Maternal MTHFR polymorphism (677 C T) and risk of Down s syndrome child: meta-analysis

Transcription

1 c Indian Academy of Sciences RESEARCH ARTICLE Maternal MTHFR polymorphism (677 C T) and risk of Down s syndrome child: meta-analysis AMANDEEP KAUR and ANUPAM KAUR Department of Human Genetics, Guru Nanak Dev University, Amritsar , India Abstract Methylenetetrahydrofolate reductase (MTHFR) is the most important gene that participates in folate metabolism. Presence of valine instead of alanine at position 677 and elevated levels of homocystein causes DNA hypomethylation which in turn favours nondisjunction. In this study, we conducted a meta-analysis to establish link between maternal single-nucleotide polymorphism (SNP) and birth of Down s syndrome (DS) child. A total of 37 case control studies were selected for analysis including our own, in which we investigated 110 cases and 111 control mothers. Overall, the result of meta-analysis showed significant risk of DS affected by the presence of maternal SNP (MTHFR 677 C T OR = 0.816, 95% CI = , P < ). Heterogeneity of high magnitude was observed among the studies. The chi-square value suggested a highly significant association between homozygous mutant TT genotype and birth of DS child (χ 2 = 23.63, P = 0.000). Genetic models suggested that T allele possesses high risk for DS whether present in dominant (OR = 1.23, 95% CI = ); codominant (OR = 1.17, 95% CI = ) or recessive (OR = 1.21, 95% CI = ) form. The analysis from all 37 studies combined together suggested that MTHFR 677 C T is a major risk factor for DS birth. [Kaur A. and Kaur A Maternal MTHFR polymorphism (677 C T) and risk of Down s syndrome child: meta-analysis. J. Genet. 95, ] Introduction Down s syndrome (DS) is the most common chromosomal abnormality that occurs with the prevalence rate of about 1 in 1200 live births (Patel and Adhia 2005), due to nondisjunction during meiosis I or II. Many studies have reported that increased homocystein levels and presence of MTHFR 677 C T polymorphism impairs the folate metabolism and results in DNA hypomethylation, which in turn favours nondisjunction of chromosome 21 (James et al. 1999; Fenech 2001). The results seen in literature are quite contradictory, may be due to small sample size, making it difficult to elucidate the role of single-nucleotide polymorphism (SNP) for the risk of DS child. The present meta-analysis was conducted to understand whether mutation in MTHFR gene is a risk factor for DS or not. Materials and methods Literature regarding DS was searched on electronic PubMed database and Google scholar from 1999 to January 2015 for the studies on MTHFR 677 C T polymorphism (figure 1). For correspondence. anupamkaur@yahoo.com. Keywords. MTHFR gene; Down s syndrome; folate metabolism. All the selected reports were case control studies. The keywords used were: MTHFR 677 C T, Down s syndrome, methylenetetrahydrofolate reductase and folate metabolism. A total of 37 studies that evaluated the association between the presence of MTHFR 677 C T polymorphism and risk of DS child were included. Only research article papers were considred for this study. For each data, information about author, country and year of publication along with total number of cases and controls were extracted. Odds ratio, chisquare values along with frequencies were calculated (table 1). Inclusion criteria The following criteria were considered. (i) Articles published in English language; (ii) articles evaluated MTHFR 677 C T SNP; (iii) only those articles in which the mother had at least two normal children, without miscarriages and abnormalities; and (iv) reports with genotypic and allelic frequencies, odds ratio and chi-square. Statistical analysis The meta-analysis was performed using StatsDirect 3. Risk for the birth of DS child and presence of maternal Journal of Genetics, DOI /s , Vol. 95, No. 3, September

2 Amandeep Kaur and Anupam Kaur polymorphism was assessed by odds ratio with 95% CI. Presence of heterogeneity was estimated using chi-square based Q-statistics and I 2 metrics. In case of higher heterogeneity (I 2 > 50%), random effect model (DerSimonian and Laird 1986) was used else fixed effect model (Mantel and Haenszel 1959) was applied. To estimate publication bias, Begg and Mazumdar rank correlation (Begg and Mazumdar 1994) and Egger s regression intercept (Egger et al. 1997) tests were performed. P = 0.003). Similar results were obtained from American population suggesting association between 677 C T SNP and DS child birth (fixed model OR = 0.658, 95% CI = , P = ) (figure 5). However, heterogeneity was found to be of low magnitude (I 2 = 39.9%, Q = 11.64, P = 0.113, df = 7). On the other hand, no association and heterogeneity were found among European studies (fixed model OR = 0.982, 95% CI = , P = 0.838, I 2 = 0%, Q = 8.264, P = 0.764, df = 12) (figure 6). Results In this study, we identified 37 eligible reports from a total of 182 articles published between 1999 and 2015 (figure 1). The reports were from different ethnic populations, i.e. Asians, Americans, Europeans and others. A total of 3401 mothers having DS children and 5277 mothers having normal children were included in the study. Meta-analysis indicated a highly significant difference between cases and controls (χ 2 = 23.63, P = 0.000). The three genetic models (dominant: OR = 1.23, 95% CI = ; codominant: OR = 1.17, 95% CI = ; recessive: OR = 1.21, 95% CI = ) suggested that presence of TT genotype in mothers significantly increased the risk of DS birth (table 2). The frequency of heterozygote was highest among mothers of Down s syndrome children (MDS) ( %) as compared to control mothers (0 55.3%) and a presence of TT genotype was observed to be higher among cases (0 36.6%) than the control mothers (0 21.5%) (table 1). The present analysis revealed that occurrence of maternal polymorphism 677 C T significantly increases the risk of birth of DS child (fixed effect model (OR = 0.816, 95% CI = , P < ) (figure 2); random effect model (OR = 0.776, 95% CI = , P = 0.003) (figure 3)). The random model was used since the results demonstrated high magnitude of heterogeneity among studies (I 2 = 62.6%, P < , Q = 96.26, df = 36). Subgroup analysis revealed that MTHFR 677 C T also found to be significantly associated with the birth of DS child among Asians (random model OR = 0.527, 95% CI = , P = 0.001) (figure 4). Higher heterogeneity was observed among Asian studies (I 2 = 58.9%, Q = 29.17, df = 12, Total articles searched=182 Mice models=5, Reviews=5, Case reports=7, Newsletters=10 Irrelevant articles, abstracts, foreign language articles, Insufficient data=118 After exclusion Articles included in the meta-analysis=37 Figure 1. Flow chart showing selection criteria for meta-analysis. Publication bias Publication bias was not observed among the studies as indicated by Begg-Mazumdar bias P = 0.23 and Egger s bias P = for CC versus CT+TT (figure 7). Discussion Literature search of individual reports suggest that the frequency of MTHFR 677 TT distribution varies greatly worldwide. In our previous study (Kaur and Kaur 2013), we observed that 1.8% of cases exhibited TT genotype, while control mothers did not exhibit homozygous mutant genotype. No significant association was observed among cases and controls (χ , P = 0.359) due to the absence of homozygous mutant allele. Similar findings were observed in other studies by Cyril et al. (2009), Mohanty et al. (2012), Divyakolu et al. (2013) and Pandey et al. (2013). Further, studies from other countries also corroborates with our study like James et al. (1999), Acacia et al. (2005), Biselli et al. (2008), Boduroglu et al. (2004), Bozovic et al. (2011), Brandalize et al. (2009), Chadefaux-Vekeman et al. (2002), Chango et al. (2005), Coppede et al. (2006, 2009, 2010), Cretu et al. (2010), Da Silva et al. (2005), El-Gharib et al. (2012), Elsayed et al. (2014), Kokotas et al. (2009), Martinez-Frias (2008), Meguid et al. (2008), O Leary et al. (2002), Pozzi et al. (2009), Santos-Rebaucas et al. (2008), Scala et al. (2006), Stuppia et al. (2002), Tayeb (2012), Vranekovic et al. (2010), Liao (2010) and Zampeiri et al. (2012). The nonsignificant associations in these studies could be due to small sample size and further a single polymorphism was not sufficient to establish any association. James et al. (1999) was the first to report that mutant genotype TT is associated with the increased risk of DS. In their study, the frequency of heterozygote was much higher (59.6%) when compared to controls (44%). It was suggested that predominance of heterozygote among MDS was due to foetal viability that may be lower in mothers with the TT genotype. Several studies observed nonsignificant association but the presence of mutant allele increased the risk of DS ranging from % (table 1). The high intake of folate may neutralize the metabolic impact of MTHFR polymorphism. Thus, SNPs in other genes in folate pathway, combined with MTHFR and homocystein levels need to be evaluated to see the overall association. 506 Journal of Genetics, Vol. 95, No. 3, September 2016

3 MTHFR 677 C T: a meta-analysis Table 1. Comparison of genotypic frequencies, odds ratio and chi-square values. Total no. Total no. Chi-square Odds Study group of cases CC CT TT of controls CC CT TT (sig.) ratio 95% CI P Kaur and Kaur (2013), (78.2) 22 (20.0) 2 (1.8) (80.1) 22 (19.8) 0 (0.0) (0.359) present study (India) James et al. (1999), USA (26.3) 34 (59.6) 8 (14.0) (48) 22 (44) 4 (8) (0.062) to Hobbs et al. (2000), USA (32.4) 84 (53.5) 22 (14.0) (47.8) 59 (42.1) 14 (10) (0.025) to O Leary et al. (2002), Ireland (43.9) 21 (51.2) 2 (4.8) (46.8) 84 (43.7) 18 (9.3) (0.527) to Stuppia et al. (2002), Italy (31.2) 32 (50) 12 (18.7) (24.1) 62 (55.3) 23 (20.5) (0.587) to Chadefaux-Vekeman et al. (2002), (42.3) 42 (49.4) 7 (8.2) (41.4) 30 (42.8) 11 (15.7) (0.330) to France Boduroglu et al. (2004), Turkey (56.5) 55 (36.1) 11 (7.2) (63.7) 30 (32.9) 3 (3.2) (0.333) to Chango et al. (2005), France (36.1) 64 (53.7) 12 (10.0) (41.1) 58 (48.7) 12 (10.0) (0.709) to Da Silva et al. (2005), Brazil (43.5) 72 (46.7) 15 (9.7) (53.1) 67 (42.4) 7 (4.4) (0.084) to Acacia et al. (2005), Brazil (50) 30 (42.8) 5 (7.1) (61.3) 25 (28.4) 9 (10.2) (0.161) to Liang et al. (2005), China 30 7 (23.3) 20 (66.67) 3 (10) (22.8) 34 (48.57) 20 (28.57) (0.109) to Coppede et al. (2006), Italy (25.3) 43 (54.3) 16 (20.2) (35.1) 54 (48.6) 18 (16.2) (0.34) to Scala et al. (2006), Italy (32.9) 39 (41.4) 24 (25.5) (28.0) 125 (47.3) 57 (21.5) (0.475) to Rai et al. (2006), India (65.1) 40 (26.8) 12 (8.0) (75.1) 39 (23.6) 2 (1.2) (0.008) to Wang et al. (2007), China (28) 52 (52) 20 (20) (48) 42 (42) 10 (10) (0.008) to Kohli et al. (2008), India (71.8) 29 (28.1) 0 (0.0) (65.1) 32 (29.3) 6 (5.5) (0.048) Meguid et al. (2008), Egypt (47.6) 17 (40.4) 5 (11.9) (68.7) 12 (25.0) 3 (6.2) (0.156) to Wang et al. (2008), China (21.8) 32 (50) 18 (28.1) (51.4) 29 (41.4) 5 (7.1) (0.000) to Biselli et al. (2008), Brazil (40.2) 35 (48.6) 8 (11.1) (51.5) 77 (39.6) 17 (8.7) (0.262) to Martinez-Frias (2008), Spain (41.7) 61 (41.7) 24 (16.4) (40.4) 85 (45.2) 27 (14.3) (0.786) to Santos-Rebaucas et al. (2008), (49.5) 43 (41.7) 9 (8.7) (45.3) 47 (43.5) 12 (11.1) (0.767) to Brazil Cyril et al. (2009), India (91.6) 3 (8.3) (100) (0.095) Kokotas et al. (2009), Denmark (51.9) 72 (40.6) 13 (7.3) (50.2) 449 (41.4) 90 (8.3) (0.870) to Coppede et al. (2009), Italy (26.5) 52 (55.3) 17 (18.0) (35.3) 55 (48.6) 18 (15.9) (0.379) to Brandalize et al. (2009), Brazil (39.3) 113 (47.2) 32 (13.3) (43.6) 93 (47.2) 18 (9.1) (0.334) to Pozzi et al. (2009), Danish (37.8) 30 (40.5) 16 (21.6) (33.6) 93 (50.5) 29 (15.7) (0.300) to Coppede et al. (2010), Italy 29 5 (17.2) 19 (65.5) 5 (17.2) (34.3) 17 (53.1) 4 (12.5) (0.31) to Cretu et al. (2010), Rome (53.8) 10 (38.4) 2 (7.6) (39.1) 21 (45.6) 7 (15.2) (0.414) to Vranekovic et al. (2010), Crotia (44.1) 49 (44.1) 13 (11.7) (47.1) 64 (45.3) 11 (7.8) (0.572) to Liao et al. (2010), China (20) 26 (43.3) 22 (36.6) (33.8) 33 (48.5) 12 (17.6) (0.345) to Sadiq et al. (2011), Jordan (43.3) 27 (50.9) 3 (5.6) (79.3) 5 (17.2) 1 (3.4) (0.006) to Bozovic et al. (2011), Crotia (41.0) 55 (49.1) 11 (9.8) (45.7) 97 (43.8) 23 (10.4) (0.660) to El-Gharib et al. (2012), Egypt (22.5) 48 (60) 14 (17.5) (43.3) 12 (40) 5 (16.6) (0.083) to Tayeb (2012), Saudi Arabia (53.3) 10 (33.3) 4 (13.3) (55) 14 (35) 4 (10) (0.909) to Mohanty et al. (2012), India (84.6) 8 (15.4) (94.2) 3 (5.9) (0.202) Zampeiri et al. (2012), Brazil (38.1) 55 (52.4) 10 (9.5) (51.0) 73 (39.4) 18 (9.7) (0.087) to Pandey et al. (2013), India (83.7) 12 (15) 2 (2.5) (87) 9 (9) 3 (3) (0.486) to Divyakolu et al. (2013), India (84) 4 (16) (84) 8 (16) (0.738) Elsayed et al. (2014), Egypt (42.3) 12 (46.1) 3 (11.5) (49.1) 24 (39.3) 7 (11.4) (0.824) to Total (44.89) 1472 (43.28) 402 (11.82) (50.08) 2106 (39.94) 528 (10.00) (0.000) to Significant association. Journal of Genetics, Vol. 95, No. 3, September

4 Amandeep Kaur and Anupam Kaur Indian reports by Kohli et al. (2008) (χ , P 0.048) and Rai et al. (2006) (χ , P 0.008) showed a significant association among cases and controls. The frequency of TT genotype in a study by Rai et al. (2006) was 8% and showed a 7.13-fold increase risk of DS birth in mothers less than 31 years of age. Similarly, other reports also Table 2. Genetic models showing odds ratio and chi-square values. Model Odds ratio 95% CI Chi-square Dominant (CT/TT versus CC) , P = Codominant (TT/CT versus CT/CC) , P = Recessive (TT versus CC/CT) , P = James et al. (1999), USA 0.39 (0.16, 0.94) Hobbs et al. (2000), USA 0.52 (0.32, 0.86) O Leary et al. (2002), Ireland 0.89 (0.42, 1.84) Stuppia et al. (2002), Italy 1.43 (0.68, 2.98) Chadeaux-Vekemans et al. (2002), France 1.04 (0.52, 2.08) Boduroglu et al. (2004), Turkey 0.74 (0.42, 1.31) Chango et al. (2005), France 0.81 (0.46, 1.41) Da Silva et al. (2005), Brazil 7.38 (3.23, 18.83) Acacio et al. (2005), Brazil 0.39 (0.20, 0.75) Liang et al. (2006), China 1.03 (0.31, 3.09) Scala et al. (2006), Italy 1.21 (0.70, 2.06) Rai et al. (2006), India 0.62 (0.37, 1.03) Wang et al. (2007), C hina 0.42 (0.22, 0.79) Kohli et al. (2008), India 1.37 (0.73, 2.56) Meguid et al. (2008), Egypt 0.44 (0.17, 1.12) Wang et al. (2007), C hina 0.26 (0.11, 0.60) Biselli et al. (2008), Brazil 0.63 (0.35, 1.14) Martinez-Frias et al. (2008), Spain 1.06 (0.67, 1.68) Santos et al. (2008), Brazil 1.18 (0.66, 2.10) Cyril et al. (2009), India 0.08 (0.00, 1.42) Kokotas et al. (2009), Denmark 1.07 (0.77, 1.49) Coppede et al. (2009), Italy 0.66 (0.35, 1.25) Brandalize et al. (2009), Brazil Pozzi et al. (2009), Danish 0.84 (0.56, 1.25) 1.20 (0.65, 2.17) Cretu et al. (2010), Rome 1.81 (0.62, 5.37) Vrenekovic et al. (2010), Crotia 0.90 (0.53, 1.53) Lio et al. (2010), China 0.49 (0.20, 1.17) Sadiq 2011 (Jordan) 0.20 (0.06, 0.62) Bozovic et al. (2011), Crotia 0.83 (0.51, 1.34) Gharib et al. (2012), Egypt 0.38 (0.14, 1.03) Tayeb 2012 (Saudi Arabia) 0.94 (0.33, 2.69) Mohanty et al. (2012), India 0.34 (0.05, 1.53) Zampeiri et al. (2012), Brazil Pandey et al. (2013), India Divyakolu et al. (2013), India 0.60 (0.35, 1.00) 0.66 (0.26, 1.65) 1.00 (0.23, 5.07) Elsayed et al. (2014), Egypt 0.76 (0.27, 2.10) Kaur and Kaur 2013 (India)our study 0.89 (0.44, 1.79) combined [fixed] 0.82 (0.74, 0.90) Figure 2. Forest plot (fixed effect) showing significant association between maternal SNP MTHFR 677 C T and birth of DS child (CC vs CT+TT). 508 Journal of Genetics, Vol. 95, No. 3, September 2016

5 MTHFR 677 C T: a meta-analysis James et al. (1999), USA Hobbs et al. (2000), USA O Leary et al. (2002), Ireland Stuppia et al. (2002), Italy Chadeaux-Vekemans et al. (2002), France Boduroglu et al. (2004), Turkey Chango et al. (2005), France Da Silva et al. (2005), Brazil Acacio et al. (2005), Brazil Liang et al. (2006), China Scala et al. (2006), Italy Rai et al. (2006), India Wang et al. (2007), China Kohli et al. (2008), India Meguid et al. (2008), Egypt Wang et al. (2008), China Biselli et al. (2008), Brazil Martinez-Frias et al. (2008), Spain Santos et al. (2008), Brazil Cyril et al. (2009), India Kokotas et al. (2009), Denmark Coppede et al. (2009), Italy Brandalize et al. (2009), Brazil Pozzi et al. (2009), Danish Cretu et al. (2010), Rome Vrenekovic et al. (2010), Crotia Lio et al. (2010), China Sadiq 2011 (Jordan) Bozovic et al. (2011), Crotia Gharib et al. (2012), Egypt Tayeb 2012 (Saudi Arabia) Mohanty et al. (2012), India Zampeiri et al. (2012), Brazil Pandey et al. (2013), India Divyakolu et al. (2013), India Elsayed et al. (2014), Egypt Kaur and Kaur 2013 (India)our study 0.39 (0.16, 0.94) 0.52 (0.32, 0.86) 0.89 (0.42, 1.84) 1.43 (0.68, 2.98) 1.04 (0.52, 2.08) 0.74 (0.42, 1.31) 0.81 (0.46, 1.41) 7.38 (3.23, 18.83) 0.39 (0.20, 0.75) 1.03 (0.31, 3.09) 1.21 (0.70, 2.06) 0.62 (0.37, 1.03) 0.42 (0.22, 0.79) 1.37 (0.73, 2.56) 0.44 (0.17, 1.12) 0.26 (0.11, 0.60) 0.63 (0.35, 1.14) 1.06 (0.67, 1.68) 1.18 (0.66, 2.10) 0.08 (0.00, 1.42) 1.07 (0.77, 1.49) 0.66 (0.35, 1.25) 0.84 (0.56, 1.25) 1.20 (0.65, 2.17) 1.81 (0.62, 5.37) 0.90 (0.53, 1.53) 0.49 (0.20, 1.17) 0.20 (0.06, 0.62) 0.83 (0.51, 1.34) 0.38 (0.14, 1.03) 0.94 (0.33, 2.69) 0.34 (0.05, 1.53) 0.60 (0.35, 1.00) 0.66 (0.26, 1.65) 1.00 (0.23, 5.07) 0.76 (0.27, 2.10) 0.89 (0.44, 1.79) combined [random] 0.78 (0.65, 0.92) Figure 3. Forest plot (random effect) showing significant association between MTHFR 677 C T and DS child (CC vs CT+TT). suggested that homozygous mutant (TT) genotype is a risk factor for DS and its presence increases the risk of DS by folds (Hobbs et al. 2000; Wang et al. 2007, 2008; Sadiq et al. 2011). Some Indian and international reports failed to show any significant association between cases and controls when analysed individually. However, the genetic models were best fit for this analysis, demonstrating the presence of MTHFR 677 C T polymorphism either in dominant or recessive form, playing a significant role as one of the risk factor for the birth of DS (table 2). The variation in frequency of T allele may be due to ethnicity, lifestyle and availability of dietary folate that minimizes the effect of MTHFR 677 C T polymorphism. Large-scale studies are needed to rule out the exact mechanism behind the role of homocystein, folate, vitamin B levels in combination with other polymorphisms in folate pathway. Literatures of various meta-analysis suggest significant association between presence of maternal polymorphism and birthofdschild.wuet al. (2014) provided the result on 28 publications that included 2806 cases and 4597 control mothers for MTHFR 677 C T analysis. They also performed Journal of Genetics, Vol. 95, No. 3, September

6 Amandeep Kaur and Anupam Kaur Rai et al. ( 2006), India 0.62 (0.37, 1.03) Liang et al. (2006), China 1.03 (0.31, 3.09) Wang et al. ( 2007), China 0.42 (0.22, 0.79) Kohli et al. (2008), India 1.37 (0.73, 2.56) Wang et al. (2008), China 0.26 (0.11, 0.60) Cyril et al. (2009), India 0.08 (0.00, 1.42) Lio et al. ( 2010), China 0.49 (0.20, 1.17) Sadiq 2011 (Jordan) 0.20 (0.06, 0.62) Tayeb 2012 (Saudi Arabia) 0.94 (0.33, 2.69) Mohanty et al. ( 2012), India 0.34 (0.05, 1.53) Pandey et al. (2013), India Divyakolu et al. (2013), India Kaur and Kaur 2013 (India) our study 0.66 (0.26, 1.65) 1.00 (0.23, 5.07) 0.02 (0.00, 0.17) combined [random] 0.53 (0.36, 0.78) Figure 4. Forest plot (random plot) showing significant association between MTHFR 677 C T and DS child among Asians (CC vs CT+TT). James et al. (1999), USA 0.39 (0.16, 0.94) Hobbs et al. (2000), USA 0.52 (0.32, 0.86) Da Silva et al. (2005), Brazil 0.68 (0.42, 1.09) Acacio et al. (2005), Brazil 0.39 (0.20, 0.75) Biselli et al. (2008), Brazil 0.63 (0.35, 1.14) Santos et al. (2008), Brazil 1.18 (0.66, 2.10) Brandalize et al. (2009), Brazil 0.84 (0.56, 1.25) Zamoeiri et al. (2012), Brazil 0.60 (0.35, 1.00) combined [fixed] 0.66 (0.55, 0.79) Figure 5. Forest plot (fixed effect) showing significant association between MTHFR 677 C T and risk of DS child in American studies (CC vs Ct+TT). subgroup analysis and revealed significant association. Other analyses by Medica et al. (2009), Rai et al. (2014) and Victorino et al. (2014) have suggested similar findings. Yang et al. (2013) also reported significant association but did not perform subgroup analysis. In our meta-analysis, a strong association of MTHFR 677 C T was observed and we have also performed subgroup analysis and genetic models to estimate the association. On the other hand, Zintazaras (2007) did not observe significant involvement of MTHFR 677 C T polymorphism. Similarly, Costa-Lima et al. (2013) conducted meta-analysis on 20 publications and reported moderate relationship for maternal MTHFR 677 C T using codominant model only. The difference in the results may be due to difference in the number of publications included and various approaches used to demonstrate the association. Our study have few limitations: we analysed only single polymorphism, unadjusted OR in the analysis was used, no environmental factors were considered, foreign language and unpublished reports were not included. However, advantages were: there were more number of studies in this metaanalysis (37 studies), overlapping studies were excluded and subgroup analysis was also conducted. In conclusion, our meta-analysis suggests that exhibiting TT genotype significantly increases the risk for DS. However, this risk varies across different ethnicities and DS results from the interaction of genetic and environmental 510 Journal of Genetics, Vol. 95, No. 3, September 2016

7 MTHFR 677 C T: a meta-analysis O leary et al. (2002), Ireland 0.89 (0.42, 1.84) Stuppia et al. (2002), Italy 1.43 (0.68, 2.98) Chadeaux-Vekemans et al. (2002), France 1.04 (0.52, 2.08) Boduroglu et al. (2004), Turkey 0.74 (0.42, 1.31) Chango et al. (2005), France 0.81 (0.46, 1.41) Scala et al. (2006), Italy 1.21 (0.70, 2.06) Martinez-Frias et al. (2008), Spain 1.06 (0.67, 1.68) Kokotas et al. (2009), Denmark 1.07 (0.77, 1.49) Coppede et al. (2009), Italy 0.66 (0.35, 1.25) Pozzi et al. (2009), Danish 1.20 (0.65, 2.17) Cretu et al. (2010), Rome 1.81 (0.62, 5.37) Vrenekovic et al. (2010), Crotia 0.90 (0.53, 1.53) Bozovic et al. (2011), Crotia 0.83 (0.51, 1.34) combined [fixed] 0.98 (0.85, 1.13) Figure 6. Forest plot (fixed effect) showing significant association between MTHFR 677 C T and birth of DS child among European studies (CC versus CT+TT). Figure 7. Standard error by log odds ratio for dominant model. factors as well. Considering all such factors in future studies will lead to better understanding of association between SNPs and birth of DS child. Acknowledgements We gratefully acknowledge the support from DST grant and fellowship (SR/WOS-A/LS-348/2013) awarded to Amandeep Kaur and in part (UGC) grant no. F.37190/2009 (SR) awarded to Anupam Kaur. References Acacia G. L., Barini R., Bertuzzo C., Couto E. C., Annichino- Bizzachi J. M. and Junior W. P Methylenetetrahydrofolate reductase gene polymorphisms and their association with trisomy 21. Prenat. Diagn. 25, Begg C. B. and Mazumdar M Operating characteristics of a rank correlation test for publication bias. Biometrics 50, Biselli J. M., Golonani-Betollo E. C., Zampieri B. L., Haddad R., Eberlin M. N. and Pavarino-Bertelli E. C Genetics polymorphisms involved in folate metabolism and elevated plasma concentration of homocystein: maternal risk factors for Down syndrome in Brazil. Genet. Mol. Res. 7, Boduroglu K., Alanay Y., Koldan B. and Tuncbilek E Methylenetetrahydrofolate reductase enzyme polymorphisms as maternal risk for Down syndrome among Turkish women. Am. J. Med. Genet. A 127A, Bozovic I. B., Vranekovic J., Cizmarevic N. S., Mahulja- Stamenkovic V., Prpic I. and Brajenovic-Milic B MTHFR C677T and A1298C polymorphisms as a risk factor for congenital heart defects in Down syndrome. Pediatr. Int. 53, Brandalize A. P., Bandinelli E., Dos Santos P. A., Roisenberg I. and Schuller-Faccini L Evaluation of C677T and A1298C polymorphisms of the MTHFR gene as maternal risk factor for Journal of Genetics, Vol. 95, No. 3, September

8 Amandeep Kaur and Anupam Kaur Down syndrome and congenital heart defects. Am. J. Med. Genet. A 149A, Chadefaux-Vekeman B., Caude M., Muller F., Oury J. F., Chabli A., Jais J. et al Methylenetetrahydrofolate reductase polymorphism in the etiology of Down syndrome. Pediatr. Res. 51, Chango A., Fillon-Emery N., Mircher C., Blehaut H., Lambert D., Herbeth B. et al No association between common polymorphisms in genes of folate and homocystein metabolism and risk of Down syndrome among French mothers. Br. J. Nutr. 96, Coppede F., Margini G., Bargagna S., Stuppia L., Minichilli F., Fontana I., Coloqnato R. et al Folate gene polymorphisms an risk of Down syndrome pregnancies in young Italian women. Am. J. Med. Genet. A 140, Coppede F., Migheli F., Bargagna S., Siciliano G., Antonucci I., Stuppia L. et al Association of maternal polymorphisms in folate metabolizing genes with chromosome damage and risk of Down syndrome offspring. Neurosci. Lett. 499, Coppede F., Grossi E., Migheli F. and Migliore L Polymorphisms in folate metabolizing genes, chromosome damage and risk of Down syndrome in Italian mothers: identification of key factors using artificial neural networks. BMC Med. Genet. 3, 42. Costa-Lima M. A., Amorim M. R. and Orioli I. M Association of methylenetetrahydrofolate reductase gene 677 C>T polymorphism and Down syndrome. Mol. Biol. Rep. 40, Cretu R., Neagos D., Tutulan-Cunita A., Bohiltea L.C. and Stolian V MTHFR gene polymorphisms and prenatal risk of Down syndrome. Alexandra Loan Guza. Genet. Mol. Biol. 19, Cyril C., Rai P., Chandra N., Gopinath P. M. and Satyamoorthy K MTHFR gene variants C677T, A1298C and association with Down syndrome: a case control study from South India. Ind. J. Hum. Genet. 15, Da Silva L., Vergani N., Galdieri L. C., Ribeiro P. M., Longhitano S. B., Brunoni D. et al Relationship between polymorphisms in genes involved in homocystein metabolism and maternal risk factor for Down syndrome in Brazil. Am. J. Med. Genet. A 135, DerSimonian R. and Laird N Meta-analysis in clinical trials. Control. Clin. Trials 7, Divyakolu S., Tejaswani Y., Thomas W., Thumoju S., Sreekanth V. R., Vasavi M. et al Evaluation of C677T polymorphism of the methylenetetrahydrofolate reductase (MTHFR) gene in various neurological disorders. J. Neurol. Disord. 2, 142. Egger M., Davey S.G., Schneider M. and Minder C Bias in meta-analysis detected by a simple, graphical test. BMJ 315, El-Gharib M. N., Mahfouz A. E. and Hammoudah S. A Maternal MTHFR gene polymorphisms and risk of Down syndrome offspring. Int. J. Med. Biomed. Res. 1, Elsayed G. M., Elsayed S. M. and Ezz-Elarab S. S Maternal MTHFR C677T genotype and septal defects in offsprings with Down syndrome: a pilot study. Egyptian J. Med. Hum. Genet. 15, Fenech M The role of folic acid and vitamin B12 in genomic stability of human cells. Mutat. Res. 475, Hobbs C. A., Sherman S. L., Yi P., Hopkins S. E., Torfs C. P., Hine R. J. et al Polymorphisms in genes involved in folate metabolism as maternal risk factors for Down syndrome. Am. J. Hum. Genet. 67, James S. J., Pogribna M., Pogribny I. P., Melnyk S., Hine R. J., Gibson J. B. et al Abnormal folate metabolism and mutation in methylenetetrahydrofolate reductase gene may be maternal risk factor for Down syndrome. Am.J.Clin.Nutr.70, Kaur A. and Kaur A Prevalence of methylenetetrahydrofolate reductase 677 C T polymorphism among mothers of Down syndrome children. Ind. J. Hum. Genet. 19, Kohli U., Arora S., Kabra M., Ramakrishanan I., Gulati S. and Pandey R. M Prevalence of MTHFR C677T polymorphism in north Indian mothers having babies with Trisomy 21 Down syndrome. Downs Syndr. Res. Pract. 12, Kokotas H., Grigoriadou M., Mikkelson M., Gannaoulia-Karantana A. and Petersen M. B Investigating the impact of Down syndrome related common MTHFR 677 C>T polymorphism in Danish population. Dis. Markers 27, Liang X., Feng Z., Lan-Fang Z., Guo X. X., Xiao G. F. et al Analysis of Down syndrome screening and antenatal diagnosis of 3195 cases in the middle period of pregnancy. China J. Modern Med. 20,11. Liao Y. P., Bao M. S., Liu C. Q., Liu H. and Zhang D Folate gene polymorphism and the risk of Down syndrome pregnancies in young Chinese women. Yi Chuan. 32, Mantel N. and Haenszel W Statistical aspects of the analysis of data from retrospective studies of disease. J. Natl. Cancer Inst. 22, Martinez-Frias M. L The biochemical structure and function of methylenetetrahydrofolate reductase provide the rationale to interpret the epidemiological results on the risk for infants with Down syndrome. Am. J. Med. Genet. A 146A, Medica I., Maver A., Augusto G. and Peterlin B Polymorphisms in genes involved in folate metabolism as maternal risk factors for Down syndrome-meta analysis. Open Med. 4, Meguid N. A., Dardir A. A., Khass M., Hossieny L. E., Ezzat A. and El-Awady M. K MTHFR genetic polymorphism as a risk factor in Egyptian mothers in Down syndrome children. Dis. Markers 28, Mohanty P. K., Kapoor S., Dubey A. P., Pandey S., Shah R., Nayak H. K. et al Evaluation of C677T polymorphism of the methylenetetrahydrofolate reductase gene and its association with levels of serum homocystein folate and vitamin B12 as maternal risk factors for Down syndrome. Ind. J. Hum. Genet. 18, O Leary V. B., Paele-Mcdermott A., Molloy A. M., Kirke P. N., Johnson Z., Conley M. et al MTRR and MTHFR polymorphisms: link to Down syndrome? Am.J.Med.Genet.10, Pandey S. K., Mohanty P. K., Polipalli S. K. and Kapoor S Genetic polymorphisms of MTHFR (C677T and A1298C) and homocystein metabolism as a risk factor for Down s syndrome patients in north Indian mothers. Int. J. Pharma. Biosci. 4, Patel Z. M. and Adhia R. A Birth defects surveillance study. Ind. J. Pediatr. 72, Pozzi E., Vergani P., Dalpra L., Combi R., Silvestri D., Crosti F. et al Maternal polymorphism for methylenetetrahydrofoalte reductase and methionine synthase reductase and risk of children with Down syndrome. Am. J. Obstet. Gynecol. 200, e1 e6. Rai A. K., Singh S., Mehra S., Kumar A., Pandey I. K. and Raman R MTHFR C677T and A1298C polymorphisms are risk factors for Down syndrome in Indian mothers. J. Hum. Genet. 51, Rai V., Yadav U., Kumar P., Yadav S. K. and Mishra O. P Maternal methylenetetrahydrofolate reductase C677T polymorphism and Down syndrome risk: a meta-analysis from 34 studies. PLoS One 9, e Sadiq M. F., Al-Refai E. A., Al-Nasser A., Khassawneh M. and Al-Batayneh Q Methylenetetrahydrofolate reductase polymorphisms C677T and A1298C as maternal risk factors for 512 Journal of Genetics, Vol. 95, No. 3, September 2016

9 MTHFR 677 C T: a meta-analysis Down syndrome in Jordan. Genet. Test Mol. Biomarkers 15, Santos-Rebaucas C. B., Correa J. C., Bonomo A., Fintelman- Rodrigues N., Moura K. C., Rodriguez C. S. et al The impact of folate pathway polymorphisms combined to nutritional deficiency as maternal predisposition factor for Down syndrome. Dis. Markers 25, Scala I., Granese B., Sellitto M., Salome S., Sammartino A., Pepe A. et al Analysis of seven maternal polymorphisms of genes involved in homocystein/folate metabolism and risk of Down syndrome offspring. Genet. Med. 8, Stuppia L., Gatta V., Gaspari A. R., Antonucci I., Morizio E., Calabrese G. et al C677T mutation in the 5,10 MTHFR gene and risk of Down syndrome in Italy. Eur. J. Hum. Genet. 10, Tayeb M. T The methylenetetrahydrofolate reductase gene variant (C677T) in risk mothers with Down syndrome among Saudi population. Egyptian J. Med. Hum. Genet. 13, Victorino D. B., Godoy M. F., Goloni-Bertollo E. M. and Pavarino E. C Meta-analysis of methylenetetrahydrofolate reductase maternal gene in Down syndrome: increased susceptibility in women carriers of the MTHFR 677T allele. Mol. Biol. Rep. 41, Vranekovic J., Babic Bozovic I., Starcevic Cizmarevic N., Buretic- Tomljanovic A., Ristic S., Petrovic O. et al Functional inference of methylenetetrahydrofolate reductase gene polymorphisms on enzyme stability as a potential risk factor for Down syndrome in Croatia. Dis. Markers 28, Wang W., Xei W. and Wang X The relationship between polymorphism of gene involved in folate metabolism, homocystein level and risk of Down syndrome. Zhonghua Yi Xue Yi Chuan Xue Za Zhi 24, Wang S. S., Qiao F. Y. and Lv J. J Polymorphisms in genes involved in folate metabolism as maternal risk factors for Down syndrome in China. J. Zhejiang Univ. Sci. B 9, Wu X., Wang X., Chan Y., Jia S., Luo Y and Tang W Folate metabolism gene polymorphisms MTHFR C677T and A1298C and risk of for Down syndrome offspring: a meta analysis. Eur. J. Obstet. Gynec. Rep. Biol. 167, Yang M., Gong T., Lin X., Qi L., Guo Y., Cao Z. et al Maternal gene polymorphisms involved in folate metabolism and the risk of having Down syndrome offspring: a meta analysis. Mutagenesis 28, Yaping Liao M. P. C. L Folate gene polymorphism and the risk of Down syndrome pregnancies in young Chinese women. Yi Chuan 32, Zampeiri B. L., Biselli L. M., Goloni-Bertollo E. M., Vannuchi H., Carvatho V. M., Cordeiro J. A. et al Maternal risk for Down syndrome is modulated by genes involved in folate metabolism. Dis. Markers 32, Zintazaras E Maternal gene polymorphisms involved in folate metabolism and risk of Down syndrome offspring: a meta analysis. J. Hum. Genet. 52, Received 23 June 2015, in revised form 23 September 2015; accepted 24 November 2015 Unedited version published online: 30 November 2015 Final version published online: 4 July 2016 Journal of Genetics, Vol. 95, No. 3, September