Maternal Risk for Down Syndrome Is Modulated by Genes Involved in Folate Metabolism

Studies have shown that the maternal risk for Down syndrome (DS) may be modulated by alterations in folate metabolism. The aim of this study was to evaluate the influence of 12 genetic polymorphisms involved in folate metabolism on maternal risk for DS. In addition, we evaluated the impact of these polymorphisms on serum folate and plasma methylmalonic acid (MMA, an indicator of vitamin B12 status) concentrations. The polymorphisms transcobalamin II (TCN2) c.776C>G, betaine-homocysteine S-methyltransferase (BHMT) c.742A>G, methylenetetrahydrofolate reductase (NAD(P)H) (MTHFR) c.677 C>T and the MTHFR 677C-1298A-1317T haplotype modulate DS risk. The polymorphisms MTHFR c.677C>T and solute carrier family 19 (folate transporter), member 1 (SLC19A1) c.80 A>G modulate folate concentrations, whereas the 5-methyltetrahydrofolate-homocysteine methyltransferase reductase (MTRR) c.66A>G polymorphism affects the MMA concentration. These results are consistent with the modulation of the maternal risk for DS by these polymorphisms.


Introduction
Down syndrome (DS), or trisomy 21 (MIM 190685), is the most common genetic disorder with a prevalence of 1 in 660 live births [27]. The only well-established risk factor for DS is advanced maternal age [7]. However, many DS children are born to mothers younger than 35 years, suggesting that other factors can also influence DS etiology. James et al. [48] hypothesized that pericentromeric hypomethylation, result-ing from impaired folate metabolism secondary to a polymorphism on methylenetetrahydrofolate reductase (NAD(P)H) (MTHFR) gene, could impair chromosomal segregation and increase the risk for chromosome 21 nondisjunction in young mothers. Since then, several studies have revealed that polymorphisms in genes involved in the folate pathway modulate the maternal risk for DS [6,17,35,39,49] and the concentrations of metabolites involved in the folate pathway [14,30,43].
Folate metabolism vitally participates in the biosynthesis of nucleotides and S-adenosyl-methionine (SAM), the major methyl donor for DNA methylation reactions (Fig. 1). A folate deficiency has been associated with DNA hypomethylation, DNA damage, chromosomal instability, abnormal chromosome segregation and aneuploidy of chromosome 21 [45,47].

Material and methods
The study protocol was approved by the Research Ethics Committee of São José do Rio Preto Medical School (CEP-FAMERP), State of São Paulo, and by the National Research Commission (CONEP), Brazil. Fasting peripheral blood samples were obtained from 105 women (case mothers) with karyotypically confirmed full trisomy 21 (translocation or mosaicism were not included) liveborn offspring and from 185 mothers with at least one healthy offspring and no history of miscarriage (control mothers). Case mothers were identified at the time of their offspring's initial consultation at the General Genetics Outpatient Service of Hospital de Base de São José do Rio Preto, SP, Brazil, and were enrolled in the study during the following consultation. The control group was composed of volunteers from the FAMERP Campus and of women submitted to routine exams at the Clinical Analysis Laboratory of Hospital de Base de São José do Rio Preto. Informed consent was obtained from all study participants.
The maternal age of the case group was calculated using the age of the mothers at the birth of the DS child and using the age of the mothers at the birth of the last child for the control group. The median child age at the time of the mother's recruitment was 14.8 years (0.15-39.09) for the control group and 1.1 years (0.02-30.35) for the case group. Given that the Brazilian population is ethnically heterogeneous, a result of centuries of admixture in the immigrant population, separating it into distinct ethnic groups becomes a challenge. This process of admixture has contributed to the specific characteristics of the Brazilian population [12]. All participants were from the same region of São Paulo (in the northwest). Thus, they likely share similar sociodemographic characteristics and racial backgrounds.
Genomic DNA was isolated from leucocytes in peripheral blood according to Miller et al. [46] or using the GFX TM Genomic Blood DNA Purification Kit (GE Healthcare, USA). The polymorphisms MTHFR c.677C>T, MTR c.2756A>G, SLC19A1 c.80A>G, TCN2 c.776C>G, CBS c.844ins68, CBS c.833T>C and MTHFD1 c.1958G>A were determined as previously described [11,17,23,33,41]. The variant TCN2 c.776C>G was analyzed via the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method using primers from Pietrzyk & Bik-Multanowski (2003) [20], and the PCR products were digested with the Scrf1 enzyme. The polymorphisms MTRR c.66A>G, TCN2 c.67A>G and BHMT c.742G>A were analyzed by real time PCR allelic discrimination (Taqman SNP Genotyping Assays, C 3068176 10, C 25967461 10 and C 11646606 20 respectively, Applied Biosystems, Foster City, CA, USA). Automated sequencing was used to investigate the MTHFR c.1298A>C and c.1317T>C polymorphisms as previously described [37]. The analysis, except for the purification procedure, was performed using the enzymes Exonuclease I and Shrimp Alkaline Phosphatase (Fermentas Life Sciences, Brazil) according to the manufacturer's instructions. Plasma MMA concentrations were determined by liquid chromatography-tandem mass spectrometry as previously described [51], and folate concentrations were determined by chemiluminescence (Immulite Kit, DPC Medlab, Brazil).

Statistical analysis
The Hardy-Weinberg (HW) equilibrium was assessed via a chi-square test using the BioEstat program, and the genotype frequencies were compared between case and control mothers by the likelihood ratio test. The relationship between the number of deleterious alleles for the 12 loci tested and the maternal risk for DS was assessed by logistic regression analysis. For this analysis, the sample was divided in two subsets (0-7 and 8-14 alleles for the total group and for the subgroup of women with maternal age 35 years old) according to the median value of deleterious alleles (median = 7). Previously published data [2,9,16] and the results in Table 1 Deleterious alleles considered for each of the 12 genetic polymorphisms investigated G * The results of the CBS c. 844ins68 genotypes were defined as I for the allele with the 68bp insertion and W for the wild -type allele.
this study were used to assess the deleterious status of both alleles for each polymorphism, shown in Table 1. The haplotype frequencies of the MTHFR, TCN2 and CBS genes were inferred using the Haploview program (version 4.0).
Multiple logistic regression analyses, with forward stepwise selection by the likelihood ratio, were performed using SPSS19 software. Genotype data for the 12 polymorphisms, maternal age and folate and MMA concentrations were included in the original models to verify the influence of each of these factors on the maternal risk for DS. Only the women that provided all the data were included in this analysis. The genotype data used in the logistic regression model was analyzed in two different ways: considering either (1) the dominant model (heterozygous + mutant homozygous versus wild-type homozygous) or (2) the recessive model (wild-type homozygous + heterozygous versus mutant homozygous). Once advanced maternal age is shown to be an important risk factor for DS, the analyses were also performed in a sub-group formed only by women 35 years old (case: 54; control:173). These tests were applied to generate an odds ratio (OR) and 95% confidence intervals (CI).
Stepwise forward multivariate logistic regression analyses were also performed for the total group (case and control together) to verify the factors that influence biochemical parameters. For MMA analysis, the concentrations were categorized considering the values above (case mothers = 21; control mothers = 41) or equal to/below (case mothers = 66; control mothers = 137) the 75 th percentile, and the genotype data for the 12 polymorphisms (recessive and dominant models) as well as the folate concentrations were used as predictors. For folate analysis, the concentrations were cate- gorized considering values below (case mothers = 21; control mothers = 44) or equal to/above (case mothers = 66; control mothers = 137) the 25 th percentile, and the genotype data for the 12 polymorphisms (recessive and dominant models) as well as the MMA concentrations were used as predictors.
The computer-assisted statistical analyses were carried out using Minitab for Windows program (Release 14) and SPSS19 software. Values of P 0.05 were considered significant.

Results
According to the likelihood ratio test, the genotype  Table 2). The genotype frequencies were in HW equilibrium in both groups, except for the polymorphisms CBS c.833T>C and c.844ins68 (P = 0.032 for both polymorphisms), and a Bonferroni adjustment revealed that the variant homozygous genotypes were more frequent in the case group (P < 0.0005 for both polymorphisms). The median value of the folate concentration in DS mothers (12.2 ng/mL, 3.7-36.5) was significantly lower (P = 0.028) than in the case group (14.6 ng/mL; 5-74). Conversely, the median value of the MMA concentration in DS mothers (0.17 µmol/L, 0.07-1.46) was significantly higher (P = 0.003) that in the control group (0.14 µmol/L, 0.05-0.81). The haplotype frequencies of the MTHFR, TCN2 and CBS genes are presented in Table 3 . There was no difference in the haplotype frequencies for the TCN2 and CBS genes between the groups. The CBS haplotypes 833T/844I and 833C/844W were not present in either group.
The median number of deleterious alleles did not differ between the groups, both in the total group and in the group of women 35 years old.

Discussion
Although advanced maternal age at conception represents an important and well-established risk factor for DS [7], as confirmed in this study, the occurrence of DS births by young mothers suggests that other risk factors are also involved in the etiology of this syndrome. Abnormal folate metabolism has been identified as a maternal risk factor for DS in several populations [9]. This study reveals that polymorphisms in folate metabolism genes modulate the maternal risk for bearing a child with DS. This is the first study evaluating the role of MTHFR c.1317T>C, CBS c.833T>C, TCN2 c.67A>G, MTHFD1 c.1958G>A and BHMT c.742G>A polymorphisms in mothers of DS children in a Brazilian population. In addition, the influence of the polymorphisms BHMT c.742G>A and TCN2 c.67A>G on DS risk have never been studied until now.
The polymorphism TCN2 c.776 GG, which has been previously investigated for DS risk by our group in a smaller sample [21] and by Fintelman-Rodrigues et al. [38], with negative results, was associated with increased DS risk in this study. Moreover, we observed that the TCN2 c.776 GG genotype influenced the risk for DS in both the total group and in the group with the conception age of women 35 years. The presence of the TCN2 c.776 GG genotype has been shown to negatively affect the serum concentration of the TCN2 protein-vitamin B 12 complex [28] and to be associated with low concentrations of SAM in childbearingage women [43]. Considering that SAM is the major methyl donor for DNA methylation reactions, the variant TCN2 c.776C>G may influence maternal risk for DS by modifying the DNA methylation pattern. We are the first group to study the influence of the TCN2 c.67A>G polymorphism on maternal risk for DS, where no association was observed with DS risk. The LD between the variants TCN2 c.67A>G and c.776C>G observed in this study is consistent with a previous study [26].
Our group is the first to evaluate the role of the BHMT c.742G>A polymorphism on the risk of bearing a DS child, where an association between the BHMT c.742 AA genotype and decreased maternal risk for DS was observed. The BHMT protein catalyzes an alternative route of homocysteine (Hcy) remethylation (Fig. 1). The polymorphism produces two distinct alloenzymes, which exhibit significant differences in Km values for Hcy and betaine [10]. The Km values are lower for the variant alloenzyme compared to the wildtype. The low Km of the alloenzyme may be responsible for the increased efficiency of Hcy remethylation using betaine as a methyl group donor [42]. The decreased effect of the BHMT c.742A allele on DS risk could be expected when the maternal AA genotype was protective against neural tube defects (NTD) in the offspring [2,16]. Moreover, NTDs and DS are influenced by the same genetic factors involved in folate metabolism [13].
Polymorphisms in the MTHFR gene have been extensively analyzed for influences on folate and methyl metabolisms in maternal risk for DS. The MTHFR enzyme plays an important role in regulating DNA methylation reactions through the reduction of 5,10-methylentetrahydrofolate (5,10-MTHF) to 5-methylTHF (Fig. 1). A common polymorphism in the MTHFR gene, c.677 C>T, is known to decrease the affinity of the enzyme for its flavin adenine dinucleotide (FAD) cofactor, thereby decreasing the enzyme activity [4,25]. The heterozygous MTHFR c.677 CT genotype reduces the enzyme activity by approximately 35%, and the homozygous TT genotype reduces activity by 70% [41]. In this study, the presence of the MTHFR c.677 CT or TT genotypes was associated with increased maternal risk for DS, which corroborates previous associations between the MTHFR c.677 C>T polymorphism and the modulation of the maternal risk for DS [32,39,49]. Coppedè et al. [8] have observed an association between the MTHFR c.677 T allele and the occurrence of chromosome damage and missegregation events in mothers of DS individuals, supporting the role of this allele in the etiology of trisomy 21. Previously, these authors observed a significant increase in the rate of aneuploidy of chromosome 21 in these mothers [31].
The LD between the MTHFR polymorphisms c.677C>T, c.1298A>C and c.1317T>C observed in this study is consistent with the literature, which has illustrated LD between the MTHFR c.677 C>T and c.1298A>C [9,34]. Moreover, the silent polymorphism at position 1317 is near the one at 1298 position. The higher frequency of the MTHFR 677C-1298A-1317T haplotype in the control group confirms the protective maternal effect of these alleles against DS, which is indicated by the rare alleles 677T and 1298C that have been associated with increased maternal risk for DS in several studies [22,32,39,49]. In addition, this result corroborates the association between the 677T-1298C haplotype and the maternal risk for DS observed by Scala et al. [17].
MTRR, an enzyme codified by the MTRR gene, is responsible for the maintenance of the activated form of the MTR enzyme [29]. Several studies have observed an association between the MTRR c.66A>G polymorphism, alone and combined with other genetic variants, and DS risk and an elevated Hcy concentration [3,5,6,36,40,44,49]. Additionally, a steady state kinetic analysis revealed a significant decrease in the affinity for MTRR accompanying a c.66A>G substitution, revealing a significant difference in the relative efficacies of the common MTRR polymorphism c.66A>G [15]. These findings further validate the association between the MTRR c.66 AG and GG genotypes and higher concentrations of MMA, likely a consequence of the variant enzyme activity that results in a higher Hcy concentration and consequently higher MMA concentrations.
The SLC19A1 gene encodes an enzyme that participates in folic acid absorption, transporting for 5-methylTHF, an important determinant of folate concentration, to the interior of a variety of cells [50,52]. The SLC19A1 gene is polymorphic at nucleotide 80 (A>G), and an assessment of the impact of this polymorphism on protein function has demonstrated a difference in its affinity for subtracts and/or its efficiency in transport compared to the wild type enzyme [24]. Chango et al. [1] observed that SLC19A1 c.80AA/ MTHFR c.677CT individuals exhibited higher plasma folate levels than SLC19A1 c.80GG/ MTHFR c.677CT individuals, which corroborates our observations of lower folate concentrations in the presence of the SLC19A1 c.80 GG genotype.
Because most polymorphisms, with the exception of CBS c.844ins68 and c.833 T>C, did not deviate from the HW equilibrium, our sample set was appropriately ascertained [18,19]. Departure from the HW equilibrium may have resulted from random selection or a small sample size.
A major strength of our study was the number of polymorphisms in folate metabolism genes investigated. Out of 12 polymorphisms, five have never been analyzed in a Brazilian population until now, including the BHMT c.742G>A and TCN2 c.67A>G polymorphisms that influence the maternal risk for DS. A potential limitation of our study is that folate and MMA concentrations were not measured at the child's delivery in both case and control mothers. Although the measurement of the concentrations at the time of conception would have been more relevant, the current quantification is a likely reflection of adult dietary patterns, once an adult's dietary tends to have a similar pattern over the time. Also, the influence of the polymorphisms on MMA and folate concentrations was analyzed in the total group, including DS and control mothers, to investigate the polymorphism influence on the concentrations regardless of the presence of a DS child. The small size of the case group, which could reduce the power of the statistical analysis and complicate an investigation of possible genotype combinations that may influence the maternal risk for DS, was an additional limitation of this study. However, studies that have investigated an association between folate gene polymorphisms and the risk of DS offspring have been conducted with relatively small sample sizes [9], primarily due to difficulties in the recruitment of these mothers, with significant results.
In conclusion, the results of this study indicate that the TCN2 c.776C>G, BHMT c.742A>G, and MTH-FR c.677 C>T polymorphisms and the MTHFR 677C-1298A-1317T haplotype modulate the risk for DS. The polymorphisms MTHFR c.677C>T and SLC19A1 c.80 A>G modulate folate concentrations, whereas the MTRR c.66A>G polymorphism affects MMA concentrations. These findings contribute to future research aimed at identifying metabolic interventions that will aid in preventing nondisjunction of the 21 chromosome. Future studies may benefit from sample sizes that are large enough to identify specific gene-gene interactions.