = 0.52; Pandey et al. [43], p = 0.44) did not decrease heterogeneity but increased odds ratio (OR = 1.29, 95% CI = 1.1821.41, p = ,0.0001). p = 0.14 for T vs. C; Begg’s p = 0.38, Egger’s p = 0.56 for TT vs. CC; Begg’s p = 0.13, Egger’s p = 0.05 for TT+CT vs. CC and Begg’s p = 0.19, Egger’s p = 0.0.05 for TT vs. CC+CT) but publication bias was observed in co-dominant model (Begg’s p = 0.04, Egger’s p = 0.02 for CT vs. CC) of overall by using Begg’s and Egger’s test (Table 3). Funnel plots were showed in Figures 8 and 9. Discussion In 1999, James et al [3] reported that genetic polymorphism of folate and homocysteine pathway enzymes predispose a woman to abnormal chromosome segregation, which act as risk factor for DS pregnancy. In subsequent years, several in vivo studies in humans suggested that chronic folate deficiency has been associated with abnormal DNA methylation [11,53,54], and aberrant chromosome segregation [6,55259]. Population-based studies have shown that folic acid intake during fetal development has a protective effect, resulting in a significant reduction in the Publication bias Publication bias was not observed in allele contrast, homozygote, dominant and recessive models (Begg’s p = 0.28, Egger’s PLOS ONE | www.plosone.org 9 September 2014 | Volume 9 | Issue 9 | e108552 MTHFR C677T Polymorphism as Risk Factor for Down Syndrome Figure 6. Forest plots (Random effect) showed no association between MTHFR C677T polymorphism and risk of Down syndrome in American studies using allele contrast model (T versus C). Results of individual and summary OR estimates and 95% CI of each study were shown. doi:10.1371/journal.pone.0108552.g006 Figure 7. Forest plots (Fixed effect) showed no association between MTHFR C677T polymorphism and risk of Down syndrome in European studies using allele contrast model (T versus C). Results of individual and summary OR estimates and 95% CI of each study were shown. Horizontal lines represented 95% CI, and dotted vertical lines represent the value of the summary OR. doi:10.1371/journal.pone.0108552.g007 PLOS ONE | www.plosone.org 10 September 2014 | Volume 9 | Issue 9 | e108552 MTHFR C677T Polymorphism as Risk Factor for Down Syndrome Figure 8. Funnel plots a2f. a. Precision by log odds ratio for additive model; b. standard error by log odds ratio for additive model; c. precision by log odds ratio for co-dominant model; d. standard error by log odds ratio for co-dominant model; e. precision by log odds ratio for dominant model; f. standard error by log odds ratio for Dominant model. doi:10.1371/journal.pone.0108552.g008 occurrence of developmental defects, like neural tube defects (NTD), congenital heart defects, limb defects, and orofacial clefts [60]. PLOS ONE | www.plosone.org Meta-analysis is a powerful tool for analyzing cumulative data with small and low power studies. Several meta-analyses were published accessing MTHFR as risk factor to various diseases/ 11 September 2014 | Volume 9 | Issue 9 | e108552 MTHFR C677T Polymorphism as Risk Factor for Down Syndrome Figure 9. Funnel plots a2f. a. Precision by log odds ratio for additive model; b. standard error by log odds ratio for additive model for Asian studies; c. precision by log odds ratio for additive model; d. standard error by log odds ratio for additive model for American studies; e. precision by log odds ratio for additive model; f. standard error by log odds ratio for additive model for European studies. doi:10.1371/journal.pone.0108552.g009 PLOS ONE | www.plosone.org 12 September 2014 | Volume 9 | Issue 9 | e108552 Reported Allelic contrast 1.26 (1.09–1.46), 0.001 ,0.0001 69.42 doi:10.1371/journal.pone.0108552.t004 3048 Present Study, 2014 34 PLOS ONE | www.plosone.org disorders like- neural tube defects [61,62], cleft lip and palate [63], stroke [64], psychiatric disorders [65]. During literature search, we identified four meta-analyses [66–69] published between 2007 and 2013. They examined the effect of maternal MTHFR C677T as DS risk, but no consistent conclusion was achieved. Zintzaras [66] performed a meta-analysis based on eleven studies and did not find any significant association between the maternal MTHFR polymorphisms and DS risk. Medica et al. [67] aggregated sixteen studies and reported significant relationship between the maternal mutant genotypes (TT+CT vs CC) and risk of DS child. Recently, Wu et al. [68] published a meta-analysis (included twenty eight studies with 2806 cases/4597 controls), and found statistical association with dominant model (OR = 1.305, 95% CI = 1.125– 1.514, p = 0, p = 0.003). Yang et al. [69] performed a metaanalysis which was based on twenty six studies (2458 cases/3144 controls) and found statistically significant association in allele contrast model (OR = 1.28; 95% CI: 1.11–1.47) (Table 4). Several newly published studies were not included in the previous published meta-analyses. So authors conducted a comprehensive meta-analysis with the largest number of studies (34 studies). In the present meta-analysis significant association was found between maternal C677T polymorphism and DS risk in total 34 studies using all five genetic models. Whereas in stratified analysis, except allele contrast model in American population, no significant association was observed in European and American population but significant higher risk was found in Asian population. Such phenomenon probably could be ascribed to the folate metabolism profile and dietary structure of different regions. There are few limitations of the present meta-analysis like- i) we used crude ORs in the pooled analysis without adjustment; ii) the relatively small sample size in some of the included studies, especially those from Asia; iii) we considered only one gene polymorphism (MTHFR C677T) of folate pathway. Present metaanalysis had several advantages/strength to the previous published meta-analyses like- (i) the publication bias was not detected in present meta-analysis, (ii) pooled number of cases and controls from different studies significantly increased the statistical power of the analysis, (iii) largest number of studies (34 studies) with largest sample size (3,098 cases and 4,852 controls) was included in the present meta-analysis, (iv) controls included in the present metaanalysis was mothers of healthy child, (v) distribution of genotypes in control mothers except two studies was in Hardy-Weinberg equilibrium, (vi) significant association was found between maternal MTHFR C677T polymorphism and DS risk in allelic contrast, homozygote, co-dominant and dominant genetic models and (vii) in addition we did sub-group analysis according to geographical regions. In conclusion, results of present meta-analysis suggest that the maternal MTHFR 677T allele is a risk factor for development of DS pregnancy. However the results of present meta-analysis were based on single gene polymorphism and significant heterogeneity was also observed; hence results should be interpreted with caution. 4852 Reported Reported Allelic contrast Dominant model 1.224 (1.085–1.38), 0.001 1.28 (1.11–1.47) ,0.01 0.0 48.0 58.2 2806 4597 2458 28 Wu et al., 2013 Yang et al., 2013 26 3144 Not reported Not reported Allelic contrast Dominant model 1.40 (1.16–1.70), 0.0006 1.20 (1.06–1.35) 0.03 – – 49 1545 2052 1129 16 Medica et al., 2009 Zintaras, 2007 11 1489 Subgroup analysis Model OR (95% CI), p-value Heterogeneity p-value (Q test) I2 (%) Controls Cases Number of Studies Study Table 4. 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