Original Article

European Journal of Clinical Nutrition (2008) 62, 1010–1021; doi:10.1038/sj.ejcn.1602810; published online 23 May 2007

Association between decreased vitamin levels and MTHFR, MTR and MTRR gene polymorphisms as determinants for elevated total homocysteine concentrations in pregnant women

Contributors: PRB was a Master Degree student at the Faculty of Pharmaceutical Science, University of Sao Paulo and she determined the clinical chemistry and the gene polymorphism genotypes at the Hematology laboratory of the Clinical and Toxicology Analysis Department of the University of São Paulo. ALKM and RB provided technical assistance and were recipients of fellowships from Projeto 4 of University of São Paulo and PIBIC-CNPq, Brazil, respectively. MHH and RDCH contributed with the strategies and interpretation of results from genotyping analysis as well as in preparation of the manuscript. LFSN is the obstetrician who participated in planning and executing this study at the Hospital Regional of Conjunto Hospitalar and at the Hospital Santa Lucinda. The Brazilian authors have no conflicts of interest. SPS and RHA were responsible for the assays of the amino acids, SAM and SAH in their laboratories. They also provided interpretation of the values and participated in the preparation of the manuscript. The University of Colorado and SPS and RHA hold patents on various aspects of the use of homocysteine and methylmalonic acid in the diagnosis of cobalamin or folate deficiency. A company (called Metabolite Laboratories Inc.) has been formed at the University of Colorado to perform the assays. EMG-S was the coordinator of this study in Brazil, responsible for all of its phases (planning, collecting the samples, interviews, analysis of the data, interpretation of the values and preparation of the manuscript).

P R Barbosa1, S P Stabler2, A L K Machado1, R C Braga1, R D C Hirata1, M H Hirata1, L F Sampaio-Neto3, R H Allen2 and E M Guerra-Shinohara1

  1. 1Departamento de Análises Clínicas e Toxicológicas, Faculdade de Ciências Farmacêuticas da Universidade de São Paulo, São Paulo, SP, Brazil
  2. 2Department of Medicine, University of Colorado Health Sciences Center, Denver, CO, USA
  3. 3Faculdade de Medicina, Pontificia Universidade Católica de São Paulo, São Paulo, SP, Brazil

Correspondence: Professor EM Guerra-Shinohara, Department of Clinical Chemistry and Toxicology, Faculty of Pharmaceutical Sciences, University of Sao Paulo, Av. Prof. Lineu Prestes, 580 Bloco 17, Sao Paulo, SP CEP 05508-900, Brazil. E-mail: emguerra@usp.br

Received 17 October 2006; Revised 19 March 2007; Accepted 18 April 2007; Published online 23 May 2007.

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Abstract

Objectives:

 

To examine the association between methylenetetrahydrofolate reductase (MTHFR) (C677T and A1298C), methionine synthase (MTR) A2756G and methionine synthase reductase (MTRR) A66G gene polymorphisms and total homocysteine (tHcy), methylmalonic acid (MMA) and S-adenosylmethionine/S-adenosylhomocysteine (SAM/SAH) levels; and to evaluate the potential interactions with folate or cobalamin (Cbl) status.

Subjects/Methods:

 

Two hundred seventy-five healthy women at labor who delivered full-term normal babies. Cbl, folate, tHcy, MMA, SAM and SAH were measured in serum specimens. The genotypes for polymorphisms were determined by PCR-restriction fragment length polymorphism (RFLP).

Results:

 

Serum folate, MTHFR 677T allele and MTR 2756AA genotypes were the predictors of tHcy levels in pregnant women. Serum Cbl and creatinine were the predictors of SAM/SAH ratio and MMA levels, respectively. The gene polymorphisms were not determinants for MMA levels and SAM/SAH ratios. Low levels of serum folate were associated with elevated tHcy in pregnant women, independently of the gene polymorphisms. In pregnant women carrying MTHFR 677T allele, or MTHFR 1298AA or MTRR 66AA genotypes, lower Cbl levels were associated with higher levels of tHcy. Lower SAM/SAH ratio was found in MTHFR 677CC or MTRR A2756AA genotypes carriers when Cbl levels were lower than 142pmol/l.

Conclusions:

 

Serum folate and MTHFR C677T and MTR A2576G gene polymorphisms were the determinants for tHcy levels. The interaction between low levels of serum Cbl and MTHFR (C677T or A1298C) or MTRR A66G gene polymorphisms was associated with increased tHcy.

Keywords:

cobalamin, folate, polymorphisms, homocysteine, methylmalonic acid, pregnant women

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Introduction

Cobalamin (Cbl) and folate are essential for nucleic acid synthesis and are required for homocysteine metabolism and methylation reactions (Chanarin, 1990).

Cbl and folate deficiencies may impair the synthesis of methionine and determine the concentrations of S-adenosylmethionine (SAM) (Scott et al., 1981). In a previous study, we demonstrated that concentrations of S-adenosylhomocysteine (SAH) were higher and methionine and SAM concentrations and SAM/SAH ratios were lower in pregnant women in the lowest Cbl quartile (Cblless than or equal to102pmol/l) (Guerra-Shinohara et al., 2004). In addition, total homocysteine (tHcy) and methylmalonic acid (MMA) levels and SAM/SAH ratio were also abnormal in newborns of these mothers (Guerra-Shinohara et al., 2004). These findings are disquieting, since SAM is a major cellular methyl donor for nucleic acid, protein and phospholipid methylation (Clarke and Banfield, 2001).

Folate deficiency was also associated with genomic DNA hypomethylation in a male child with Down's syndrome and neural tube defects (NTDs) (Al-Gazali et al., 2001). Moreover, DNA methylation was correlated positively with folate levels and inversely with plasma tHcy in adults (Friso et al., 2002, 2005). In these studies, the associations found between hypomethylation and folate status were dependent on the methylenetetrahydrofolate reductase (MTHFR) C677T gene polymorphism.

It has been described that serum tHcy levels are 30–60% lower in pregnant women than in the women of child-bearing age and the lowest tHcy values are found in the second trimester (Andersson et al., 1992; Walker et al., 1999; Holmes et al., 2005). Murphy et al. (2002) demonstrated that this decrease in tHcy is a physiologic effect of the pregnancy and it is independent of folic acid supplementation, plasma-volume expansion, or a decrease in serum albumin. On the other hand, Holmes et al. (2005) showed that plasma tHcy levels were lower in pregnant women taking folic acid supplements than in nonsupplemented pregnant women, and this difference was statistically significant in the third trimester. Moreover, maternal tHcy concentrations at labor are strongly correlated with newborn values (Guerra-Shinohara et al., 2002; Molloy et al., 2002). Thus, maternal tHcy serum levels at labor reflect the concentrations displayed during gestation and if elevated these may be harmful to the newborns. Murphy et al. (2004) showed that neonates of mothers in the highest tertile of tHcy at labor weighed, on average, 227.98g less than those of mothers in the low and medium tertiles.

Several key enzymes, including MTHFR, methionine synthase (MTR) and methionine synthase reductase (MTRR), are important in homocysteine metabolism and therefore in methylation reactions. MTHFR reduces 5,10 methylenetetrahydrofolate to 5-methyltetrahydrofolate. MTR is a Cbl-dependent enzyme that uses the methyl group from 5-methyltetrahydrofolate for remethylation of tHcy to methionine. In this reaction cob(I)alamin is readily oxidized to cob(II)alamin, which inactivates the Cbl-MTR-enzyme complex. The MTRR restores MTR activity by reductive methylation of cob(II)alamin, using SAM as methyl donor (Gaughan et al., 2001; Yamada et al., 2006).

Polymorphisms at the MTHFR (C677T), MTR (A2756G) and MTRR (A66G) genes have been associated with high tHcy concentrations in some populations (Harmon et al., 1999; Gaughan et al., 2001; Jacques et al., 2003; Russo et al., 2003; Alessio et al., 2004; Pereira et al., 2004). Genetic variants of enzymes involved in the homocysteine remethylation pathway might act as predisposing factors contributing to NTDs. The C677T polymorphism in the MTHFR gene has been associated with reduced enzyme activity and increased tHcy levels (Frosst et al., 1995) and risk for NTDs (Christensen et al., 1999; Shields et al., 1999; Wilson et al., 1999; Cunha et al., 2002). The MTHFR A1298C polymorphism does not alter plasma tHcy concentrations, although subjects with heterozygous genotypes for MTHFR C677T and A1298C polymorphisms may be at the risk of mild elevation of tHcy levels (van der Put et al., 1998). These polymorphisms were also associated with increased risk of spontaneous abortion (Zetterberg et al., 2002; Zetterberg, 2004).

The gene–nutrient interactive effect, rather than genotype alone, has been suggested to increase the risk of NTDs (Christensen et al., 1999; Wilson et al., 1999) and DNA methylation (Friso et al., 2002, 2005). Christensen et al. (1999) found an increased risk of having an NTD case when MTHFR 677TT genotype was associated with RBC folate in the lowest quartile. MTHFR 677TT genotype and lower levels of plasma folate were also associated with reduced DNA methylation (Friso et al., 2002). Moreover, hypomethylation in DNA was associated with MTHFR 1298AA genotype and reduced folate levels (Friso et al., 2005). However, the high prevalence of the 677TT genotype within the 1298AA group (79%) and similar biochemical features of 1298AA/677CC and 1298CC/677CC haplotypes suggest that the gene–nutrient interaction affecting DNA methylation in 1298AA is mainly due to the coexistence of 677TT genotype.

The interaction between MTRR 66GG genotype and low Cbl levels was associated with fivefold increase in risk for mothers of children with spina bifida, compared with those women who have other genotypes and Cbl levels in the other three quartiles (Wilson et al., 1999).

High frequency of low Cbl, increased tHcy and other metabolic abnormalities were found in pregnant women and their newborns from a sample of our population (Guerra-Shinohara et al., 2004). However, the effects of the interaction between vitamin deficiencies and MTHFR, MTR and MTRR gene polymorphisms on vitamin-dependent metabolite concentrations are still unknown.

The aims of the present study were to examine the association between MTHFR (C677T and A1298C), MTR A2756G and MTRR A66G gene polymorphisms and tHcy, MMA and SAM/SAH levels; as well as to evaluate the potential interactions between genotypes and folate or Cbl levels and their relations with tHcy and other metabolites.

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Subjects and methods

Subjects

Blood was collected from 405 pregnant women at two public hospitals of Sorocaba city, Brazil, after admission for labor and up to 12h before delivery, from April to May in 2001 and 2003.

All deliveries occurred within the gestational age ranging from 37 to 42 weeks. Women with clinical diagnosis of metabolic diseases, renal insufficiency, increased serum liver enzymes, hemoglobinopathies (HbS and HbC trait, elevated HbA2 and HbF concentrations), multiple gestations, preterm deliveries and complications during delivery such as birth of a newborn with congenital malformation and anoxia were excluded from the study (N=28). Women who took supplements during pregnancy (N=94) and those with no information about the use of supplements (N=8) were also excluded from this study.

Socioeconomic data (family monthly per capita income, schooling and occupation), obstetrical status and supplemental vitamin intake were assessed by questionnaire. The ethnic classification was based on phenotype (pigmentation of skin, hair type, conformation of nose and lips and by descent of family). The protocol was approved by the local ethics committees (Brazil) and by the investigational review board at the University of Colorado (USA), and written informed consent was obtained from all pregnant women before participation.

Methods

Blood sampling and biochemical measurements
 

Detailed descriptions of blood sampling of this study have been published in a previous study (Guerra-Shinohara et al., 2004).

Serum folate concentration was determined by the ion capture method (IMx System, Abbott Laboratories, Abbott Park, IL, USA) and by the chemiluminescent method (Immulite, DPC MedLab, Los Angeles, CA, USA). The serum Cbl concentration was measured using the Immulite kit, Diagnostic Products Corporation – DPC).

Indices of kidney and liver function were investigated with measurements of serum creatinine, AST and ALT by kits (Dimension AR, Dade Behringer, Marburg, HE, DE). Pregnant women with abnormalities in these parameters were excluded from our study.

The measurements of tHcy and MMA were performed using stable isotope dilution capillary gas chromatography/mass spectrometry (Stabler et al., 1986, 1987). SAM and SAH were measured using a stable isotope dilution liquid chromatography/mass spectrometry method (Stabler and Allen, 2004).

Genetics analysis
 

Genomic DNA was isolated from whole blood (Salazar et al., 1998). Genotyping for each polymorphism was accomplished by polymerase chain reaction followed by restriction digests (PCR-RFLP) as described previously for MTHFR C677T (Frosst et al., 1995) and MTHFR A1298C (van der Put et al., 1998), using the restriction enzymes: HinfI and MboII.

The primer sequences for genotyping MTR A2756G and MTRR A66G polymorphisms were modified, in the present study, using Primer Premier version 5.0 program (Sigma Chemical Co., St Louis, USA) based on the sequences previously published (Harmon et al., 1999; Jacques et al., 2003). The MTR A2756G polymorphism was detected using new sequences or primers (forward: 5′-GGTGCCAGGTATACAGTGACTCT-3′ and reverse: 5′-GATCCAAAGCCTTTTACACTCCTC-3′) and HaeIII endonuclease in PCR-RFLP assays. MTRR A66G was genotyped using primers (forward: 5′-AAGGCCATCGCAGAAGACAT-3′ and reverse: 5′-CACTTCCCAACCAAAATTCTTCAAAG-3′) and NdeI restriction enzyme.

Statistical analysis
 

All statistical analyses were carried out using statistical analysis software (SAS-Statistical Analysis System for Windows, version 6.12, SAS Institute Inc., 1989–1996, Cary, NC, USA), with the level of significance set at P<0.05.

Hardy–Weinberg equilibrium was determined for all of the genotypes using χ2 testing. χ2 test was also used to compare the frequencies of genotypes for each polymorphism according to ethnic groups (Caucasian vs African descent).

Because the distribution of serum values of Cbl, folate, tHcy, MMA, SAM, SAH, SAM/SAH and creatinine had a positive skew, all analyses were carried out after logarithmic transformation, and back-transformed results are shown.

The median value of per capita income was used as cutoff for characterizing two groups of women (lower per capita income less than or equal toUS$69.37 and higher per capita income >US$69.37). Student's t-test was used for comparing the means of biochemical variables between groups formed according to per capita income. Student's t-test adjusted by per capita income was used for comparing the means of biochemical variables from Caucasian-descent and African-descent.

The data from two Asian-descent women were not included in the statistical analysis when the ethnic groups were compared.

One-way analysis of covariance (ANCOVA) was used to compare the biochemical data from groups formed according to genotypes for each polymorphism (MTHFR C677T, MTHFR A1298C, MTR A2756G and MTRR A66G) and haplotypes (MTHFR C677T and MTHFR A1298C) and combination of genotypes (MTHFR C677T and MTR A2756G; MTHFR C677T and MTRR A66G). The covariates used were age, parity, ethnic group (Caucasian vs African descent) and monthly per capita income. When significant differences among groups were observed, Tukey–Kramer post hoc testing was performed to identify the significantly different group means.

To assess the simultaneous relations between the various predictors of tHcy, MMA and SAM/SAH ratio in the pregnant women (as dependent variables), three models of multiple linear regression analysis (saturated models) were used. The reference genotypes were: CC for MTHFR C677T, AC plus CC for MTHFR A1298C, AG plus GG for MTR A2756G and AA for MTRR A66G polymorphisms. The models were adjusted by the same covariates: age, parity, ethnic group (Caucasian vs African descent) and monthly per capita income.

The interaction between MTHFR C677T genotypes and vitamin status for determining the increase on tHcy levels were performed by Student's t-test adjusted by the covariates: age, parity, ethnic group (Caucasian vs African descent) and monthly per capita income. The medians of Cbl and serum folate were used as cutoff values. The interaction between other genotypes for MTHFR A1298C, MTR A2756G and MTRR A66G polymorphisms and vitamin levels was also evaluated by Student's t-test adjusted by the same covariates including the genotypes for MTHFR C677T polymorphism (CC vs CT+TT).

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Results

Population characteristics

Of the 405 eligible subjects, 275 pregnant women met the entry criteria of this study. The mean (±s.d.) age of the pregnant women was 25.2 (±6.5) years (range: 13–44 years), gestational age: 39.0 (±1.2) weeks (range: 37–42 weeks) and parity 2.6±1.7, including the actual pregnancy. Low socioeconomic status without occupations (75.7%) and without basic schooling (44.3%) was found in this sample. The geometric mean (95% CI) per capita family income was US$66.18 (60.30–72.62) per month.

Information about ethnic group was not available for three women. The ethnic backgrounds of the women were Caucasian-descent (58.5%), African-descent (40.8%) and Asian descent (0.7%).

No difference was found between frequencies of numbers of years of school and ethnic groups (Caucasian-descent vs African-descent) (P=0.158).

The Caucasian-descent women had higher per capita income (US$73.80) than African-descent women (US$56.30), P=0.005. No difference was observed in the ages of women between two groups (P=0.203). However, there was a trend (P=0.062) for increasing parity in African-descent women (3.5) than Caucasian-descent (3.2).

The women with lower per capita income had higher parity (3.7) than those with higher per capita income (2.9) (P<0.001). Low Cbl levels (P=0.041) and higher tHcy (P=0.012) and MMA levels (P=0.049) were observed in pregnant women with lower per capita income than those with higher per capita income. There was high frequency of Caucasian-descent in the group with high per capita income (68.2%) than African-descent (31.8%), P=0.007.

The Student's t-test adjusted by per capita income showed that the Caucasian-descent women had lower Cbl (trend, P=0.074) and higher MMA (P=0.005) levels than African-descent women.

The genotype distributions of the MTHFR C677T, MTHFR A1298C, MTR A2756G and MTRR A66G polymorphisms were in Hardy–Weinberg equilibrium (P>0.05).

The allele frequencies for MTHFR 677T, MTHFR 1298C, MTR 2756G and MTRR 66G were, respectively, 31.0, 26.1, 19.3 and 43.3% in Brazilian pregnant women (Table 1).


No difference was observed between the frequencies of genotypes of MTHFR C677T, MTR A2756G and MTRR A66G according to ethnic groups (Caucasian-descent vs African-descent) (Table 1). However, in the group of African-descent, there was higher frequency of MTHFR 1298AA genotype carriers than Caucasian-descent group (P=0.004) (Table 1).

The frequency of MTHFR 677T allele in black pregnant women was 19.6%.

Associations between polymorphisms and vitamin and metabolite concentrations

Women with renal disease were excluded from this study. The geometric mean (95% CI) of serum creatinine was 0.61mg/dl (0.60, 0.62).

Pregnant women carrying the MTHFR 677T allele (CT+TT genotypes) had lower serum folate levels and higher tHcy concentrations than the CC genotype carriers (Table 2). Lower Cbl was found in MTHFR 1298AA genotypes carriers when compared with the women carrying MTHFR 1298C allele (AC+CC genotypes). The pregnant women with MTR 2756AA genotype had lower Cbl and higher tHcy levels than those with MTR 2756AG and GG genotypes (Table 2). No differences in vitamin and metabolite concentrations were observed between pregnant women carrying different genotypes for MTRR A66G polymorphisms (Table 2).


The genotypes for MTHFR C677T, MTHFR A1298C, MTR A2756G and MTRR A66G polymorphisms were not associated with variation in MMA levels and SAM/SAH ratios (Table 2).

The effects of haplotypes of the MTHFR gene (C677T and A1298C), and combinations of genotypes for MTHFR C677T and MTR A2756G, and MTHFR C677T and MTRR A66G polymorphisms on vitamin and metabolite concentrations are shown in Table 3. The one-way analysis adjusted by covariates (age, parity, ethnic group and monthly per capita income) showed that pregnant women with the MTHFR 677TT/MTHFR 1298AA haplotype had lower serum folate and higher tHcy concentrations than those with 677CC/1298AA and 677CC/1298AC haplotypes (Table 3). The pregnant women with MTHFR 677CT/MTHFR 1298AC haplotype had similar serum folate as those with other haplotypes and tHcy concentrations lower than those pregnant women with 677TT/1298AA haplotype (Table 3).


Women carrying the combination of genotypes MTHFR 677TT/MTR 2756AG had lower Cbl levels than those with 677CC/2756AG. However, pregnant women with the combination of genotypes MTHFR 677TT/MTR 2756AA had lower serum folate levels than those with 677CC/2756AA, 677CC/2756AG and 677CT/2756AG. Higher values of tHcy were found in pregnant women with the combination of genotypes MTHFR 677TT/MTR 2756AA when compared with the combination of genotypes: CC/AA, CC/AG, CC/GG, CT/AA, CT/AG and TT/AG. Higher MMA levels were found in pregnant women with the combination of genotypes MTHFR 677TT/MTR 2756AG as compared to 677CT/2756AG carriers (Table 3).

In addition, pregnant women with the combination of genotypes MTHFR 677TT/MTRR 66AG and MTHFR 677TT/MTRR 66GG had lower serum folate than those with the 677CC/66AA haplotype. The carriers with the combination of genotypes 677TT/66GG had elevated tHcy levels compared with all combinations of genotypes, except 677TT/66AG carriers (Table 3).

Predictors of metabolite variables

Serum folate levels, MTHFR 677T allele (CT+TT genotypes) and MTR 2756AA genotypes were predictors of tHcy concentrations in pregnant women. The range of serum folate values observed in this study sample explains 15.5% of the variance in tHcy, and the MTHFR 677T allele and MTR 2756AA genotype explain, respectively, 1.7 and 1.6% of the variance in tHcy (Table 4). SAM/SAH ratios were predicted by Cbl levels (R2=0.0164). The MMA levels were predicted by serum creatinine (partial R2=0.0207) (Table 4).


Gene–nutrient interaction

The interaction effect between genotypes for MTHFR C677T, MTHFR A1298C, MTR A2756G and MTRR A66G polymorphisms, and vitamin status on tHcy and MMA levels and SAM/SAH ratios were evaluated. The medians for Cbl (142pmol/l) and folate (12.5nmol/l) were used for characterizing two groups according to vitamin status (above median and below or equal median).

Table 5 shows results from interaction between genotypes for gene polymorphisms and vitamin status on tHcy levels. Low values of serum folate were associated with increased tHcy levels independently of genotypes for MTHFR C677T, MTHFR A1298C, MTR A2756G and MTRR A66G polymorphisms. In pregnant women with high serum folate levels, the MTHFR 677T allele was associated with high levels of tHcy (P=0.020) when the model was adjusted for covariates (age, parity, ethnic group (Caucasian vs African descent) and monthly per capita income). Similar results were found for MTR 2756AA genotype carriers when the model was adjusted for the same covariates including genotypes for MTHFR C677T polymorphism.


The interaction between gene polymorphisms and Cbl status on tHcy levels was also evaluated (Table 5). In pregnant women carrying the MTHFR 677T allele, lower Cbl levels were associated with higher tHcy levels (P=0.030). Moreover, the MTHFR 677CT+TT genotypes carriers have higher tHcy levels than 677CC carriers, independently of the Cbl status (P<0.05).

In MTHFR 1298AA genotype carriers, elevated tHcy levels were found in pregnant women with low Cbl levels compared with those with high Cbl (P=0.040) after adjusting for covariates including genotypes for the MTHFR C677T polymorphism. Similar results were found in pregnant women carrying the MTRR 66AA genotype (P=0.049).

Effects of interactions between gene polymorphisms and vitamin status were also evaluated on MMA levels and SAM/SAH ratios. No differences in MMA levels were associated with the gene polymorphisms independently of serum folate or Cbl levels.

Analysis of SAM/SAH ratio showed that in MTHFR 677CC genotype carriers, lower ratios were associated with lower Cbl levels (geometric mean 3.8 and 95% CI 3.2, 4.5) than those with higher Cbl levels (geometric mean 4.7 and 95% CI 4.2, 5.3) (P=0.049). Similar results were found in pregnant women carrying the MTR 2756AA genotypes as pregnant women with low Cbl levels had lower SAM/SAH (geometric mean 3.9 and 95% CI 3.5, 4.4) than those with higher Cbl levels (geometric mean 4.7 and 95% CI 4.2, 5.3) (trend, P=0.053) after adjusting for covariates including genotypes for the MTHFR C677T polymorphism.

Previous history of miscarriage

Information about the mother's previous history of miscarriage was not available for three women. A total of 224 (82.4%) of the pregnant women had no previous history of miscarriage and 48 (17.6%) had a previous history with one or more miscarriages. Only 2 (0.7%) women had history of three or more miscarriages.

No difference was observed between the frequency of mothers with and without previous history of miscarriage according to ethnic group (Caucasian-descent vs African-descent) (P=0.682, data not shown).

The pregnant women with previous history of miscarriage had higher age and parity than those without history of miscarriage. However, no differences in vitamins (Cbl, serum folate) and metabolite (tHcy, MMA, SAM, SAH) concentrations were observed between pregnant women from the two groups (data not shown).

No difference was observed in frequencies of women with and without previous history of miscarriage according to genotypes for MTHFR C677T, MTHFR A1298C and MTRR A66G. However, the pregnant women with previous history of miscarriage had a trend (P=0.098) for having higher frequency of MTR 2756AA genotype than pregnant women with no previous history of miscarriage (data not shown).

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Discussion

In the current study, 75% of Brazilian pregnant women had Cbl concentrations lower than 179pmol/l. This cutoff is similar to that cited by Bruinse and van den Berg (1995) as indicative of marginal or deficient stores (values <180pmol/l). The much lower Cbl levels and the alterations in metabolite levels found in this study may be the result of the interaction between environmental factors (such as lower socioeconomic conditions and nutritional deficiency) and genetic factors (presence of one polymorphism or combinations of several polymorphisms in genes of key enzymes of homocysteine metabolism).

It is noteworthy that this study was carried out before the food folic acid fortification program became effective in Brazil in 2004. Moreover, the Brazilian women did not use supplementation with folic acid or vitamin B12 during their pregnancy. One strength of our investigation was that the samples were carefully collected before the delivery, stored and shipped frozen, and assayed promptly so that artifactual increases or decreases in metabolites did not occur.

Pregnant women with renal insufficiency were excluded from this study, and the serum concentrations of MMA were used as a marker of impaired Cbl status and the serum tHcy concentrations and SAM/SAH ratio were also used as markers of Cbl and folate deficiencies.

We found that many variables affected the metabolites and vitamin concentrations such as socioeconomic status, median per capita income and racial-ethnic group. In addition, there were interactions between the racial-ethnic groups and socioeconomic variables that impacted the biochemical variables. The lower Cbl and higher MMA values we found in pregnant women of Caucasian-descent were similar to previously reported racial differences (Carmel, 1999; Stabler et al., 1999). Therefore, we controlled for race, ethnicity, parity and per capita income when analyzing our data.

The frequency of MTHFR 677TT genotype (10.2%) in pregnant women in the current investigation was similar to that found in adults and children from Brazil (9–10%) (Arruda et al., 1998; Alessio et al., 2004; Pereira et al., 2004). The T allele frequency found in pregnant women of African-descent pregnant women (32.4%) was higher than that found in Brazilian black people from three distinct regions of Brazil (20.0%) (Arruda et al., 1998) and in Brazilian black blood donors (12.5%) (Pereira et al., 2004), P<0.05. However, in black pregnant women, the frequency of MTHFR 677T allele (19.6%) was similar to the frequencies found in previous studies with Brazilian individuals (Arruda et al., 1998; Pereira et al., 2004). These results suggest that our sample is representative of the Brazilian population.

In this study, the MTHFR 1298C allele was more frequent (31.3%) in Caucasian Brazilian pregnant women than in those of African-descent (19.3%, P=0.003). The frequency of the MTHFR 1298C allele in African-derived pregnant women was similar to that previously reported in mixed (22.0%) and black (13.7%) healthy blood donors in Sao Paulo city, Brazil (Pereira et al., 2004). The frequencies of rare genotypes for MTR A2756G and MTRR A66G were similar to those found in other studies (Alessio et al., 2004; Pereira et al., 2004).

The effects of each gene polymorphism alone or in haplotypes on vitamin and metabolite levels were evaluated in this study. The MTHFR C677T gene polymorphism is the most important genetic factor that influences blood tHcy levels in different populations (Russo et al., 2003; Alessio et al., 2004; Pereira et al., 2004). In the present study, pregnant women carrying the MTHFR 677T allele (CT plus TT genotypes) had lower serum folate and Cbl and higher tHcy levels than the CC genotype carriers. Previous studies have demonstrated that the MTHFR C677T polymorphism, which causes an alanine-to-valine substitution at position 222 of the enzyme, confers MTHFR thermolability and significant reduction of its activity in vitro (Frosst et al., 1995; Chango et al., 2000) affecting directly the folate status.

We found that serum Cbl concentration was lower in MTHFR 1298A allele carriers (AA, common-genotype) than in those carrying the 1298C allele (AC plus CC genotypes) in contrast to findings previously reported in blood donors from a Brazilian sample (Pereira et al., 2004). It is conceivable that the result found in this study may be due to the effect of interactions between MTHFR C677T and A1298C polymorphisms on Cbl levels as well as tHcy. Lower Cbl levels were found in TT/AA genotype carriers when compared with CC/CC genotype carriers. Interestingly, the interaction between the 1298AA genotype and low Cbl determined a 20.6% increase in tHcy levels.

The MTHFR A1298C mutation, located in the enzyme regulatory domain does not result in either a thermolabile protein or increased tHcy (van der Put et al., 1998; Hanson et al., 2001). However, MTHFR 677CT/1298AC compound heterozygosity reportedly has similar impact as C677T homozygosity (Chango et al., 2000). In the current investigation, the one-way analysis adjusted by covariates showed that the CT/AC haplotype is not associated with increased tHcy and decreased serum folate and Cbl levels in pregnant women. On the other hand, the pregnant women carrying TT/AA haplotype had lower serum folate and higher tHcy levels than the CC/AA and CC/AC haplotypes carriers. It appears that the TT/AA haplotype is more likely to influence homocysteine metabolism than the CT/AC haplotype.

In our study, the MTRR A66G polymorphism alone was not associated with alterations in vitamin and metabolite concentrations and this finding was similar to those described in other studies (Wilson et al., 1999; Gaughan et al., 2001; Jacques et al., 2003). However, when the combinations of genotypes were taken into account, the carriers of combined genotypes MTHFR 677TT/MTRR 66GG had elevated tHcy levels compared with all combinations of genotypes for these polymorphisms, except 677TT/66AG. Similar results were found in nonpregnant healthy American women (Vaughn et al., 2004). It appears that the interaction between MTHFR 677TT and MTRR 66GG genotypes is associated with high tHcy levels.

It has been suggested that the MTRR A66G (I22M) variant is located in the putative flavin mononucleotide-binding domain of the MTRR enzyme that interacts with MTR (Leclerc et al., 1998). Substitution of an isoleucine by a methionine in the 66G allele could thus disrupt the binding of MTRR to the MTR-cob(I)alamin-complex, thereby decreasing the rate of homocysteine remethylation (Olteanu et al., 2002). Therefore, it is possible that the combination of MTHFR 677T and MTRR 66G alleles affects substantially the homocysteine status in pregnant women.

Pregnant women with MTR 2756AA genotype had lower Cbl and higher tHcy levels than those with the G allele (AG plus GG genotypes). This finding is similar to those found in a working male Irish population (Harmon et al., 1999) and healthy adult Canadian controls (Miriuka et al., 2005) and different from the finding in healthy Dutch controls (Klerk et al., 2003). It is conceivable that the presence of the G allele in the methionine synthase gene is a protective factor against increasing tHcy concentrations.

An interesting finding was observed in carriers of 677TT/2756AA genotypes, who had higher tHcy levels than all combinations of these genotypes, except for 677CT/2756GG. This result is consistent with the predictors of tHcy by multiple linear regression analysis in which serum folate concentration, MTHFR 677CT plus TT genotypes and MTR 2756AA genotypes were the determinants of tHcy concentrations in Brazilian pregnant women. Our results are consistent with those of Harmon et al. (1999), but are different from other studies which reported no effects of MTHFR C677T and/or MTR A2756G polymorphism on tHcy levels (Christensen et al., 1999; Molloy et al., 2002; Alessio et al., 2004).

The MTR A2756G polymorphism, which results in substitution of glycine for aspartic acid at amino acid position 919, is located in a domain of the protein that interacts with SAM and auxiliary proteins that are required for the reductive methylation and reactivation of the Cbl cofactor, which can be inactivated by oxidation during catalysis (Leclerc et al., 1998; Harmon et al., 1999). It has been speculated that the MTR 2756A allele might impair the binding of SAM and/or auxiliary proteins, or possibly impair the stability of protein (Harmon et al., 1999). However, this effect was not demonstrated in in vitro studies (Harmon et al., 1999).

In the gene–nutrient interaction analysis, elevated tHcy levels were associated with low serum folate status in pregnant women independently of MTHFR, MTR and MTRR gene polymorphisms. These findings show that reduced serum folate is the strongest environmental factor for elevated tHcy in pregnant women. Interestingly, increased tHcy was also dependent on the interaction between MTHFR 677T allele or MTHFR 1298AA or MTRR 66AA genotypes and low levels of serum Cbl. Therefore, gene–nutrient interaction found in pregnant women may be important in determining the risk for increased tHcy during pregnancy and in neonates considering its risk for NTDs and other fetal abnormalities.

The MMA levels were also evaluated in pregnant women due to its relevance as a sensitive marker for functional Cbl deficiency (Allen et al., 1993). We could not demonstrate a relation between MMA levels and isolated gene polymorphisms in pregnant women. On the other hand, the MTHFR 677TT genotype in combination with MTR 2756AG genotype was associated with increased MMA levels when compared with 677CT plus 2756AG. This result may be influenced by the low number of individuals with these genetic characteristics in our sample. Moreover, in our study, we did not find that Cbl levels or the variants for polymorphisms were an independent predictor of the MMA levels, in contrast to the serum creatinine. Therefore, additional studies with larger population samples should be conducted in order to confirm these results.

In a previous study, we found that the patients with severe Cbl deficient megaloblastic anemia had elevated serum SAH levels and low SAM/SAH ratio, which were corrected with Cbl therapy (Guerra-Shinohara et al., 2007). Lower SAM/SAH ratio was found in pregnant women in the lowest Cbl quartile (Cblless than or equal to102pmol/l) (Guerra-Shinohara et al., 2004). Thus, in the present study, we used the SAM/SAH ratio as potential marker of Cbl deficiency. MTHFR C677T, MTHFR A1298C, MTR A2756G and MTRR A66G gene polymorphisms were not associated with variations in SAM/SAH ratio in our sample. On the other hand, reduced SAM/SAH ratio was determined by the interaction between low Cbl levels and 677CC or 2756AA genotypes. It is possible that this finding is dependent on the reduced levels of Cbl considering that this vitamin was the variable selected in models by the multiple linear regression analysis.

Cbl is the second nutrient that has been implicated in increasing risk of NTDs. Several studies have reported the decreased Cbl concentrations in the circulation or amniotic fluid in mothers of children with NTDs (Kirke et al., 1993; Adams et al., 1995; Steen et al., 1998). A consequence of this vitamin deficiency is elevated tHcy and MMA levels, which have been reported to be a risk factor for NTDs.

In the current study, only two pregnant women had a history of three or more pregnancy losses. The pregnant women with a previous history of one or more miscarriages did not have elevated tHcy levels when compared with those having no history of miscarriage. We could not find an increased frequency of gene polymorphisms in these two groups. Our results were different from other studies (Nelen et al., 2000; Unfried et al., 2002; Holmes et al., 2005; Mtiraoui et al., 2006). It is possible that the small number of Brazilian pregnant women with previous history of three or more miscarriages could be responsible for these differences.

Our findings contribute to understanding the role of nutritional and genetic factors on maternal metabolism, which may impact their newborns. The effect of the combination between nutrition and gene polymorphisms during pregnancy on vitamin dependent metabolite (tHcy and MMA) levels and DNA methylation index should be investigated further.

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Acknowledgements

This study was supported financially by Fundação de Amparo à Pesquisa do Estado de São Paulo – FAPESP, Brazil (Proc. 00/12467-0 and 01/09836-7) and PHS Grant NIA-AG-09834. Andre LK Machado and Renata C Braga had fellowships of Projeto 4 (Pró Reitoria de Pesquisa) da Universidade de São Paulo and PIBIC CNPq, respectively. Elvira M Guerra-Shinohara is a recipient of a fellowship from CNPq, Brasilia, DF, Brazil. We thank the Hospital Regional of Conjunto Hospitalar de Sorocaba and Hospital Santa Lucinda and the pregnant women who participated in the study.

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