European Journal of Clinical Nutrition (2008) 62, 1010–1021 & 2008 Macmillan Publishers Limited All rights reserved 0954-3007/08 $30.00 www.nature.com/ejcn

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

PR Barbosa1, SP Stabler2, ALK Machado1, RC Braga1, RDC Hirata1, MH Hirata1, LF Sampaio-Neto3, RH Allen2 and EM Guerra-Shinohara1

1Departamento de Ana´lises Clı´nicas e Toxicolo´gicas, Faculdade de Cieˆncias Farmaceˆuticas da Universidade de Sa˜o Paulo, Sa˜o Paulo, SP, Brazil; 2Department of Medicine, University of Colorado Health Sciences Center, Denver, CO, USA and 3Faculdade de Medicina, Pontificia Universidade Cato´lica de Sa˜o Paulo, Sa˜o Paulo, SP, Brazil

Objectives: To examine the association between methylenetetrahydrofolate reductase (MTHFR) (C677T and A1298C), synthase (MTR) A2756G and 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 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 142 pmol/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. European Journal of Clinical Nutrition (2008) 62, 1010–1021; doi:10.1038/sj.ejcn.1602810; published online 23 May 2007

Keywords: cobalamin; folate; polymorphisms; homocysteine; methylmalonic acid; pregnant women

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: [email protected] 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 Sa˜o Paulo. ALKM and RB provided technical assistance and were recipients of fellowships from Projeto 4 of University of Sa˜o 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). Received 17 October 2006; revised 19 March 2007; accepted 18 April 2007; published online 23 May 2007 Association between vitamin deficiency and polymorphisms PR Barbosa et al 1011 Introduction cob(II)alamin, which inactivates the Cbl-MTR- complex. The MTRR restores MTR activity by reductive Cobalamin (Cbl) and folate are essential for nucleic acid methylation of cob(II)alamin, using SAM as methyl donor synthesis and are required for homocysteine and (Gaughan et al., 2001; Yamada et al., 2006). methylation reactions (Chanarin, 1990). Polymorphisms at the MTHFR (C677T), MTR (A2756G) Cbl and folate deficiencies may impair the synthesis of and MTRR (A66G) have been associated with high methionine and determine the concentrations of S-adenosyl- tHcy concentrations in some populations (Harmon et al., methionine (SAM) (Scott et al., 1981). In a previous study, we 1999; Gaughan et al., 2001; Jacques et al., 2003; Russo et al., demonstrated that concentrations of S-adenosylhomocys- 2003; Alessio et al., 2004; Pereira et al., 2004). Genetic teine (SAH) were higher and methionine and SAM concentra- variants of involved in the homocysteine remethyl- tions and SAM/SAH ratios were lower in pregnant women in ation pathway might act as predisposing factors contributing the lowest Cbl quartile (Cblp102 pmol/l) (Guerra-Shinohara to NTDs. The C677T polymorphism in the MTHFR gene has et al., 2004). In addition, total homocysteine (tHcy) and been associated with reduced enzyme activity and increased methylmalonic acid (MMA) levels and SAM/SAH ratio tHcy levels (Frosst et al., 1995) and risk for NTDs (Christen- were also abnormal in newborns of these mothers (Guerra- sen et al., 1999; Shields et al., 1999; Wilson et al., 1999; Shinohara et al., 2004). These findings are disquieting, since Cunha et al., 2002). The MTHFR A1298C polymorphism does SAM is a major cellular methyl donor for nucleic acid, not alter plasma tHcy concentrations, although subjects with and phospholipid methylation (Clarke and Banfield, 2001). heterozygous genotypes for MTHFR C677T and A1298C Folate deficiency was also associated with genomic DNA polymorphisms may be at the risk of mild elevation of tHcy hypomethylation in a male child with Down’s syndrome and levels (van der Put et al., 1998). These polymorphisms were neural tube defects (NTDs) (Al-Gazali et al., 2001). Moreover, also associated with increased risk of spontaneous abortion DNA methylation was correlated positively with folate levels (Zetterberg et al., 2002; Zetterberg, 2004). and inversely with plasma tHcy in adults (Friso et al., 2002, The gene–nutrient interactive effect, rather than genotype 2005). In these studies, the associations found between alone, has been suggested to increase the risk of NTDs hypomethylation and folate status were dependent on the (Christensen et al., 1999; Wilson et al., 1999) and DNA methylenetetrahydrofolate reductase (MTHFR) C677T gene methylation (Friso et al., 2002, 2005). Christensen et al. polymorphism. (1999) found an increased risk of having an NTD case when It has been described that serum tHcy levels are 30–60% MTHFR 677TT genotype was associated with RBC folate in lower in pregnant women than in the women of child- the lowest quartile. MTHFR 677TT genotype and lower levels bearing age and the lowest tHcy values are found in the of plasma folate were also associated with reduced DNA second trimester (Andersson et al., 1992; Walker et al., 1999; methylation (Friso et al., 2002). Moreover, hypomethylation Holmes et al., 2005). Murphy et al. (2002) demonstrated that in DNA was associated with MTHFR 1298AA genotype and this decrease in tHcy is a physiologic effect of the pregnancy reduced folate levels (Friso et al., 2005). However, the high and it is independent of folic acid supplementation, plasma- prevalence of the 677TT genotype within the 1298AA group volume expansion, or a decrease in serum albumin. On the (79%) and similar biochemical features of 1298AA/677CC other hand, Holmes et al. (2005) showed that plasma tHcy and 1298CC/677CC haplotypes suggest that the gene– levels were lower in pregnant women taking folic acid nutrient interaction affecting DNA methylation in 1298AA supplements than in nonsupplemented pregnant women, is mainly due to the coexistence of 677TT genotype. and this difference was statistically significant in the third The interaction between MTRR 66GG genotype and low trimester. Moreover, maternal tHcy concentrations at Cbl levels was associated with fivefold increase in risk for labor are strongly correlated with newborn values (Guerra- mothers of children with spina bifida, compared with those Shinohara et al., 2002; Molloy et al., 2002). Thus, maternal women who have other genotypes and Cbl levels in the tHcy serum levels at labor reflect the concentrations other three quartiles (Wilson et al., 1999). displayed during gestation and if elevated these may be High frequency of low Cbl, increased tHcy and other harmful to the newborns. Murphy et al. (2004) showed that metabolic abnormalities were found in pregnant women and neonates of mothers in the highest tertile of tHcy at labor their newborns from a sample of our population (Guerra- weighed, on average, 227.98 g less than those of mothers in Shinohara et al., 2004). However, the effects of the interac- the low and medium tertiles. tion between vitamin deficiencies and MTHFR, MTR and Several key enzymes, including MTHFR, methionine MTRR gene polymorphisms on vitamin-dependent metabo- synthase (MTR) and methionine synthase reductase (MTRR), lite concentrations are still unknown. are important in homocysteine metabolism and therefore in The aims of the present study were to examine the methylation reactions. MTHFR reduces 5,10 methylene- association between MTHFR (C677T and A1298C), MTR tetrahydrofolate to 5-methyltetrahydrofolate. MTR is a A2756G and MTRR A66G gene polymorphisms and tHcy, Cbl-dependent enzyme that uses the methyl group from 5- MMA and SAM/SAH levels; as well as to evaluate the methyltetrahydrofolate for remethylation of tHcy to methio- potential interactions between genotypes and folate or Cbl nine. In this reaction cob(I)alamin is readily oxidized to levels and their relations with tHcy and other metabolites.

European Journal of Clinical Nutrition Association between vitamin deficiency and polymorphisms PR Barbosa et al 1012 Subjects and methods followed by restriction digests (PCR-RFLP) as described previously for MTHFR C677T (Frosst et al., 1995) and MTHFR Subjects A1298C (van der Put et al., 1998), using the restriction Blood was collected from 405 pregnant women at two public enzymes: HinfI and MboII. hospitals of Sorocaba city, Brazil, after admission for labor The primer sequences for genotyping MTR A2756G and and up to 12 h before delivery, from April to May in 2001 and MTRR A66G polymorphisms were modified, in the present 2003. study, using Primer Premier version 5.0 program (Sigma All deliveries occurred within the gestational age ranging Chemical Co., St Louis, USA) based on the sequences from 37 to 42 weeks. Women with clinical diagnosis of previously published (Harmon et al., 1999; Jacques et al., metabolic diseases, renal insufficiency, increased serum liver 2003). The MTR A2756G polymorphism was detected using enzymes, hemoglobinopathies (HbS and HbC trait, elevated new sequences or primers (forward: 50-GGTGCCAGGTATA 0 0 HbA2 and HbF concentrations), multiple gestations, preterm CAGTGACTCT-3 and reverse: 5 -GATCCAAAGCCTTTTA deliveries and complications during delivery such as birth CACTCCTC-30) and HaeIII endonuclease in PCR-RFLP assays. of a newborn with congenital malformation and anoxia MTRR A66G was genotyped using primers (forward: 50- were excluded from the study (N ¼ 28). Women who took AAGGCCATCGCAGAAGACAT-30 and reverse: 50-CACTTCC supplements during pregnancy (N ¼ 94) and those with no CAACCAAAATTCTTCAAAG-30) and NdeI restriction information about the use of supplements (N ¼ 8) were also enzyme. excluded from this study. Socioeconomic data (family monthly per capita income, schooling and occupation), obstetrical status and supple- Statistical analysis. All statistical analyses were carried out mental vitamin intake were assessed by questionnaire. The using statistical analysis software (SAS-Statistical Analysis ethnic classification was based on phenotype (pigmentation System for Windows, version 6.12, SAS Institute Inc., 1989– of skin, hair type, conformation of nose and lips and by 1996, Cary, NC, USA), with the level of significance set at descent of family). The protocol was approved by the local Po0.05. ethics committees (Brazil) and by the investigational review Hardy–Weinberg equilibrium was determined for all of the board at the University of Colorado (USA), and written genotypes using w2 testing. w2 test was also used to compare informed consent was obtained from all pregnant women the frequencies of genotypes for each polymorphism before participation. 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 Methods positive skew, all analyses were carried out after logarithmic Blood sampling and biochemical measurements. Detailed de- transformation, and back-transformed results are shown. scriptions of blood sampling of this study have been The median value of per capita income was used as cutoff published in a previous study (Guerra-Shinohara et al., for characterizing two groups of women (lower per capita 2004). income pUS$69.37 and higher per capita income Serum folate concentration was determined by the ion 4US$69.37). Student’s t-test was used for comparing the capture method (IMx System, Abbott Laboratories, Abbott means of biochemical variables between groups formed Park, IL, USA) and by the chemiluminescent method according to per capita income. Student’s t-test adjusted (Immulite, DPC MedLab, Los Angeles, CA, USA). The serum by per capita income was used for comparing the means Cbl concentration was measured using the Immulite kit, of biochemical variables from Caucasian-descent and Diagnostic Products Corporation – DPC). African-descent. Indices of kidney and liver function were investigated The data from two Asian-descent women were not with measurements of serum creatinine, AST and ALT by kits included in the statistical analysis when the ethnic groups (Dimension AR, Dade Behringer, Marburg, HE, DE). Pregnant were compared. women with abnormalities in these parameters were ex- One-way analysis of covariance (ANCOVA) was used to cluded from our study. compare the biochemical data from groups formed accord- The measurements of tHcy and MMA were performed ing to genotypes for each polymorphism (MTHFR C677T, using stable isotope dilution capillary gas chromatography/ MTHFR A1298C, MTR A2756G and MTRR A66G) and mass spectrometry (Stabler et al., 1986, 1987). SAM and SAH haplotypes (MTHFR C677T and MTHFR A1298C) and were measured using a stable isotope dilution liquid combination of genotypes (MTHFR C677T and MTR chromatography/mass spectrometry method (Stabler and A2756G; MTHFR C677T and MTRR A66G). The covariates Allen, 2004). used were age, parity, ethnic group (Caucasian vs African descent) and monthly per capita income. When significant Genetics analysis. Genomic DNA was isolated from whole differences among groups were observed, Tukey–Kramer post blood (Salazar et al., 1998). Genotyping for each polymor- hoc testing was performed to identify the significantly phism was accomplished by polymerase chain reaction different group means.

European Journal of Clinical Nutrition Association between vitamin deficiency and polymorphisms PR Barbosa et al 1013 To assess the simultaneous relations between the various The Student’s t-test adjusted by per capita income showed predictors of tHcy, MMA and SAM/SAH ratio in the pregnant that the Caucasian-descent women had lower Cbl (trend, women (as dependent variables), three models of multiple P ¼ 0.074) and higher MMA (P ¼ 0.005) levels than African- linear regression analysis (saturated models) were used. The descent women. reference genotypes were: CC for MTHFR C677T, AC plus CC The genotype distributions of the MTHFR C677T, MTHFR for MTHFR A1298C, AG plus GG for MTR A2756G and AA for A1298C, MTR A2756G and MTRR A66G polymorphisms were MTRR A66G polymorphisms. The models were adjusted by in Hardy–Weinberg equilibrium (P40.05). the same covariates: age, parity, ethnic group (Caucasian vs The allele frequencies for MTHFR 677T, MTHFR 1298C, African descent) and monthly per capita income. MTR 2756G and MTRR 66G were, respectively, 31.0, 26.1, The interaction between MTHFR C677T genotypes and 19.3 and 43.3% in Brazilian pregnant women (Table 1). vitamin status for determining the increase on tHcy levels No difference was observed between the frequencies of were performed by Student’s t-test adjusted by the covariates: genotypes of MTHFR C677T, MTR A2756G and MTRR A66G age, parity, ethnic group (Caucasian vs African descent) and according to ethnic groups (Caucasian-descent vs African- monthly per capita income. The medians of Cbl and serum descent) (Table 1). However, in the group of African-descent, folate were used as cutoff values. The interaction between there was higher frequency of MTHFR 1298AA genotype other genotypes for MTHFR A1298C, MTR A2756G and carriers than Caucasian-descent group (P ¼ 0.004) (Table 1). MTRR A66G polymorphisms and vitamin levels was also The frequency of MTHFR 677T allele in black pregnant evaluated by Student’s t-test adjusted by the same covariates women was 19.6%. including the genotypes for MTHFR C677T polymorphism (CC vs CT þ TT). Associations between polymorphisms and vitamin and metabolite concentrations Women with renal disease were excluded from this study. Results The geometric mean (95% CI) of serum creatinine was 0.61 mg/dl (0.60, 0.62). Population characteristics Pregnant women carrying the MTHFR 677T allele (CT þ TT Of the 405 eligible subjects, 275 pregnant women met the genotypes) had lower serum folate levels and higher tHcy entry criteria of this study. The mean (7s.d.) age of the concentrations than the CC genotype carriers (Table 2). pregnant women was 25.2 (76.5) years (range: 13–44 years), Lower Cbl was found in MTHFR 1298AA genotypes carriers gestational age: 39.0 (71.2) weeks (range: 37–42 weeks) and when compared with the women carrying MTHFR 1298C parity 2.671.7, including the actual pregnancy. Low socio- allele (AC þ CC genotypes). The pregnant women with MTR economic status without occupations (75.7%) and without 2756AA genotype had lower Cbl and higher tHcy levels than basic schooling (44.3%) was found in this sample. The those with MTR 2756AG and GG genotypes (Table 2). No geometric mean (95% CI) per capita family income was differences in vitamin and metabolite concentrations were US$66.18 (60.30–72.62) per month. observed between pregnant women carrying different geno- Information about ethnic group was not available for types for MTRR A66G polymorphisms (Table 2). three women. The ethnic backgrounds of the women were The genotypes for MTHFR C677T, MTHFR A1298C, MTR Caucasian-descent (58.5%), African-descent (40.8%) and A2756G and MTRR A66G polymorphisms were not asso- Asian descent (0.7%). ciated with variation in MMA levels and SAM/SAH ratios No difference was found between frequencies of numbers (Table 2). of years of school and ethnic groups (Caucasian-descent vs The effects of haplotypes of the MTHFR gene (C677T and African-descent) (P ¼ 0.158). A1298C), and combinations of genotypes for MTHFR C677T The Caucasian-descent women had higher per capita and MTR A2756G, and MTHFR C677T and MTRR A66G income (US$73.80) than African-descent women (US$56.30), polymorphisms on vitamin and metabolite concentrations P ¼ 0.005. No difference was observed in the ages of women are shown in Table 3. The one-way analysis adjusted by between two groups (P ¼ 0.203). However, there was a trend covariates (age, parity, ethnic group and monthly per capita (P ¼ 0.062) for increasing parity in African-descent women income) showed that pregnant women with the MTHFR (3.5) than Caucasian-descent (3.2). 677TT/MTHFR 1298AA haplotype had lower serum folate The women with lower per capita income had higher and higher tHcy concentrations than those with 677CC/ parity (3.7) than those with higher per capita income (2.9) 1298AA and 677CC/1298AC haplotypes (Table 3). The (Po0.001). Low Cbl levels (P ¼ 0.041) and higher tHcy pregnant women with MTHFR 677CT/MTHFR 1298AC (P ¼ 0.012) and MMA levels (P ¼ 0.049) were observed in haplotype had similar serum folate as those with other pregnant women with lower per capita income than those haplotypes and tHcy concentrations lower than those with higher per capita income. There was high frequency of pregnant women with 677TT/1298AA haplotype (Table 3). Caucasian-descent in the group with high per capita income Women carrying the combination of genotypes MTHFR (68.2%) than African-descent (31.8%), P ¼ 0.007. 677TT/MTR 2756AG had lower Cbl levels than those with

European Journal of Clinical Nutrition Association between vitamin deficiency and polymorphisms PR Barbosa et al 1014 Table 1 Frequencies of genotypes and alleles for MTHFR C677T, MTHFR A1298C, MTR A2756G and MTRR A66G gene polymorphisms in the total group of pregnant women and according to ethnic groups

Polymorphism Total group Caucasians-descent Africans-descent Asians-descent

MTHFR C677T Genotypes N (%) CC 132 (48.0) 79 (49.7) 52 (46.9) CT 115 (41.8) 66 (41.5) 46 (41.4) 2 (100) TT 28 (10.2) 14 (8.8) 13 (11.7) Total 275 (100) 159 (100) 111 (100) Allele 677C (%) 69.0 70.4 67.6 Allele 677T (%) 31.0 29.6 32.4

MTHFR A1298C Genotypes N (%) AA 143 (52.5) 70 (44.3) 69 (63.3) 1 (50.0) AC 116 (42.7) 77 (48.7) 38 (34.9) 1 (50.0) CC 13 (4.8) 11 (7.0) 2 (1.8) Total 272 (100) 158 (100) 109 (100) Allele 1298A (%) 73.9 68.7 80.7 Allele 1298C (%) 26.1 31.3 19.3

MTR A2756G Genotypes N (%) AA 174 (64.0) 98 (62.4) 73 (66.4) 1 (50.0) AG 91 (33.4) 55 (35.0) 34 (30.9) 1 (50.0) GG 7 (2.6) 4 (2.6) 3 (2.7) Total 272 (100) 157 (100) 110 (100) Allele 2756A (%) 80.7 79.9 81.8 Allele 2756G (%) 19.3 20.1 18.2

MTRR A66G Genotypes N (%) AA 92 (33.4) 56 (35.2) 36 (32.4) AG 128 (46.6) 71 (44.7) 56 (50.5) 1 (50.0) GG 55 (20.0) 32 (20.1) 19 (17.1) 1 (50.0) Total 275 (100) 159 (100) 111 (100) Allele 66A (%) 56.7 57.5 57.7 Allele 66G (%) 43.3 42.5 42.3

Abbreviations: MTHFR, methylenetetrahydrofolate reductase; MTR, methionine synthase; MTRR, methionine synthase reductase. Information about ethnic group and genotypes for MTHFR A1298C and MTR A2756G was not available for three women. No difference was found between genotype frequencies in Caucasian and African-descent women by w2-test for MTHFR C677T (P ¼ 0.719), MTR A2756G (P ¼ 0.781) and MTRR A66G (P ¼ 0.628) polymorphisms. However, there was a difference in frequencies of genotype MTHFR 1298AA between Caucasian and African-descent (P ¼ 0.004).

677CC/2756AG. However, pregnant women with the com- Predictors of metabolite variables bination of genotypes MTHFR 677TT/MTR 2756AA had Serum folate levels, MTHFR 677T allele (CT þ TT genotypes) lower serum folate levels than those with 677CC/2756AA, and MTR 2756AA genotypes were predictors of tHcy 677CC/2756AG and 677CT/2756AG. Higher values of tHcy concentrations in pregnant women. The range of serum were found in pregnant women with the combination of folate values observed in this study sample explains 15.5% of genotypes MTHFR 677TT/MTR 2756AA when compared with the variance in tHcy, and the MTHFR 677T allele and MTR the combination of genotypes: CC/AA, CC/AG, CC/GG, CT/ 2756AA genotype explain, respectively, 1.7 and 1.6% of the AA, CT/AG and TT/AG. Higher MMA levels were found in variance in tHcy (Table 4). SAM/SAH ratios were predicted by pregnant women with the combination of genotypes MTHFR Cbl levels (R2 ¼ 0.0164). The MMA levels were predicted by 677TT/MTR 2756AG as compared to 677CT/2756AG carriers serum creatinine (partial R2 ¼ 0.0207) (Table 4). (Table 3). In addition, pregnant women with the combination of genotypes MTHFR 677TT/MTRR 66AG and MTHFR 677TT/ Gene–nutrient interaction MTRR 66GG had lower serum folate than those with the The interaction effect between genotypes for MTHFR C677T, 677CC/66AA haplotype. The carriers with the combination MTHFR A1298C, MTR A2756G and MTRR A66G polymor- of genotypes 677TT/66GG had elevated tHcy levels com- phisms, and vitamin status on tHcy and MMA levels and SAM/ pared with all combinations of genotypes, except 677TT/ SAH ratios were evaluated. The medians for Cbl (142 pmol/l) 66AG carriers (Table 3). and folate (12.5 nmol/l) were used for characterizing two

European Journal of Clinical Nutrition Association between vitamin deficiency and polymorphisms PR Barbosa et al 1015 Table 2 Geometric means (95% CI) of biochemical variables according to genotypes for MTHFR C677T, MTHFR A1298C, MTR A2756G and MTRR A66G gene polymorphisms

Genotypes Cbl a (pmol/l) Serum folate a (nmol/l) tHcy a (mmol/l) SAM/SAH a MMAa (nmol/l)

MTHFR C677T CC (N ¼ 132) 145 (137, 153) 13.7 (12.8, 14.6) 6.3 (6.0, 6.7) 4.2 (3.8, 4.7) 229 (210, 248) CT þ TT (N ¼ 143) 140 (132, 149) 11.5 (10.8, 12.3) 7.3 (6.8, 7.8) 4.3 (4.0, 4.7) 237 (218, 259) ANCOVA P-value 0.665 0.002 0.001 0.495 0.362

MTHFR A1298C AA (N ¼ 143) 135 (127, 143) 12.7 (11.8, 13.5) 7.0 (6.6, 7.5) 4.3 (3.9, 4.6) 240 (220, 262) AC þ CC (N ¼ 129) 150 (142, 159) 12.4 (11.5, 13.4) 6.6 (6.2, 7.0) 4.3 (3.9, 4.7) 226 (208, 247) ANCOVA P-value 0.041 0.684 0.521 0.700 0.221

MTR A2756G AA (N ¼ 174) 136 (130, 143) 12.5 (11.7, 13.3) 7.0 (6.6, 7.4) 4.3 (3.9, 4.6) 233 (216, 251) AG þ GG (N ¼ 97) 154 (142, 166) 12.6 (11.6, 13.7) 6.4 (5.9, 6.9) 4.3 (3.9, 4.7) 230 (207, 255) ANCOVA P-value 0.002 0.662 0.049 0.860 0.675

MTRR A66G AA (N ¼ 92) 148 (137, 159) 13.0 (12.0, 14.2) 6.6 (6.1, 7.1) 4.4 (4.0, 4.9) 250 (224, 280) AG þ GG (N ¼ 183) 140 (133, 147) 12.3 (11.5, 13.0) 6.9 (6.5, 7.3) 4.2 (3.9, 4.6) 225 (210, 242) ANCOVA P-value 0.237 0.261 0.296 0.610 0.116

Abbreviations: Cbl, cobalamin; CI, confidence interval; MMA, methylmalonic acid; MTHFR, methylenetetrahydrofolate reductase; MTR, methionine synthase; MTRR, methionine synthase reductase; SAH, S-adenosylhomocysteine; SAM, S-adenosylmethionine; tHcy, total homocysteine. All values are geometric mean; 95% CIs in parentheses. aANCOVA performed on the log-transformed variables. The models were adjusted by covariates: age, ethnic group, parity and per capita income. groups according to vitamin status (above median and below SAM/SAH ratios. No differences in MMA levels were or equal median). associated with the gene polymorphisms independently of Table 5 shows results from interaction between genotypes serum folate or Cbl levels. for gene polymorphisms and vitamin status on tHcy levels. Analysis of SAM/SAH ratio showed that in MTHFR 677CC Low values of serum folate were associated with increased genotype carriers, lower ratios were associated with lower tHcy levels independently of genotypes for MTHFR C677T, Cbl levels (geometric mean 3.8 and 95% CI 3.2, 4.5) than MTHFR A1298C, MTR A2756G and MTRR A66G polymor- those with higher Cbl levels (geometric mean 4.7 and 95% phisms. In pregnant women with high serum folate levels, CI 4.2, 5.3) (P ¼ 0.049). Similar results were found in the MTHFR 677T allele was associated with high levels of pregnant women carrying the MTR 2756AA genotypes as tHcy (P ¼ 0.020) when the model was adjusted for covariates pregnant women with low Cbl levels had lower SAM/SAH (age, parity, ethnic group (Caucasian vs African descent) and (geometric mean 3.9 and 95% CI 3.5, 4.4) than those with monthly per capita income). Similar results were found for higher Cbl levels (geometric mean 4.7 and 95% CI 4.2, 5.3) MTR 2756AA genotype carriers when the model was adjusted (trend, P ¼ 0.053) after adjusting for covariates including for the same covariates including genotypes for MTHFR genotypes for the MTHFR C677T polymorphism. C677T polymorphism. The interaction between gene polymorphisms and Cbl status on tHcy levels was also evaluated (Table 5). In Previous history of miscarriage pregnant women carrying the MTHFR 677T allele, lower Information about the mother’s previous history of mis- Cbl levels were associated with higher tHcy levels (P ¼ 0.030). carriage was not available for three women. A total of 224 Moreover, the MTHFR 677CT þ TT genotypes carriers have (82.4%) of the pregnant women had no previous history of higher tHcy levels than 677CC carriers, independently of the miscarriage and 48 (17.6%) had a previous history with one Cbl status (Po0.05). or more miscarriages. Only 2 (0.7%) women had history of In MTHFR 1298AA genotype carriers, elevated tHcy levels three or more miscarriages. were found in pregnant women with low Cbl levels No difference was observed between the frequency of compared with those with high Cbl (P ¼ 0.040) after mothers with and without previous history of miscarriage adjusting for covariates including genotypes for the according to ethnic group (Caucasian-descent vs African- MTHFR C677T polymorphism. Similar results were found descent) (P ¼ 0.682, data not shown). in pregnant women carrying the MTRR 66AA genotype The pregnant women with previous history of miscarriage (P ¼ 0.049). had higher age and parity than those without history of Effects of interactions between gene polymorphisms miscarriage. However, no differences in (Cbl, and vitamin status were also evaluated on MMA levels and serum folate) and metabolite (tHcy, MMA, SAM, SAH)

European Journal of Clinical Nutrition Association between vitamin deficiency and polymorphisms PR Barbosa et al 1016 Table 3 Geometric means (95% CI) of vitamin and metabolite concentrations according to haplotypes for MTHFR C677T and MTHFR A1298C; and combination of genotypes for MTHFR C677T and MTR A2756G; and MTHFR C677T and MTRR A66G gene polymorphisms

Cbl* (pmol/l) Serum folate* (nmol/l) tHcy* (mmol/l) MMA* (nmol/l) SAM/SAH* ratio

MTHFR C677T/MTHFR A1298C CC/AA (N ¼ 59) 143 (131, 157) 14.8a (13.5, 16.3) 6.1a (5.5, 6.6) 244 (213, 281) 4.2 (3.5, 4.9) CC/AC (N ¼ 61) 145 (133, 158) 13.2a (11.8, 14.8) 6.4a (5.9, 6.9) 216 (193, 242) 4.2 (3.6, 4.7) CC/CC (N ¼ 11) 147 (123, 177) 10.5a,b (9.4, 11.8) 7.5a,b (6.0, 9.5) 220 (162, 298) 4.6 (3.0, 7.1) CT/AA (N ¼ 60) 135 (122, 149) 12.1a,b (10.9, 13.4) 7.4a,b (6.8, 8.0) 237 (208, 270) 4.4 (3.9, 4.9) CT/AC (N ¼ 52) 155 (141, 171) 11.9a,b (10.6, 13.4) 6.5a (5.9, 7.2) 227 (196, 263) 4.3 (3.7, 4.9) TT/AA (N ¼ 24) 118 (106, 130) 9.6b (8.4, 10.9) 9.0b (6.9, 11.7) 237 (189, 296) 4.3 (3.5, 5.1) TT/AC (N ¼ 3) 183 (150, 222) 11.7a,b (1.7, 81.2) 10.1a,b (2.8, 36.5) 442 (204, 956) 5.6 (2.4, 13.1) ANCOVA P-value 0.148 0.001 o0.001 0.114 0.875

MTHFR C677T/MTR A2756G CC/AA (N ¼ 84) 137a,b (127, 146) 13.9a (12.7, 15.2) 6.4a (6.0, 6.8) 228a,b (204, 255) 4.2 (3.6, 4.8) CC/AG (N ¼ 41) 162a (146, 181) 13.2a (11.8, 14.9) 6.1a (5.4, 7.0) 226a,b (196, 259) 4.4 (3.8, 5.2) CC/GG (N ¼ 4) 154a,b (103, 230) 14.3a,b (8.8, 23.2) 4.7a (1.9, 11.8) 187a,b (68, 516) 3.7 (1.7, 8.2) CT/AA (N ¼ 75) 139a,b (128, 150) 11.8a,b (10.8, 12.9) 7.1a (6.5, 7.7) 242a,b (216, 271) 4.5 (4.0, 4.9) CT/AG (N ¼ 37) 157a,b (135, 183) 12.7a (10.8, 14.9) 6.5a (6.0, 7.1) 198a (166, 235) 4.0 (3.4, 4.7) CT/GG (N ¼ 3) 132a,b (70, 249) 10.6a,b (6.8, 16.6) 9.7a,b (2.5, 38.0) 444a,b (155, 1274) 4.9 (1.7, 14.6) TT/AA (N ¼ 15) 125a,b (111, 141) 8.8b (7.2, 10.7) 11.2b (7.8, 16.1) 214a,b (161, 283) 3.8 (3.1, 4.7) TT/AG (N ¼ 13) 125b (102, 154) 10.7a,b (8.6, 13.4) 6.9a (5.3, 8.9) 324b (235, 446) 5.0 (3.7, 6.6) ANCOVA P-value 0.014 0.006 o0.001 0.026 0.904

MTHFR C677T/MTRR A66G CC/AA (N ¼ 45) 154 (140, 169) 14.3a (12.9, 15.8) 5.9a (5.4, 6.5) 236 (205, 271) 4.4 (3.7, 5.2) CC/AG (N ¼ 55) 139 (126, 153) 13.5a,b (11.9, 15.2) 6.5a (5.9, 7.1) 233 (204, 267) 3.9 (3.3, 4.6) CC/GG (N ¼ 32) 142 (126, 159) 13.2a,b (11.5, 15.0) 6.6a,b (5.8, 7.4) 212 (178, 252) 4.7 (3.9, 5.7) CT/AA (N ¼ 38) 148 (129, 170) 12.1a,b (10.4, 14.0) 7.5a,b (6.8, 8.3) 265 (217, 323) 4.4 (3.8, 5.0) CT/AG (N ¼ 60) 138 (127, 150) 11.8a,b (10.7, 13.1) 6.7a,b (6.1, 7.4) 217 (192, 246) 4.3 (3.8, 4.8) CT/GG (N ¼ 17) 158 (123, 203) 12.8a,b (10.4, 15.8) 6.7a,b (5.8, 7.7) 214 (179, 255) 4.2 (3.2, 5.7) TT/AA (N ¼ 9) 120 (94, 154) 11.3a,b (8.3, 15.4) 6.5a,b (4.5, 9.5) 260 (179, 377) 4.8 (3.8, 5.9) TT/AG (N ¼ 13) 131 (111, 155) 9.2b (7.6, 11.1) 9.5b,c (7.2, 12.4) 306 (197, 475) 4.8 (3.6, 6.5) TT/GG (N ¼ 6) 121 (95, 156) 8.4b (5.2, 13.4) 12.5c (4.9, 31.9) 207 (154, 278) 3.1 (2.1, 4.4) ANCOVA P-value 0.222 0.005 o0.001 0.343 0.672

Abbreviations: Cbl, cobalamin; CI, confidence interval; MMA, methylmalonic acid; MTHFR, methylenetetrahydrofolate reductase; MTR, methionine synthase; MTRR, methionine synthase reductase; tHcy, total homocysteine. All values are geometric means; 95% CIs in parentheses. *ANCOVA performed on the log-transformed variables. The models were adjusted by covariates: age, ethnic group, parity and per capita income. Values in a row with different superscript letters are significantly different, Po0.05 (Tukey’s test).

concentrations were observed between pregnant women nomic conditions and nutritional deficiency) and genetic from the two groups (data not shown). factors (presence of one polymorphism or combinations of No difference was observed in frequencies of women with several polymorphisms in genes of key enzymes of homo- and without previous history of miscarriage according to cysteine metabolism). genotypes for MTHFR C677T, MTHFR A1298C and MTRR It is noteworthy that this study was carried out before the A66G. However, the pregnant women with previous history food folic acid fortification program became effective in of miscarriage had a trend (P ¼ 0.098) for having higher Brazil in 2004. Moreover, the Brazilian women did not use frequency of MTR 2756AA genotype than pregnant women supplementation with folic acid or during their with no previous history of miscarriage (data not shown). 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 Discussion increases or decreases in metabolites did not occur. Pregnant women with renal insufficiency were excluded In the current study, 75% of Brazilian pregnant women had from this study, and the serum concentrations of MMA were Cbl concentrations lower than 179 pmol/l. This cutoff is used as a marker of impaired Cbl status and the serum tHcy similar to that cited by Bruinse and van den Berg (1995) as concentrations and SAM/SAH ratio were also used as markers indicative of marginal or deficient stores (values o180 pmol/l). of Cbl and folate deficiencies. The much lower Cbl levels and the alterations in metabolite We found that many variables affected the metabolites and levels found in this study may be the result of the interaction vitamin concentrations such as socioeconomic status, between environmental factors (such as lower socioeco- median per capita income and racial-ethnic group. In addition,

European Journal of Clinical Nutrition Association between vitamin deficiency and polymorphisms PR Barbosa et al 1017 Table 4 Association between biochemical variables and genotypes for MTHFR C677T, MTHFR A1298C, MTR A2756G and MTRR A66G gene polymorphisms as independent variables in three models of multiple linear regression analysis

Dependent variables (N)a Independent variablesa B (s.e.m.) R2 P-value

Intercept 1.45 (0.19) — o0.001 Cbl (pmol/l) À0.06 (0.07) 0.0028 0.407 Serum folate (nmol/l) À0.34 (0.06) 0.1548 o0.001 Creatinine (mg/dl) 0.19 (0.12) 0.0081 0.102 Model 1 SAM/SAH À0.01 (0.04) 0.0001 0.978 tHcy C677T (CC vs CT þ TT) 0.05 (0.02) 0.0172 0.017 (N ¼ 249) A1298C (AC þ CC vs AA) 0.01 (0.02) 0.0004 0.726 A2756G (AG þ GG vs AA) 0.04 (0.02) 0.0162 0.049 A66G (AA vs AG þ GG) 0.01 (0.02) 0.0005 0.712

Intercept 0.28 (0.31) — 0.369 Cbl (pmol/l) 0.19 (0.10) 0.0164 0.063 Serum folate (nmol/l) 0.12 (0.09) 0.0086 0.167 Creatinine (mg/dl) À0.20 (0.17) 0.0061 0.239 Model 2 tHcy (mmol/l) À0.01 (0.10) 0.0001 0.978 SAM/SAH C677T (CC vs CT þ TT) 0.03 (0.03) 0.0045 0.314 (N ¼ 249) A1298C (AC þ CC vs AA) 0.01 (0.03) 0.0007 0.650 A2756G (AG þ GG vs AA) 0.01 (0.03) 0.0005 0.692 A66G (AA vs AG þ GG) À0.01 (0.03) 0.0001 0.900

Intercept 3.27 (0.26) — o0.001 Cbl (pmol/l) À0.11 (0.10) 0.0099 0.267 Model 3 Serum folate (nmol/l) À0.05 (0.08) 0.0015 0.564 MMA Creatinine (mg/dl) 0.38 (0.16) 0.0207 0.018 (N ¼ 228) C677T (CC vs CT þ TT) 0.03 (0.03) 0.0061 0.332 A1298C (AC þ CC vs AA) 0.02 (0.03) 0.0023 0.414 A2756G (AG þ GG vs AA) 0.01 (0.03) 0.0004 0.733 A66G (AA vs AG þ GG) À0.05 (0.03) 0.0114 0.081

Abbreviations: B, regression parameter estimate; Cbl, cobalamin; MMA, methylmalonic acid; MTHFR, methylenetetrahydrofolate reductase; MTR, methionine synthase; MTRR, methionine synthase reductase; SAH, S-adenosylhomocysteine; SAM, S-adenosylmethionine; tHcy, total homocysteine. aLog-transformed variable. The three models were adjusted by maternal age, ethnic group, parity and per capita income.

there were interactions between the racial-ethnic groups the MTHFR 1298C allele in African-derived pregnant women and socioeconomic variables that impacted the biochemical was similar to that previously reported in mixed (22.0%) and variables. The lower Cbl and higher MMA values we found in black (13.7%) healthy blood donors in Sao Paulo city, Brazil pregnant women of Caucasian-descent were similar to (Pereira et al., 2004). The frequencies of rare genotypes for previously reported racial differences (Carmel, 1999; Stabler MTR A2756G and MTRR A66G were similar to those found in et al., 1999). Therefore, we controlled for race, ethnicity, other studies (Alessio et al., 2004; Pereira et al., 2004). parity and per capita income when analyzing our data. The effects of each gene polymorphism alone or in The frequency of MTHFR 677TT genotype (10.2%) in haplotypes on vitamin and metabolite levels were evaluated pregnant women in the current investigation was similar to in this study. The MTHFR C677T gene polymorphism is the that found in adults and children from Brazil (9–10%) most important genetic factor that influences blood tHcy (Arruda et al., 1998; Alessio et al., 2004; Pereira et al., 2004). levels in different populations (Russo et al., 2003; Alessio The T allele frequency found in pregnant women of African- et al., 2004; Pereira et al., 2004). In the present study, descent pregnant women (32.4%) was higher than that pregnant women carrying the MTHFR 677T allele (CT plus found in Brazilian black people from three distinct regions TT genotypes) had lower serum folate and Cbl and higher of Brazil (20.0%) (Arruda et al., 1998) and in Brazilian black tHcy levels than the CC genotype carriers. Previous studies blood donors (12.5%) (Pereira et al., 2004), Po0.05. How- have demonstrated that the MTHFR C677T polymorphism, ever, in black pregnant women, the frequency of MTHFR which causes an alanine-to-valine substitution at position 677T allele (19.6%) was similar to the frequencies found in 222 of the enzyme, confers MTHFR thermolability and previous studies with Brazilian individuals (Arruda et al., significant reduction of its activity in vitro (Frosst et al., 1998; Pereira et al., 2004). These results suggest that our 1995; Chango et al., 2000) affecting directly the folate status. sample is representative of the Brazilian population. We found that serum Cbl concentration was lower in In this study, the MTHFR 1298C allele was more frequent MTHFR 1298A allele carriers (AA, common-genotype) than (31.3%) in Caucasian Brazilian pregnant women than in in those carrying the 1298C allele (AC plus CC genotypes) in those of African-descent (19.3%, P ¼ 0.003). The frequency of contrast to findings previously reported in blood donors

European Journal of Clinical Nutrition Association between vitamin deficiency and polymorphisms PR Barbosa et al 1018 Table 5 Total homocysteine levels (mmol/l) according to genotypes for MTHFR C677T, MTHFR A1298C, MTR A2756G and MTRR A66G gene polymorphisms and serum folate or cobalamin levels (stratification by folate or cobalamin values above and below the median)

Serum folate P-value Serum cobalamin P-value

412.5 nmol/l p12.5 nmol/l 4142 pmol/l p142 pmol/l

MTHFR C677T a CC 5.8 (5.4, 6.2) 7.1 (6.4, 7.7) 0.001 6.0 (5.5, 6.6) 6.6 (6.1, 7.1) 0.259 77 55 68 64 CT þ TT 6.4 (6.0, 6.9) 8.1 (7.2, 9.0) 0.002 6.8 (6.2, 7.4) 7.9 (7.1, 8.7) 0.030 62 79 68 74 P-value 0.020 0.110 0.046 0.010

MTHFR A1298Cb AA 6.1 (5.7, 6.6) 8.3 (7.4, 9.4) o0.001 6.3 (5.7, 7.0) 7.6 (6.9, 8.4) 0.040 78 65 60 83 AC þ CC 6.1 (5.7, 6.5) 7.0 (6.4, 7.6) 0.017 6.4 (5.9, 7.0) 6.7 (6.2, 7.3) 0.541 60 68 74 55 P-value 0.708 0.237 0.465 0.660

MTR A2756Gb AA 6.3 (6.0, 6.6) 7.9 (7.1, 8.8) 0.002 6.6 (6.1, 7.1) 7.4 (6.8, 8.0) 0.116 91 81 77 96 AG þ GG 5.7 (5.1, 6.3) 7.1 (6.4, 7.9) o0.001 6.1 (5.6, 6.8) 6.8 (6.0, 7.7) 0.437 47 51 58 40 P-value 0.006 0.534 0.189 0.193

MTRR A66Gb AA 6.1 (5.6, 6.6) 7.2 (6.4, 8.1) 0.030 6.2 (5.6, 6.8) 7.2 (6.5, 7.9) 0.049 49 43 52 40 AG þ GG 6.1 (5.7, 6.5) 7.8 (7.1, 8.6) o0.001 6.5 (6.0, 7.0) 7.3 (6.7, 8.0) 0.244 90 91 84 98 P-value 0.962 0.311 0.349 0.821

Abbreviations: MTHFR, methylenetetrahydrofolate reductase; MTR, methionine synthase; MTRR, methionine synthase reductase. All values are geometric means; 95% CIs in parentheses, and number of subjects. The cutoff values of serum cobalamin and serum folate are medians. aStudent’s t-test performed on the log-transformed variables adjusted by covariates age, ethnic group, parity and per capita income. bStudent’s t-test adjusted by covariates age, ethnic group, parity, per capita income and genotypes for MTHFR C677T polymorphism.

from a Brazilian sample (Pereira et al., 2004). It is conceivable In our study, the MTRR A66G polymorphism alone was not that the result found in this study may be due to the effect associated with alterations in vitamin and metabolite con- of interactions between MTHFR C677T and A1298C poly- centrations and this finding was similar to those described in morphisms on Cbl levels as well as tHcy. Lower Cbl levels other studies (Wilson et al., 1999; Gaughan et al., 2001; Jacques were found in TT/AA genotype carriers when compared with et al., 2003). However, when the combinations of genotypes CC/CC genotype carriers. Interestingly, the interaction were taken into account, the carriers of combined genotypes between the 1298AA genotype and low Cbl determined a MTHFR 677TT/MTRR 66GG had elevated tHcy levels com- 20.6% increase in tHcy levels. pared with all combinations of genotypes for these poly- The MTHFR A1298C mutation, located in the enzyme morphisms, except 677TT/66AG. Similar results were found in regulatory domain does not result in either a thermolabile nonpregnant healthy American women (Vaughn et al., 2004). protein or increased tHcy (van der Put et al., 1998; Hanson It appears that the interaction between MTHFR 677TT and et al., 2001). However, MTHFR 677CT/1298AC compound MTRR 66GG genotypes is associated with high tHcy levels. heterozygosity reportedly has similar impact as C677T It has been suggested that the MTRR A66G (I22M) variant homozygosity (Chango et al., 2000). In the current investi- is located in the putative flavin mononucleotide-binding gation, the one-way analysis adjusted by covariates showed domain of the MTRR enzyme that interacts with MTR that the CT/AC haplotype is not associated with increased (Leclerc et al., 1998). Substitution of an isoleucine by a tHcy and decreased serum folate and Cbl levels in pregnant methionine in the 66G allele could thus disrupt the binding women. On the other hand, the pregnant women carrying of MTRR to the MTR-cob(I)alamin-complex, thereby decreas- TT/AA haplotype had lower serum folate and higher tHcy ing the rate of homocysteine remethylation (Olteanu et al., levels than the CC/AA and CC/AC haplotypes carriers. It 2002). Therefore, it is possible that the combination of appears that the TT/AA haplotype is more likely to influence MTHFR 677T and MTRR 66G alleles affects substantially the homocysteine metabolism than the CT/AC haplotype. homocysteine status in pregnant women.

European Journal of Clinical Nutrition Association between vitamin deficiency and polymorphisms PR Barbosa et al 1019 Pregnant women with MTR 2756AA genotype had lower we did not find that Cbl levels or the variants for Cbl and higher tHcy levels than those with the G allele (AG polymorphisms were an independent predictor of the plus GG genotypes). This finding is similar to those found in MMA levels, in contrast to the serum creatinine. Therefore, a working male Irish population (Harmon et al., 1999) and additional studies with larger population samples should be healthy adult Canadian controls (Miriuka et al., 2005) and conducted in order to confirm these results. different from the finding in healthy Dutch controls (Klerk In a previous study, we found that the patients with severe et al., 2003). It is conceivable that the presence of the G allele Cbl deficient megaloblastic anemia had elevated serum SAH in the methionine synthase gene is a protective factor levels and low SAM/SAH ratio, which were corrected with against increasing tHcy concentrations. Cbl therapy (Guerra-Shinohara et al., 2007). Lower SAM/SAH An interesting finding was observed in carriers of 677TT/ ratio was found in pregnant women in the lowest Cbl 2756AA genotypes, who had higher tHcy levels than all quartile (Cblp102 pmol/l) (Guerra-Shinohara et al., 2004). combinations of these genotypes, except for 677CT/2756GG. Thus, in the present study, we used the SAM/SAH ratio as This result is consistent with the predictors of tHcy by potential marker of Cbl deficiency. MTHFR C677T, MTHFR multiple linear regression analysis in which serum folate A1298C, MTR A2756G and MTRR A66G gene polymor- concentration, MTHFR 677CT plus TT genotypes and MTR phisms were not associated with variations in SAM/SAH ratio 2756AA genotypes were the determinants of tHcy concen- in our sample. On the other hand, reduced SAM/SAH ratio trations in Brazilian pregnant women. Our results are was determined by the interaction between low Cbl levels consistent with those of Harmon et al. (1999), but are and 677CC or 2756AA genotypes. It is possible that this different from other studies which reported no effects of finding is dependent on the reduced levels of Cbl consider- MTHFR C677T and/or MTR A2756G polymorphism on tHcy ing that this vitamin was the variable selected in models by levels (Christensen et al., 1999; Molloy et al., 2002; Alessio the multiple linear regression analysis. et al., 2004). Cbl is the second nutrient that has been implicated in The MTR A2756G polymorphism, which results in sub- increasing risk of NTDs. Several studies have reported the stitution of glycine for aspartic acid at position decreased Cbl concentrations in the circulation or amniotic 919, is located in a domain of the protein that interacts fluid in mothers of children with NTDs (Kirke et al., 1993; with SAM and auxiliary that are required for the Adams et al., 1995; Steen et al., 1998). A consequence of this reductive methylation and reactivation of the Cbl , vitamin deficiency is elevated tHcy and MMA levels, which which can be inactivated by oxidation during catalysis have been reported to be a risk factor for NTDs. (Leclerc et al., 1998; Harmon et al., 1999). It has been In the current study, only two pregnant women had a speculated that the MTR 2756A allele might impair the history of three or more pregnancy losses. The pregnant binding of SAM and/or auxiliary proteins, or possibly impair women with a previous history of one or more miscarriages the stability of protein (Harmon et al., 1999). However, this did not have elevated tHcy levels when compared with those effect was not demonstrated in in vitro studies (Harmon et al., having no history of miscarriage. We could not find an 1999). increased frequency of gene polymorphisms in these two In the gene–nutrient interaction analysis, elevated tHcy groups. Our results were different from other studies (Nelen levels were associated with low serum folate status in et al., 2000; Unfried et al., 2002; Holmes et al., 2005; Mtiraoui pregnant women independently of MTHFR, MTR and MTRR et al., 2006). It is possible that the small number of Brazilian gene polymorphisms. These findings show that reduced pregnant women with previous history of three or more serum folate is the strongest environmental factor for miscarriages could be responsible for these differences. elevated tHcy in pregnant women. Interestingly, increased Our findings contribute to understanding the role of tHcy was also dependent on the interaction between MTHFR nutritional and genetic factors on maternal metabolism, 677T allele or MTHFR 1298AA or MTRR 66AA genotypes and which may impact their newborns. The effect of the low levels of serum Cbl. Therefore, gene–nutrient interaction combination between nutrition and gene polymorphisms found in pregnant women may be important in determining during pregnancy on vitamin dependent metabolite (tHcy the risk for increased tHcy during pregnancy and in neonates and MMA) levels and DNA methylation index should be considering its risk for NTDs and other fetal abnormalities. investigated further. 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 Acknowledgements relation between MMA levels and isolated gene polymor- phisms in pregnant women. On the other hand, the MTHFR This study was supported financially by Fundac¸a˜ode 677TT genotype in combination with MTR 2756AG geno- Amparo a` Pesquisa do Estado de Sa˜o Paulo – FAPESP, Brazil type was associated with increased MMA levels when (Proc. 00/12467-0 and 01/09836-7) and PHS Grant NIA-AG- compared with 677CT plus 2756AG. This result may be 09834. Andre LK Machado and Renata C Braga had influenced by the low number of individuals with these fellowships of Projeto 4 (Pro´ Reitoria de Pesquisa) da genetic characteristics in our sample. Moreover, in our study, Universidade de Sa˜o Paulo and PIBIC CNPq, respectively.

European Journal of Clinical Nutrition Association between vitamin deficiency and polymorphisms PR Barbosa et al 1020 Elvira M Guerra-Shinohara is a recipient of a fellowship from Gaughan DJ, Kluijtmans LAJ, Barbaux S, McMaster D, Young IS, CNPq, Brasilia, DF, Brazil. We thank the Hospital Regional of Yarnell JWG et al. (2001). The methionine synthase reductase Conjunto Hospitalar de Sorocaba and Hospital Santa Lucin- (MTRR) A66G polymorphism is a novel genetic determinant of plasma homocysteine concentrations. Atherosclerosis 157,451–456. da and the pregnant women who participated in the study. Guerra-Shinohara EM, Morita OE, Pagliusi RA, Blaia-d’Avila VL, Allen RH, Stabler SP (2007). Elevated serum S-adenosylhomocysteine in cobalamin-deficient megaloblastic anemia. Metabolism 56, 339–347. References Guerra-Shinohara EM, Morita OE, Peres S, Pagliusi RA, Sampaio Neto LF, D’Almeida V et al. (2004). Low S-adenosylmethionine/ S-adenosylhomocysteine ratio associated with vitamin deficiency Adams Jr MJ, Khoury MJ, Scanlon KS, Stevenson RE, Knight GJ, in Brazilian pregnant women and newborns. Am J Clin Nutr 80, Haddow JE et al. (1995). Elevated midtrimester serum methyl- 1312–1321. malonic acid levels as a risk factor for neural tube defects. Guerra-Shinohara EM, Paiva AA, Rondo´ PHC, Yamasaki K, Terzi CA, Teratology 51, 311–317. D’Almeida V (2002). Relationship between total homocysteine Alessio AC, Annichino-Bizzacchi JM, Bydlowski SP, Eberlin MN, and folate levels in pregnant women and their newborn babies Vellasco AP, Hoehr NF (2004). Polymorphisms in the methylene- according to maternal serum levels of vitamin B12. BJOG 109, tetrahydrofolate reductase and methionine synthase reductase 784–791. genes and homocysteine levels in Brazilian children. Am J Med Hanson NQ, Aras O, Yang F, Tsai MY (2001). C677T and A1298C Genet A 128, 256–260. polymorphisms of the methylenetetrahydrofolate reductase gene: Al-Gazali LI, Padmanabhan R, Melnyk S, Yi P, Pogribny IP, Pogribna incidence and effect of combined genotypes on plasma fasting and M et al. (2001). Abnormal folate metabolism and genetic post-methionine load homocysteine in vascular disease. Clin Chem polymorphism of the folate pathway in a child with Down 47, 661–666. Syndrome and Neural Tube Defects. Am J Med Genet 103, 128–132. Harmon DL, Shields DC, Woodside JV, McMaster D, Yarnell JW, Allen RH, Stabler SP, Savage DG, Lindenbaum J (1993). Metabolic Young IS et al. (1999). Methionine synthase D919G polymorphism abnormalities in cobalamin (vitamin B12) and folate deficiency. is a significant but modest determinant of circulating homo- FASEB J 7, 1344–1353. cysteine concentrations. Genet Epidemiol 17, 298–309. Andersson A, Hultberg B, Brattstrom L, Isaksson A (1992). Decreased Holmes VA, Wallace JMW, Alexander HD, Gilmore WS, Bradbury I, serum homocysteine in pregnancy. Eur J Clin Chem Clin Biochem Ward M et al. (2005). Homocysteine is lower in the third trimester 30, 377–379. of pregnancy in women with enhanced folate status from Arruda VR, Siquiera LH, Gonc¸alves MS, Von Zuben PM, Soares MC, Clin Chem Menezes R et al. (1998). Prevalence of the mutation C677T in the continued folic acid supplementation. 51, 629–634. et al. methylenetetrahydrofolate reductase gene among distinct ethnic Jacques PF, Bostom AG, Selhub J, Rich S, Ellison RC, Eckfeldt JH groups in Brazil. Am J Med Genet 78, 332–335. (2003). Effects of polymorphisms of methionine synthase and Bruinse HW, van den Berg H (1995). Changes of some vitamin levels methionine synthase reductase on total plasma homocysteine in during and after normal pregnancy. Eur J Obstet Gynecol Reprod Biol the NHLBI Family Heart Study. Atherosclerosis 166, 49–55. 61, 31–37. Kirke PN, Molloy AM, Daly LE, Burker H, Weir DG, Scott JM (1993). Carmel R (1999). Ethnic and racial factors in cobalamin and its Maternal plasma folate and vitamin B12 are independent risk disorders. Semin Hematol 36, 88–100. factors for neural tube defects. QJMed86, 703–708. Chanarin I (1990). The Megaloblastic Anemias, 3rd edn. Blackwell Klerk M, Lievers KJA, Kluijtmans LAJ, Blom HJ, den Heijer M, Scientific Publications: London. Schorten EG et al. (2003). The 2756A4G variant in the gene Chango A, Boisson F, Qilliot D, Droesch S, Pfister M, Fillon-Emery N encoding methionine synthase: its relation with plasma homo- et al. (2000). The effect of 677CT and 1298AC mutations on cysteine levels and risk of coronary heart disease in a Dutch case- plasma homocysteine and 5,10-methylenetetrahydrofolate reduc- control study. Thromb Res 110, 87–91. tase activity in healthy subjects. Br J Nutr 83, 593–596. Leclerc D, Wilson A, Dumas R, Gafuik C, Song D, Watkins D et al. Christensen B, Arbour L, Tran P, Leclerc D, Sabbaghian N, Platt R et al. (1998). Cloning and mapping of a cDNA for methionine synthase (1999). Genetic polymorphisms in methylenetetrahydrofolate reductase, a flavoprotein defective in patients with homocystinuria. reductase and methionine synthase, folate levels in red blood Proc Natl Acad Sci USA 95, 3059–3064. cells, and risk of neural tube defects. Am J Med Genet 84, 151–157. Miriuka SG, Langman LJ, Evrovski J, Miner SE, D’Mello N, Delgado Clarke S, Banfield K (2001). S-Adenosylmethionine-dependent DH et al. (2005). Genetic polymorphisms predisposing to hyper- methyltransferases. In: Carmel R, Jacobsen DW (eds.). Homocys- homocysteinemia in cardiac transplant patients. Transpl Int 18, teine in Health and Disease. Cambridge University Press: Cambridge 29–35. UK. pp 63–78. Molloy AM, Mills JL, McPartlin J, Kirke PN, Scott JM, Daly S (2002). Cunha ALA, Hirata M, Kim CA, Guerra-Shinohara EM, Nonoyama K, Maternal and fetal plasma homocysteine concentrations at birth: Hirata RDC (2002). Metabolic effects of C677T and A1298C the influence of folate, vitamin B12, and the 5,10-methylenete- mutations at the MTHFR gene in Brazilian children with Neural trahydrofolate reductase 677C-T variant. Am J Obstet Gynecol Tube Defects. Clin Chim Acta 318, 139–143. 186, 499–503. Friso S, Choi SW, Girelli D, Mason JB, Dolnikowski GG, Bagley PJ Mtiraoui N, Zammiti W, Ghazouani L, Braham NJ, Saidi S, Finan RR et al. (2002). A common mutation in the 5,10-methylenetetra- et al. (2006). Methylenetetrahydrofolate reductase C677T and hydrofolate reductase gene affects genomic DNA methylation A1298C polymorphism and changes in homocysteine concentra- through an interaction with folate status. Proc Natl Acad Sci USA tions in women with idiopathic recurrent pregnancy losses. 99, 5606–5611. Reproduction 131, 395–401. Friso S, Girelli D, Trabetti E, Olivieri O, Guarini P, Pignatti PF et al. Murphy MM, Scott JM, Arija V, Molloy AM, Fernandez-Ballart JD (2005). The MTHFR 1298A4C polymorphism and genomic DNA (2004). Maternal homocysteine before conception and through- methylation in human lymphocytes. Cancer Epidemiol Biomarkers out pregnancy predicts fetal homocysteine and birth weight. Prev 14, 938–943. Clin Chem 50, 1406–1412. Frosst P, Blom HJ, Milos R, Gyette P, Sheppard CA, Matthews RG et al. Murphy MM, Scott JM, Mcarlim JM, Fernandez-Ballart JD (2002). The (1995). A candidate genetic risk factor for vascular disease: a pregnancy-related decrease in fasting plasma homocysteine is not common mutation in methylenetetrahydrofolate reductase explained by folic supplementation, hemodilution, or a decrease (MTHFR). Nat Genet 10, 111–113. in albumin in a longitudinal study. Am J Clin Nutr 76, 614–619.

European Journal of Clinical Nutrition Association between vitamin deficiency and polymorphisms PR Barbosa et al 1021 Nelen WLDM, Blom HJ, Steegers EAP, Heijer MD, Thomas CMG, Stabler SP, Marcell PD, Podell ER, Allen RH, Lindenbaum J (1986). Eskes TAAB (2000). Homocysteine and folate levels as risk factors Assay of methylmalonic acid in the serum of patients with for recurrent early pregnancy loss. Obstet Gynecol 95, 519–524. cobalamin deficiency using capillary gas chromatography–mass Olteanu H, Munson T, Banerjee R (2002). Differences in the spectrometry. J Clin Invest 77, 1606–1612. efficiency of reductive activation of methionine synthase and Steen MT, Boddie AM, Fisher AJ, MacMahon W, Saxe D, Sullivan KM exogeneous electron acceptors between the common polymorphic et al. (1998). Neural-tube defects are associated with low variants of human methionine synthase reductase. Biochemistry concentrations of cobalamin (vitamin B12) in amniotic fluid. 45, 13378–13385. Prenat Diagn 18, 545–555. Pereira AC, Schettert IS, Morandini Filho AAF, Guerra-Shinohara EM, Unfried G, Griesmacher A, Weismu¨ller W, Nagele F, Huber JC, Krieger JE (2004). Methylenetetrahydrofolate reductase (MTHFR) Tempfer CB (2002). The C677T polymorphism of the methylene- c677t gene variant modulates the homocysteine folate correlation tetrahydrofolate reductase gene and idiopathic recurrent mis- in a mild folate-deficient population. Clin Chim Acta 340, 99–105. carriage. Obstet Gynecol 99, 614–619. Russo GT, Friso S, Jacques PF, Rogers G, Cucinotta D, Wilson PW et al. van der Put NM, Gabreels F, Stevens EM, Smeitink JA, Trijbels FJ, (2003). Age and gender affect the relation between methylenete- Eskes TK et al. (1998). A second common mutation in the trahydrofolate reductase C677T genotype and fasting plasma methylenetetrahydrofolate reductase gene: An additional risk homocysteine concentrations in the Framigham Offspring Study factor for neural tube defects? Am J Hum Genet 62, 1044–1051. Cohort. J Nutr 133, 3416–3421. Vaughn JD, Bailey LB, Shelnutt KP, Von-Castel Dunwoody KM, Salazar LA, Hirata MH, Cavalli SA, Machado MO, Hirata RDC (1998). Maneval DR, Davis SR et al. (2004). Methionine synthase reductase Optimized procedure for DNA isolation from fresh and cryo- 66A-G polymorphism is associated with increased plasma preserved clotted human blood useful in clinical molecular homocysteine concentration when combined with homozygous testing. Clin Chem 44, 1748–1750. methylenetetrahydrofolate reductase 677C-T variant. J Nutr 134, Scott JM, Dinn JJ, Wilson P, Weir DG (1981). Pathogenesis of 1958–2990. subacute combined degeneration: a result of methyl group Walker MC, Smith GN, Perkins SL, Keely EJ, Garner PR (1999). deficiency. Lancet 2, 334–337. Changes in homocysteine levels during normal pregnancy. Am J Shields DC, Kirke PN, Mills JL, Ramsbottom D, Molloy AM, Burke H Obstet Gynecol 180, 660–664. et al. (1999). The ‘thermolabile’ variant of methylenetetrahydro- Wilson A, Platt R, Wu Q, Leclerc D, Christensen B, Yang H et al. folate reductase and neural tube defects: an evaluation of genetic (1999). A common variant in methionine synthase reductase risk and the relative importance of the genotypes of the embryo combined with low cobalamin (vitamin B12) increases risk for and the mother. Am J Hum Genet 64, 1045–1055. spina bifida. Mol Genet Metab 67, 317–323. Stabler SP, Allen RH (2004). Quantification of serum and urinary Yamada K, Gravel RA, Toraya T, Matthews RG (2006). Human S-adenosylmethionine and S-adenosylhomocysteine by stable- methionine synthase reductase is a molecular chaperone isotope-dilution liquid chromatography-mass spectrometry. Clin for human methionine synthase. Proc Natl Acad Sci USA 103, Chem 50, 365–372. 9476–9481. Stabler SP, Allen RH, Fried LP, Pahor M, Kittner SJ, Penninx BWJH Zetterberg H (2004). Methylenetetrahydrofolate reductase and et al. (1999). Racial differences in prevalence of cobalamin and transcobalamin genetic polymorphisms in human spontaneous folate deficiencies in disable elderly women. Am J Clin Nutr 70, abortion: biological and clinical implications. Reprod Biol Endocrinol 911–919. 2,7. Stabler SP, Marcell PD, Podell ER, Allen RH (1987). Quantitation of Zetterberg H, Regland B, Palme´r M, Ricksten A, Palmqvist L, Rymo L total homocysteine, total cysteine, and methionine in normal et al. (2002). Increased frequency of combined methylenetetra- serum and urine using capillary gas chromatography-mass hydrofolate reductase C677T and A1298C mutated alleles in spectrometry. Anal Biochem 162, 185–196. spontaneously aborted. Eur J Hum Genet 10, 113–118.

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