MTRR 66A > G Polymorphism as Maternal Risk Factorfor Down Syndrome: A Meta-Analysis

Marcia R. Amorim1 and Marcelo A. Costa Lima2

Down syndrome (DS) is the most common cause of mental retardation. Recent reports have investigated pos-sible genetic factors that may increase maternal risk for DS. Methionine synthase reductase (5-methyltetra-hydrofolate-homocysteine methyltransferase reductase MTRR) plays an important role in folic acid pathway anda common polymorphism (c.66A > G) has been associated with DS but results were controversial. This meta-analysis summarizes the available data concerning this association. Online major databases were searched toidentify case-control studies regarding MTRR 66A > G polymorphism and DS. Crude odds ratios (OR) and 95%confidence intervals (CI) were calculated for maternal risk to have a DS child both using fixed and randomeffects (RE) models. Eleven articles from six populations were identified, including 1226 DS mothers and 1533control mothers. Heterogeneity among studies was significant (Q = 29.7, DF = 10, p = 0.001; I2 = 66.3%). Thepooled OR in a RE model showed an increase in the risk of having a DS child associated with the G allele (OR1.23, 95% CI 1.02–1.49). The fixed effect pooled OR was 1.19 (95% CI 1.08–1.31). This meta-analysis indicates thatmaternal MTRR 66A > G polymorphism is associated with an increased risk of having a DS child.

Introduction

Down syndrome (DS) is the most common genetic causeof mental retardation in humans. This chromosomal

abnormality occurs in 1/700 liveborn infants, with a widespectrum of clinical phenotypes (Epstein et al., 1991). To date,only maternal age has been associated with an increased riskof DS (Epstein, 2001), however the causative mechanismsinvolved in the age-dependent occurrence of meiotic mis-segregation are still unclear. The facts that most DS childrenare born from younger women (Eskes, 2006), and the inci-dence of trisomy of chromosome 21 differs between popula-tions (Wiseman et al., 2009) indicate the concurrence of othergenetic components to increase the susceptibility to disjunc-tion errors.

It has been proposed that an altered maternal folate me-tabolism could be associated to centromeric DNA hypo-methylation and chromosomal nondisjunction ( James et al.,1999). Abnormal folate metabolism could also counteract theoverexpression of the three copies of the cystathionine betasynthase gene (CBS) in the trisomy 21 fetus, assuring folateavailability for both DNA synthesis and methylation (Hobbset al., 2000).

From the original paper of James et al. (1999) to date,several reports have investigated the influence of folatepathway polymorphisms on maternal risk for DS and the

most studied genes are methylenetetrahydrofolate reductase(MTHFR) and 5-methyltetrahydrofolate-homocysteine me-thyltransferase reductase, also known as methionine syn-thase reductase (MTRR). Results of the association betweensingle nucleotide polymorphisms within these genes and therisk of DS are controversial and discrepancies among reportshave been explained mostly by the nutritional environmentand genetic characteristics of the populations (Gueant et al.,2003).

The MTRR gene was mapped to chromosome 5p15.2–15.3(Leclerc et al., 1998) and encodes an enzyme responsible forrestoration of methionine synthase (MTR) activity by reduc-tive methylation of cobalamin, leading to methionine syn-thesis and production of S-adenosylmethionine, which is themain cellular methyl donor for transmethylation reactions(Wolthers and Scrutton, 2007). A common polymorphism inhuman MTRR is an adenine to guanine transition at position66 (c.66A > G), which results in replacement of isoleucine withmethionine at residue 22 (p.I22M) (Wilson et al., 1999). Thissubstitution decreases the ability of MTRR to restore MTRactivity in vivo (Olteanu et al., 2002).

In the last decade several reports have evaluated the as-sociation between MTRR 66A > G polymorphism and an in-creased risk of having a DS child, and the results arecontroversial. This meta-analysis summarizes published dataconcerning this association.

1Departamento de Biologia Geral, Instituto de Biologia, Universidade Federal Fluminense, Niteroi, Rio de Janeiro, Brazil.2Departamento de Genetica, Instituto de Biologia Roberto Alcantara Gomes, Universidade do Estado do Rio de Janeiro, Rio de Janeiro,

Brazil.

GENETIC TESTING AND MOLECULAR BIOMARKERSVolume 17, Number 1, 2013ª Mary Ann Liebert, Inc.Pp. 69–73DOI: 10.1089/gtmb.2012.0200

69

Methods and Analyses

Eligible studies were identified by searches in major liter-ature databases (PubMed at www.ncbi.nlm.nih.gov/pubmedand Web of Science at www.isiknowledge.com), using thefollowing keywords, alone and combined: ‘‘A66G,’’‘‘66A > G,’’ ‘‘MTRR,’’ ‘‘methionine synthase reductase,’’ ‘‘5-methyltetrahydrofolate-homocysteine methyltransferase re-ductase’’ and ‘‘Down syndrome’’ or ‘‘trisomy 21.’’ SCIELO (atwww.scielo.org) was also screened using the same keywordsin English, Portuguese and Spanish in order to assure therecovery of papers published in Latin American journals notindexed in the other databases. We included studies that metthe following criteria: (1) case-control studies; (2) use of vali-dated genotyping methods to identify the polymorphism; (3)availability of MTRR genotypes for Down syndrome mothers(DSM) and control mothers (CM), and (4) written in English,Portuguese or Spanish. Both authors have reviewed inde-pendently each article. Crude odds ratios (OR) and 95%confidence intervals (CI) for having a DS child were calculatedcomparing GG and/or AG with AA and comparing G allelewith A allele. Heterogeneity among studies was tested con-sidering that when there is heterogeneity among studies, thepooled OR is preferably estimated using the random effects(RE) model instead of fixed effects (FE) model. Q-statistics andI2 metrics were calculated as described (Cochran, 1954; Hig-gins and Thompson, 2002). Statistical analyses were per-formed using Meta-Disk (version 1.4). The frequencies wereevaluated for accordance to Hardy–Weinberg equilibrium(HWE) using the w2 test.

Results

We screened major bibliographic databases searching forarticles focusing on the association of maternal MTRR66A > G polymorphism and DS. We found 11 reports thatmet the inclusion criteria conducted in populations of dif-ferent ethnic backgrounds from seven countries: UnitedStates and Canada (Hobbs et al., 2000), Ireland (O’Leary et al.,2002), France (Chango et al., 2005), China (Wang et al., 2008),Italy (Scala et al., 2006; Coppede et al., 2009; Pozzi et al., 2009)and Brazil (da Silva et al., 2005; Santos-Reboucas et al., 2008;Brandalize et al., 2010; Zampieri et al., 2012). The reportsincluded 1233 DS and 1533 CM and details of the studies arepresented in Table 1.

The observed genotype frequencies were evaluated foraccordance to HWE. The distribution of genotypes were inagreement with HWE in all studies but one (Chango et al.,2005) (w2 = 12.19, p = 0.002).

The frequency of heterozygous genotype was the highest inboth DSM and CM, ranging from 42.7% to 60.0% in DSM and45.4% to 56.3% in CM. The genotype and allele frequencies areshown in Table 2.

Overall analysis of the association between the mutant al-lele and an increase in risk of DS has revealed significantheterogeneity among studies (Q = 29.7; DF = 10; I2 = 66.3%;p = 0.001), and the RE pooled OR was significant, indicating a20% increase (OR 1.23; 95% CI 1.02, 1.49) in the risk of havinga DS child (Fig. 1).

We also evaluated the association using different interac-tion models; recessive (GG vs. AG + AA), dominant (GG + AGvs. AA) and codominant (both GG vs. AA and AG vs. AA).The heterogeneity between-studies was significant using therecessive (Q = 25.8, DF = 10, p = 0.004, I2 = 61.2%) and domi-nant (Q = 19.1, DF = 10; p = 0.04, I2 = 47.6) models. No hetero-geneity was observed when AG genotype was compared toAA (Q = 12.2, DF = 10, p = 0.27, I2 = 17.8%) and the FE pooledOR was significant (OR 1.21, 95% CI 1.02–1.44) (Table 3).

Discussion

DS is an important public health issue and most DS indi-viduals need special services (education, medical, and social)for the duration of their lives. Since the first report of a pos-sible association between genetic polymorphisms in folate-metabolizing encoding genes and an increased DS risk ( Jameset al., 1999), several studies have investigated this hypothesis.

The distributions of MTRR 66A > G genotypes vary greatlyaccording to genetic background. The higher frequencies ofMTRR GG genotype were described in Caucasians and Indi-ans ranging from 28% to 35%, and the lowest (less than 7.5%)were observed in Latin descent populations (Rady et al., 2002;Yang et al., 2008; Rai et al., 2011). We also observed differencesin MTRR GG genotype frequencies among groups of differentethnicities, ranging from 14.7% in the Chinese population to40.3% in French population.

Hobbs et al. (2000) reported the association of maternalMTRR 66A > G polymorphism with DS risk in North Ameri-can population (including samples from 16 United States and

Table 1. Characteristics of the Studies Included in the Meta-Analysis

Author Year Population/ethnicity No. of DSM No. of CM pa

Hobbs et al. 2000 North American/Caucasian 145 139 0.97O’Leary et al. 2002 Irish/Caucasian 48 192 0.67Chango et al. 2005 French/Caucasian 119 119 0.01da Silva et al. 2005 Brazilian/Mixed 154 158 0.34Scala et al. 2006 Italian/Caucasian 93 257 0.94Santos-Reboucas et al. 2008 Brazilian/Mixed 103 108 0.63Wang et al. 2008 Chinese/Asian 64 68 0.94Pozzi et al. 2009 Italian/Caucasian 74 184 0.98Coppede et al. 2009 Italian/Caucasian 81 111 0.42Brandalize et al. 2010 Brazilian/Caucasian 239 197 0.20Zampieri et al. 2012 Brazilian/Mixed 105 185 1.00

ap-Value for Hardy–Weinberg equilibrium in CM group.DSM, Down syndrome mother; CM, control mother.

70 AMORIM AND COSTA LIMA

Canada). The comparison of genotypic distribution betweenDSM and CM indicated an increase of 2.57-fold (95% CI 1.33–4.99) for women with GG genotype. The risk was increased to4.08-fold (95% CI 1.94–8.56) when the homozygous GG ge-notype was combined to MTHFR TT genotype suggesting amultiplicative effect. This pattern of association was observedin the Chinese population, where maternal GG genotype in-creased 5.2-fold (95% CI 2.06–17.50) the risk. The multiplica-

tive effect of MTHFR TT and MTRR GG genotypes were alsoobserved, leading to a sixfold (95% CI 2.06–17.50) increasedrisk (Wang et al., 2008). An impressive 15-fold (95% CI 1.94–116.0) increased risk associated with homozygous GG geno-type was observed in the Irish population (O’Leary et al., 2002).

In France, there was no significant difference betweenDSM and CM MTRR G allele frequencies (Chango et al.,2005). The authors emphasized the importance of correlating

Table 2. The Distribution of Methionine Synthase Reductase 66A > G Genotypic

and Allelic Frequencies for Down Syndrome Mothers and Control Mothers

MTRR 66A > G genotype [n (%)] MTRR 66A > G allele [n (%)]

AA AG GG A G

Reference DSM CM DSM CM DSM CM DSM CM DSM CM

Hobbs et al.(2000)

26 (17.9) 39 (28.1) 64 (44.1) 68 (48.9) 55 (37.9) 32 (23.0) 116 (40,0) 146 (52,5) 174 (60,0) 132 (47,5)

O’Leary et al.(2002)

1 (2.1) 35 (28.1) 23 (47.9) 101 (52.6) 24 (37.9) 56 (23.0) 25 (26.0) 171 (44.5) 71 (74.0) 213 (55.5)

Chango et al.(2005)

6 (5.0) 5 (4.2) 72 (60.0) 66 (55.5) 42 (35.0) 48 (40.3) 84 (35.0) 76 (31.9) 156 (65.0) 162 (68.1)

da Silva et al.(2005)

37 (24.0) 45 (28.5) 92 (59.7) 87 (55.1) 25 (16.2) 26 (16.5) 166 (53.9) 177 (56.0) 142 (46.1) 139 (44.0)

Scala et al.(2006)

28 (30.1) 69 (26.8) 46 (49.5) 131 (51.0) 19 (20.4) 57 (22.2) 102 (54.8) 269 (52.3) 84 (45.2) 245 (47.7)

Santos-Reboucaset al. (2008)

39 (37.9) 31 (28.7) 44 (42.7) 49 (45.4) 20 (19.4) 28 (25.9) 122 (59.2) 111 (51.4) 84 (40.8) 105 (48.6)

Wang et al.(2008)

10 (15.6) 24 (35.3) 28 (43.8) 34 (50.0) 26 (40.6) 10 (14.7) 48 (37.5) 82 (60.3) 80 (62.5) 54 (39.7)

Pozzi et al.(2009)

17 (23.0) 64 (34.8) 47 (63.5) 90 (48.9) 10 (13.5) 30 (16.3) 81 (54.7) 218 (59.2) 67 (45.3) 150 (40.8)

Coppede et al.(2009)

20 (24.7) 29 (26.1) 39 (48.1) 62 (55.9) 22 (27.2) 20 (18.0) 79 (48.8) 120 (54.1) 83 (51.2) 102 (45.9)

Brandalize et al.(2010)

42 (17.6) 42 (21.3) 137 (57.3) 111 (56.3) 60 (25.1) 44 (22.3) 221 (46.2) 195 (49.5) 257 (53.8) 199 (50.5)

Zampieri et al.(2012)

36 (34.3) 65 (35.1) 53 (50.5) 89 (48.1) 16 (15.2) 31 (16.8) 125 (59.5) 219 (59.2) 85 (40.5) 151 (40.8)

FIG. 1. Random effects pooled odds ratio (OR) and 95% confidence intervals forest plot for the association betweenmethionine synthase reductase 66A > G and Down syndrome in case-control studies. The OR estimates are represented bysolid black circles and the size of the symbol indicates the weight of the respective study in the meta-analysis. The pooled ORis represented by a solid black diamond.

MTRR 66A > G POLYMORPHISM AND DOWN SYNDROME 71

homocysteine (Hcy) and folate levels to increase the sensi-tivity to detect a correlation between genotype and DS risk.

Four studies were conducted in Brazil. da Silva et al. (2005)have evaluated five folate metabolizing pathway polymor-phisms (MTHFR 677C > T, MTHFR 1298A > C, MTRR66A > G, MTR 2756A > G and CBS 844ins68) and observedhigher Hcy levels among DSM when compared to CM.However, the statistical difference was associated only with aMTHFR TT genotype. For MTRR 66A > G, no difference wasobserved for both genotype and allele distributions betweenthe case and control groups, indicating that this poly-morphism did not act as an independent risk factor forDS. Santos-Reboucas et al. (2008) evaluated folate pathwaypolymorphisms combined to nutritional deficiency as ma-ternal risk factor for DS and did not found an independent orcombined association of maternal genotypes to an increaserisk of DS birth. In a sample of mothers of European descentliving in the South region of Brazil, Brandalize et al. (2010) haveevaluated four folate metabolizing polymorphisms (MTR 2756A > G, MTRR 66A > G, CBS 844ins68 and RFC 80 G > A) andfound that individual polymorphisms are not associated withDS. Zampieri et al. (2012) have studied 12 polymorphisms infolate pathway and their influence on serum folate and plasmamethylmalonic acid (MMA) concentrations as an indicator ofhigh Hcy levels. Although the authors have found that poly-morphisms in folate metabolism genes modulate the maternalrisk for bearing a child with DS, no independent associationbetween MTRR 66A > G polymorphism and DS risk was ob-served. However, this polymorphism was the only one to affectMMA concentration, validating the proposed association be-tween the presence of the mutant 66 G allele both in heterozy-gous and homozygous status with higher Hcy concentrations.

Three reports concerning MTRR 66A > G polymorphismand an increased risk of having a DS child were conducted inthe Italian population (Scala et al., 2006; Coppede et al., 2009;Pozzi et al., 2009). While Scala et al. (2006) and Coppede et al.(2009) did not found association between MTRR 66A > Gpolymorphism and DS, Pozzi et al. (2009) have found that thepresence of the mutated G allele increases two-fold the risk(95% CI 1.11–4.40) for a DS offspring after parity adjustment,with similar trends when analyses focuses on woman agedless than 35 years old. On the other hand, the association wasnot observed by Coppede et al. (2009) when a sample of Italianwoman < 35 years at conception was evaluated.

Crude data analysis has showed association of MTRR66A > G and DS in USA and Canada, Ireland and China

populations (Hobbs et al., 2000; O’Leary et al., 2002; Wanget al., 2008) while in the remaining studies, all conductedin Latin European descent populations (Chango et al.,2005; da Silva et al., 2005; Scala et al., 2006; Santos-Re-boucas et al., 2008; Coppede et al., 2009; Pozzi et al., 2009;Brandalize et al., 2010; Zampieri et al., 2012), only onestudy reported an association of MTRR 66A > G and DS,after parity adjustment (Pozzi et al., 2009). However, aprevious report on the Italian population in a sample ofgreater size has denied this association (Coppede et al.,2009).

Controversial results were also observed in meta-analysespublished to date. Zintzaras (2007) condensed the results offive reports and included 559 DSM and 866 CM and did notfind any evidence for the association between MTRR 66A > Gand DS. On the other hand, a recent report by Rai (2011), usingdata extracted from six reports with a total of 623 DSM and936 CM describe a 1.42-fold increase in DS offspring whenmothers carry G allele. We found a result similar to that de-scribed by Rai (2011), with a significant increase of 1.23-fold(95% CI 1.02–1.47) of having a DS child for woman carryingMTRR 66G allele.

It was previously proposed that the association of MTHFR677C > T polymorphism with neural tube defects could bespecific for non-Latin European descent populations (Amorimet al., 2007). We can imagine a similar scenario for the asso-ciation between MTRR 66A > G and DS. When non LatinEuropean descent reports were excluded (three studies), theRE pooled OR decreases and looses significance (data nowshown). The occurrence or absence of association between apolymorphism in a gene involved in folate metabolism withDS could be explained by difference in maternal folate intake,variation in mutant allele frequency in the population, geneticheterogeneity of studied population and different gene–nutrient interaction among populations (Amorim et al., 2007).Correlation of maternal red blood cell folate with polymor-phic genotypes will help to identify the role of these geneticvariants as risk factors for DS, and only few reports performthis correlation.

The large between-studies heterogeneity we have observedmay be due to discrepancies in study design but it could re-flect genuine differences among the studied populations(Zintzaras, 2007). Our meta-analysis indicates an independentassociation between MTRR G allele and an increased risk ofDS, and the populations without Latin European descentcould be at greater risk.

Table 3. Fixed and Random Effects Derived Odds Ratios, 95% Confidence Intervals and Heterogeneity

for the Association of Methionine Synthase Reductase 66A > G Polymorphisms and Down Syndrome

Method Model OR 95% CI Q p I2 (%)

Fixed effects Codominant (GG vs. AA) 1.40 1.14–1.71 27.1 0.003 63.1Codominant (AG vs. AA) 1.21 1.02–1.44 12.2 0.27 17.8Recessive (GG vs. AA or AG) 1.22 1.04–1.43 25.8 0.004 61.2Dominant (GG or AG vs. AA) 1.26 1.07–1.48 19.1 0.04 47.6Additive (G vs. A) 1.19 1.08–1.31 29.7 0.001 66.3

Random effects Codominant (GG vs. AA) 1.41 0.94–2.12 27.1 0.003 63.1Codominant (AG vs. AA) 1.19 0.95–1.48 12.2 0.27 17.8Recessive (GG vs. AA or AG) 1.24 0.92–1.67 25.8 0.004 61.2Dominant (GG or AG vs. AA) 1.26 0.96–1.64 19.1 0.04 47.6Additive (G vs. A) 1.23 1.02–1.49 29.7 0.001 66.3

CI, confidence interval; OR, odds ratio.

72 AMORIM AND COSTA LIMA

Acknowledgments

This study was supported by Fundacao de Amparo aPesquisa do Estado do Rio de Janeiro—FAPERJ, Brazil (No. E-26/110.427/2011).

Author Disclosure Statement

None declared.

References

Amorim MR, Costa Lima MA, Castilla EE, et al. (2007) Non LatinEuropean descent could be a requirement for association ofNTDs and MTHFR variant 677C > T: a meta-analysis. Am JMed Genet Part A 143A:1726–1732.

Brandalize APC, Bandinellia E, Santos PA, et al. (2010) Maternalgene polymorphisms involved in folate metabolism as riskfactors for Down syndrome offspring in Southern Brazil. DisMarkers 29:95–101.

Chango A, Fillon-Emery N, Mircher C, et al. (2005) No associa-tion between common polymorphisms in genes of folate andhomocysteine metabolism and the risk of Down’s syndromeamong French mothers. Br J Nutr 94:166–169.

Cochran WG (1954) The combination of estimates from differentexperiments. Biometrics 10:101–129.

Coppede F, Migheli F, Bargagna S, et al. (2009) Association ofmaternal polymorphisms in folate metabolizing genes withchromosome damage and risk of Down syndrome offspring.Neurosci Lett 449:15–19.

da Silva LRJ, Vergani N, Galdieri LC, et al. (2005) Relationshipbetween polymorphisms in genes involved in homocysteinemetabolism and maternal risk for Down syndrome in Brazil.Am J Med Genet Part A 135A:263–267.

Epstein CJ (2001) Down syndrome. In: Scriver CR, Beaudet AL,Sly WS and Valle D, (eds). The Metabolic and Molecular Basesof Inherited Disease. Vol. 1, 8th ed. McGraw-Hill, NY, pp.1223–1256.

Epstein CJ, Korenberg JR, Anneren G, et al. (1991) Protocols toestablish genotype-phenotype correlations in Down syn-drome. Am J Hum Genet 49:207–235.

Eskes TK (2006) Abnormal folate metabolism in mothers withDown syndrome offspring: review of the literature. Eur JObstet Gynecol Reprod Biol 124:130–133.

Gueant JL, Gueant-Rodriguez RM, Anello G, et al. (2003) Geneticdeterminants of folate and vitamin B12 metabolism: a com-mon pathway in neural tube defect and Down syndrome? ClinChem Lab Med 41:1473–1477.

Higgins JPT, Thompson SG (2002) Quantifying heterogeneity ina meta-analysis. Stat Med 21:1539–15358.

Hobbs CA, Sherman SL, Yi P, et al. (2000) Polymorphisms ingenes involved in folate metabolism as maternal risk factorsfor Down syndrome. Am J Hum Genet 67:623–630.

James SJ, Pogribna M, Pogribny IP, et al. (1999) Abnormal folatemetabolism and mutation in the methylenetetrahydrofolatereductase gene may be maternal risk factors for Down syn-drome. Am J Clin Nut 70:495–501.

Leclerc D, Wilson A, Dumas R, et al. (1998) Cloning and map-ping of a cDNA for methionine synthase reductase, a flavo-protein defective in patients with homocystinuria. Proc NatlAcad Sci USA 95:3059–3064.

O’Leary VB, Parle-McDermott A, Molloy AM, et al. (2002) MTRRand MTHFR polymorphism: link to Down syndrome? Am JMed Genet 107:151–155.

Olteanu H, Munson H, Banerjee R (2002) Differences in the ef-ficiency of reductive activation of methionine synthase and

exogenous electron acceptors between the common polymor-phic variants of human methionine synthase reductase. Bio-chemistry 41:13378–13385.

Pozzi E, Vergani P, Dalpra L, et al. (2009) Maternal polymor-phisms for methyltetrahydrofolate reductase and methioninesynthetase reductase and risk of children with Down syn-drome. Am J Obstet Gynecol 200:636e1–636e6.

Rady PL, Szucs S, Grady J, et al. (2002) Genetic polymorphismsof methylenetetrahydrofolate reductase (MTHFR) and methi-onine synthase reductase (MTRR) in ethnic populations inTexas: a report of a novel MTHFR polymorphic site, G1793A.Am J Med Genet 107:162–168.

Rai PS, Murali TS, Vasudevan TG et al. (2011) Genetic variationin genes involved in folate and drug metabolism in a southIndian population. Ind J Hum Genet 17:48–53. Am J MedGenet 107:162–168.

Rai V (2011) Polymorphism in folate metabolic pathway gene asmaternal risk factor for Down syndrome. Int J Biol Med Res2:1055–1060.

Santos-Reboucas CB, Correa JC, Bonomo A, et al. (2008) Theimpact of folate pathway polymorphisms combined to nutri-tional deficiency as a maternal predisposition factor for Downsyndrome. Dis Markers 25:149–157.

Scala I, Granese B, Sellitto M, et al. (2006) Analysis of sevenmaternal polymorphisms of genes involved in homocysteine/folate metabolism and risk of Down syndrome offspring.Genet Med 8:409–416.

Yang QH, Botto LD, Gallagher M et al. (2008) Prevalence and ef-fects of gene-gene and gene-nutrient interactions on serum folateand serum total homocysteine concentrations in the UnitedStates: findings from the third National Health and NutritionExamination Survey DNA Bank. Am J Clin Nutr 88:232–246.

Wang SS, Qiao FY, Feng L, et al. (2008) Polymorphisms in genesinvolved in folate metabolism as maternal risk factors forDown syndrome in China. J Zhejiang Univ Sci B 9:93–99.

Wilson A, Platt R, Wu Q, et al. (1999) A common variant in methi-onine synthase reductase combined with low cobalamin (vitaminB12) increases risk for spina bifida. Mol Genet Metabol 67:317–323.

Wiseman FK, Alford KA, Tybulewicz VJL et al. (2009) Downsyndrome—recent progress and future prospects. Hum MolGenet 18:R75–R83.

Wolthers KR, Scrutton NS (2007) Protein interactions in thehuman methionine synthase-methionine synthase reductasecomplex and implications for the mechanism of enzyme re-activation. Biochemistry 46:6696–6709.

Zampieri BL, Biselli JM, Goloni-Bertollo EM et al. (2012) Ma-ternal risk for Down syndrome is modulated by genes in-volved in folate metabolism. Dis Markers 32:73–81.

Zintzaras E (2007) Maternal gene polymorphisms involved infolate metabolism and risk of Down syndrome offspring: ameta-analysis. J Hum Genet 52:943–953.

Address correspondence to:Marcelo A. Costa Lima, Ph.D.

Departamento de GeneticaInstituto de Biologia Roberto Alcantara Gomes

Universidade do Estado do Rio de Janeiro (UERJ)Rua Sao Francisco Xavier 524, PHLC, sala 218

Maracana CEP 20550-900Rio de Janeiro

Brazil

E-mail: marlima@uerj.br; marceloacostalima@gmail.com

MTRR 66A > G POLYMORPHISM AND DOWN SYNDROME 73