Title: Maternal MTHFR polymorphism (677 C-T) and risk of Down syndrome child Metaanalysis

Transcription

1 1 Research Article Title: Maternal MTHFR polymorphism (677 C-T) and risk of Down syndrome child Metaanalysis Amandeep Kaur (M.Sc.) Women Scientist (DST, WOS-A) Department of Human Genetics Guru Nanak Dev University, Amritsar, Punjab, India Anupam Kaur (Ph.D.) (Corresponding author) Professor Department of Human Genetics Guru Nanak Dev University, Amritsar, Punjab, India Key words: MTHFR, Down syndrome, folate metabolism

2 2 Abstract Methytlenetetrahydrofolate reductase (MTHFR) is the most important gene that participates in folate metabolism. Presence of valine instead of alanine at the position 677 and elevated homocystein level causes DNA hypomethylation which in turn favors non-disjunction. We conducted a meta-analysis to establish link between maternal SNP and birth of Down syndrome child. A total of 37 case-control studies were selected for analysis including our own study, in which we investigated 110 cases and 111 control mothers. The result of meta-analysis showed significant overall 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, highly significant association between homozygous mutant TT genotype and birth of DS child (χ² , p=0.000). Genetic models suggested that T allele possess high risk for DS whether present in dominant (OR=1.23, 95% CI= ); co-dominant (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.

3 3 Introduction Down syndrome (DS) is the most commonly occurring chromosomal abnormality that occurs with the prevalence rate of 1 in 1200 live births (Patel and Adhia 2005) due to non-disjunction 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 favors non-disjunction 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 SNPs for the risk of DS child. The present metaanalysis 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). All the selected reports were case control studies. The key words used were: MTHFR 677 C-T, Down 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 full length papers were eligible for the study. For each data, author, country and year of publication along with total number of cases and controls were extracted. Odds ratio, chi square values along with frequencies were calculated (Table 1). Inclusion Criteria: a) Articles in English language, b) articles evaluating MTHFR 677 C-T SNP, c) only those articles were considered in which mothers had at least two normal children without

4 4 miscarriages and abnormalities, d) reports with genotypic and allelic frequency, Odds ratio and Chi square were considered. Statistical Analysis: The meta-analysis was done using STATS DIRECT 3. Risk for the birth of DS child and presence of maternal polymorphism was assessed by Odds Ratio (OR) with 95% CI. Presence of heterogeneity was estimated using chi-square based Q-statistics and I² metrics. In case of higher heterogeneity (I²>50%) random effect model (DerSimonian and Lard, 1986) would be used, otherwise fixed effect model (Mantel and Haenszel, 1959) would be 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. Results The flow chart shows that we identified 37 eligible reports from a total of 182 articles published between ( ). The reports were from different ethnic population i.e. Asians, Americans, Europeans and others. A total of 3401 mothers having DS children and 5277 mothers having normal children were included. Meta-analysis of the studies indicated a highly significant difference between cases and controls (χ² , p=0.000). The three genetic models (dominant: OR=1.23, 95% CI= ; co-dominant: OR=1.17, 95% CI= ; recessive: OR=1.21, 95% CI= ) suggested that presence of TT genotype in mothers significantly increases the risk for DS birth (Table II). The frequency of heterozygote was highest among MDS (8.3%- 65.5%) 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 I}. The present analysis revealed that presence of MTHFR 677 C-T in mothers is the major risk factor for the birth of DS child. Both fixed effect model (OR=0.816, 95% CI= , p< ) [Figure 2] and random effect model (OR=0.776, 95% CI= , p=0.003)

5 5 suggested that maternal polymorphism 677 C-T significantly increases the risk of birth of DS child (Figure 3). The random model was used because the results demonstrated high magnitude of heterogeneity among studies (I²=62.6%, p<0.0001, Q=96.26, df=36). Sub-group 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 (I²=58.9%, Q=29.17, df=12, 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=0.0001) (Figure 5). However, heterogeneity was found to be of low magnitude (I²=39.9%, Q=11.64, p=0.113, df=7). On the other hand, no association and no heterogeneity was found among European studies (Fixed model OR=0.982, 95% CI= , p=0.838, I²=0%, Q=8.264, p=0.764, df=12) (Figure 6). Publication Bias: Publication Bias was not observed among the studies as indicated by Begg- Mazumdar bias p=0.23, Egger s bias p=0.184 for CC vs 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 (χ²-2.047, 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).

6 6 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; DaSilva et al. 2005; El-Gharib et al. 2012; El-Sayed 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 The non-significant 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 first to report that mutant genotype TT is associated with the increased risk of DS. In his study, the frequency of heterozygote was much higher (59.6%) as compared to controls (44%). It was suggested that predominance of heterozygote among MDS was due to fetal viability that may be lower in mothers with the TT genotype. Several studies observed non-significant association, but presence of mutant allele increased the risk of DS ranging from 0.4%-2.7% (Table I). 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 overall association. Indian reports by Kohli et al. (2008) (χ²-6.054, p-0.048) and Rai et al. (2006) (χ²-9.664, 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 increased risk of DS birth in mothers less than 31 years of age. Similarly, other reports also 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. 2002; Sadiq et al. 2011; Wang et al. 2007; 2008).

7 7 Some Indian and International reports failed to show any significant association between cases and controls, when analyzed individually. However, the genetic models were best fit in this analysis demonstrating the presence of MTHFR 677 C-T polymorphism either in dominant or recessive form and playing a significant role as one of the risk factor for the birth of DS (Table II). 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 search of various meta-analysis suggest significant association between presence of maternal polymorphism and birth of DS child. Wu et al provided the result on 28 publications that included 2806 cases and 4597 control mothers for MTHFR 677 C-T analysis. They also performed sub-group analysis and revealed significant association. Other analyses by Medica et al. 2009; Rai et al and Victorino et al have suggested similar findings. Yang et al also reported significant association but did not perform sub-group analysis. In our meta-analysis, strong association of MTHFR 677 C-T was observed and we have also performed sub-group analysis and genetic models to estimate the association. On the other hand, Zintzaras 2007 did not observe significant involvement of MTHFR 677 C-T polymorphism. Similarly, Costa-Lima et al did meta-analysis on 20 publications and reported moderate relationship for maternal MTHFR 677 C-T using co-dominant 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 had few limitations: we analyzed only single polymorphism, unadjusted OR in the analysis was used, no environmental factors were considered, foreign language and unpublished

8 8 reports were not included. However, advantages were: more number of studies (37 studies) in this meta-analysis, overlapping studies were excluded and sub-group 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 interaction of genetic and environmental factors as well. Considering all such factors in future studies, will lead to better understanding of association between SNPs and birth of DS child. Acknowledgement: 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 /2009 (SR) awarded to Dr. Anupam Kaur. References: 1) Patel Z.M and Adhia R.A Birth defects surveillance study. Ind J of Pediatr. 72, ) James S.L., Pogribna M., Pogribny I.P. et al Abnormal folate metabolism and mutation in methylenetetrahydrofolate reductase gene may be maternal risk factor for Down syndrome. Am J Clin Nutr. 70, ) Fenech M The role of folic acid and vitamin B12 in genomic stability of human cells. Mutat Res. 475,

9 9 4) Kaur A., and Kaur A Prevalence of methylenetetrahydrofolate reductase 677 C-T polymorphism among mothers of Down syndrome children. Ind J Hum Genet. 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, ) 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: ) 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. Indian J Hum Genet. 18, ) 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, ) 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, ) 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, 5-10.

10 10 12) 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., Schuller-Faccini L. et al Evaluation of C677T and A1298C polymorphisms of the MTHFR gene as maternal risk factor for 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. et al Folate gene polymorphisms and 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 anf risk of Down syndrome in Italian mothers: identification of key factors using artificial neural networks. BMC Med Genet. 3, ) 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 and Mol Biol. 19,

11 11 20) 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., Laird N Meta-analysis in clinical trials. Controlled Clini Trials. 7, ) Egger M., Davey S.G., Schneider M., 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, ) 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, ) Mantel N., Haenszel W 1959 Statistical aspects of the analysis of data from retrospective studies of disease. J Natl Cancer Inst. 22, ) Martinez-Frias M.L The biochenmical structiure and funcion 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, ) 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, ) O Leary V.B., Paele-Mcdermott A., Molloy A.M., Kirke P.N., Johnson Z., Conley M. et al MTRR and MTHFR polymorphism: Link to Down syndrome? Am J Med Genet. 10,

12 12 30) 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 Medicine. 20, ) 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, 636e1-636e6. 32) 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., Pavarino E.C Meta-analysis of Methylenetetrahydrofolate reductase maternal gene in Down syndrome: increased susceptibility in women carriers of the MTHFR 677T allele. Mol boil Rep. 41, ) Vranekovic J., Babic Bozovic I., Starcevic Cizmarevic N., Buretic-Tomljanovic A., Ristic S., Petrovic O. et al Functional inference of methylenetetrahydrofolate reductase

13 13 gene polymorphisms on enzyme stability as a potential risk factor for Down syndrome in Crotia. Dis markers. 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, ) 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. Down s Syndr Res. Pract, 12, ) 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, ) 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, ) 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 Down syndrome in Jordan. Genet Test Mol Biomarkers. 15, ) 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, Received 23 June 2015, in revised form 23 September 2015; accepted 24 November 2015 Unedited version published online: 30 November 2015

14 14 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

15 15 Odds ratio meta-analysis plot [fixed effects] 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 (China) 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 (China) 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) 0.84 (0.56, 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) 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) 0.60 (0.35, 1.00) Pandey et al 2013 (India) 0.66 (0.26, 1.65) Divyakolu et al 2013 (India) 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) showed significant association between maternal SNP MTHFR 677 C-T and birth of DS child (CCvs CT+TT)

16 16 Odds ratio meta-analysis plot [random effects] 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 (China) 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 2008 (China) 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) 0.84 (0.56, 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) 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 ey al 2012 (India) 0.34 (0.05, 1.53) Zampeiri et al 2012 (Brazil) 0.60 (0.35, 1.00) Pandey e al 2013 (India) 0.66 (0.26, 1.65) Divyakolu et al 2013 (India) 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 [random] 0.78 (0.65, 0.92) Figure 3: Forest plot (Random effect) showed significant association between MTHFR 677 C-T and DS child (CCvs CT+TT)

17 17 Odds ratio meta-analysis plot [random effects] 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) 0.66 (0.26, 1.65) Divyakolu et al 2013 (India) 1.00 (0.23, 5.07) Kaur and Kaur 2013 (India) our study 0.02 (0.00, 0.17) combined [random] 0.53 (0.36, 0.78) Figure 4: Forest plot (Random Plot) showed significant association between MTHFR 677 C-T and DS child among Asians (CCvs CT+TT)

18 18 Odds ratio meta-analysis plot [fixed effects] 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) showed significant association between MTHFR 677 C-T and risk of DS child in American studies (CCvs Ct+TT)

19 19 Odds ratio meta-analysis plot [fixed effects] 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) showed significant association between MTHFR 677 C-T and birth of DS child among European studies (CCvs CT+TT)

20 Table 1: Comparison of genotypic frequencies, Odds ratio and chi square values 20

21 Study group Kaur and Kaur 2013 Present study (INDIA) 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 2005 (China) Coppede et al 2006 (Italy) 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) Coppede et al 2010 (Italy) Cretu et al 2010(Rome) Vrenekovic et al 2010(Crotia) Total no. of cases 110 CC CT TT 86 (78.2) 22 (20.0) 2 (1.8) Total no. of contr ols 111 CC CT TT 89 (80.1) 22 (19.8) Chi-Square (Sig.) Odds ratio 95% CI p-value 21 0(0.0) (0.359) to 24 (48) 22 (44) 4 (8) (0.062) (26.3) (59.6) (14.0) to 14 (10) (0.025) (32.4) (53.5) (14.0) (47.8) (42.1) to 2 (4.8) 18 (9.3) (0.527) (43.9) (51.2) (46.8) (43.7) to 32 (50) (0.587) (31.2) (18.7) (24.1) (55.3) (20.5) to 7 (8.2) (0.330) (42.3) (49.4) (41.4) (42.8) (15.7) to 3 (3.2) (0.333) (56.5) (36.1) (7.2) (63.7) (32.9) to (0.709) 1 (36.1) (53.7) (10.0) (41.1) (48.7) (10.0) to 7 (4.4) (0.084) (43.5) (46.7) (9.7) (53.1) (42.4) (50) 5 (7.1) 9 (10.2) (0.161) (42.8) (61.3) (28.4) (10) (0.109) (23.3) (66.67) (22.8) (48.57) (28.57) to (0.34) (25.3) (54.3) (20.2) (35.1) (48.6) (16.2) to (0.475) (32.9) (41.4) (25.5) (28.0) (47.3) (21.5) ( to 2 (1.2) (0.008) (65.1) (26.8) (8.0) (75.1) 6) to 28 (28) 52 (52) 20 (20) 48 (48) 42 (42) 10 (10) (0.008) (0.0) 6 (5.5) (0.048) - - (71.8) (28.1) (65.1) (29.3) to 3 (6.2) (0.156) (47.6) (40.4) (11.9) (68.7) (25.0) to 32 (50) 5 (7.1) (21.8) (28.1) (51.4) (41.4) (0.000) to 17 (8.7) (0.262) (40.2) (48.6) (11.1) (51.5) (39.6) to (0.786) (41.7) (41.7) (16.4) (40.4) (45.2) (14.3) to 9 (8.7) (0.767) (49.5) (41.7) (45.3) (43.5) (11.1) (8.3) (0.095) - - (91.6) (100) (8.3) (0.870) (51.9) (40.6) (7.3) (50.2) (41.4) to (0.379) (26.5) (55.3) (18.0) (35.3) (48.6) (15.9) ( to 18 (9.1) (0.334) (39.3).2) (13.3) (43.6) (47.2) to (0.300) (37.8) (40.5) (21.6) (33.6) (50.5) (15.7) to 4 (12.5) (0.31) (17.2) (65.5) (17.2) (34.3) (53.1) to 2 (7.6) 7 (15.2) (0.414) (53.8) (38.4) (39.1) (45.6) to 11 (7.8) (0.572) (44.1) (44.1) (11.7) (47.1) (45.3) Lio et al to 12 (20) (0.345)

22 22 Models Odds Ratio 95% CI Chi-square Dominant (CT/TT vs. CC) 1.23 ( ) , p= Co-dominant (TT/CT vs ( ) , p= CT/CC) Recessive (TT vs. CC/CT) 1.21 ( ) 7.047, p= Table II: Genetic models showing Odds ratio and chi square values