European Journal of Clinical Nutrition (2008) 62, 1010–1021

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ORIGINAL ARTICLE
Association between decreased vitamin levels and
MTHFR, MTR and MTRR gene 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

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

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 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: emguerra@usp.br
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).
Received 17 October 2006; revised 19 March 2007; accepted 18 April 2007; published online 23 May 2007

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-adenosyl-
methionine (SAM) (Scott et al., 1981). In a previous study, we
demonstrated that concentrations of S-adenosylhomocys-
teine (SAH) were higher and methionine and SAM concentra-
tions and SAM/SAH ratios were lower in pregnant women in
the lowest Cbl quartile (Cblp102 pmol/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, 227.98 g 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 methylene-
tetrahydrofolate to 5-methyltetrahydrofolate. MTR is a
Cbl-dependent enzyme that uses the methyl group from 5-
methyltetrahydrofolate for remethylation of tHcy to methio-
nine. In this reaction cob(I)alamin is readily oxidized to
Association between vitamin deficiency and polymorphisms
PR Barbosa et al
1011
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 remethyl-
ation 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 (Christen-
sen 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 interac-
tion between vitamin deficiencies and MTHFR, MTR and
MTRR gene polymorphisms on vitamin-dependent metabo-
lite 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.
European Journal of Clinical Nutrition
 Association between vitamin deficiency and polymorphisms
PR Barbosa et al
1012
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 12 h 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 ¼ 28). Women who took
supplements during pregnancy (N ¼ 94) and those with no
information about the use of supplements (N ¼ 8) were also
excluded from this study.
Socioeconomic data (family monthly per capita income,
schooling and occupation), obstetrical status and supple-
mental 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 de-
scriptions 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 ex-
cluded 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 polymor-
phism was accomplished by polymerase chain reaction
European Journal of Clinical Nutrition
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: 50 -GGTGCCAGGTATA
CAGTGACTCT-30 and reverse: 50 -GATCCAAAGCCTTTTA
CACTCCTC-30 ) and HaeIII endonuclease in PCR-RFLP assays.
MTRR A66G was genotyped using primers (forward: 50 -
AAGGCCATCGCAGAAGACAT-30 and reverse: 50 -CACTTCC
CAACCAAAATTCTTCAAAG-30 ) 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
Po0.05.
Hardy–Weinberg equilibrium was determined for all of the
genotypes using w2 testing. w2 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 pUS$69.37 and higher per capita income
4US$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 accord-
ing 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 þ TT).
Results
Population characteristics
Of the 405 eligible subjects, 275 pregnant women met the
entry criteria of this study. The mean (7s.d.) age of the
pregnant women was 25.2 (76.5) years (range: 13–44 years),
gestational age: 39.0 (71.2) weeks (range: 37–42 weeks) and
parity 2.671.7, including the actual pregnancy. Low socio-
economic 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 ¼ 0.158).
The Caucasian-descent women had higher per capita
income (US$73.80) than African-descent women (US$56.30),
P ¼ 0.005. No difference was observed in the ages of women
between two groups (P ¼ 0.203). However, there was a trend
(P ¼ 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)
(Po0.001). Low Cbl levels (P ¼ 0.041) and higher tHcy
(P ¼ 0.012) and MMA levels (P ¼ 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 ¼ 0.007.
Association between vitamin deficiency and polymorphisms
PR Barbosa et al
1013
The Student’s t-test adjusted by per capita income showed
that the Caucasian-descent women had lower Cbl (trend,
P ¼ 0.074) and higher MMA (P ¼ 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 (P40.05).
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 ¼ 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
0.61 mg/dl (0.60, 0.62).
Pregnant women carrying the MTHFR 677T allele (CT þ 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 þ 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 geno-
types for MTRR A66G polymorphisms (Table 2).
The genotypes for MTHFR C677T, MTHFR A1298C, MTR
A2756G and MTRR A66G polymorphisms were not asso-
ciated 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
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-
bination 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 com-
pared with all combinations of genotypes, except 677TT/
66AG carriers (Table 3).
Predictors of metabolite variables
Serum folate levels, MTHFR 677T allele (CT þ 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 ¼ 0.0164). The MMA levels were predicted by
serum creatinine (partial R2 ¼ 0.0207) (Table 4).
Gene–nutrient interaction
The interaction effect between genotypes for MTHFR C677T,
MTHFR A1298C, MTR A2756G and MTRR A66G polymor-
phisms, and vitamin status on tHcy and MMA levels and SAM/
SAH ratios were evaluated. The medians for Cbl (142 pmol/l)
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.
a
ANCOVA 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
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 polymor-
phisms. In pregnant women with high serum folate levels,
the MTHFR 677T allele was associated with high levels of
tHcy (P ¼ 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 ¼ 0.030).
Moreover, the MTHFR 677CT þ TT genotypes carriers have
higher tHcy levels than 677CC carriers, independently of the
Cbl status (Po0.05).
In MTHFR 1298AA genotype carriers, elevated tHcy levels
were found in pregnant women with low Cbl levels
compared with those with high Cbl (P ¼ 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 ¼ 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 ¼ 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 ¼ 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 mis-
carriage 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 ¼ 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)

European Journal of Clinical Nutrition

 Association between vitamin deficiency and polymorphisms
PR Barbosa et al
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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
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 ¼ 0.098) for having higher
frequency of MTR 2756AA genotype than pregnant women
with no previous history of miscarriage (data not shown).
Discussion
In the current study, 75% of Brazilian pregnant women had
Cbl concentrations lower than 179 pmol/l. This cutoff is
similar to that cited by Bruinse and van den Berg (1995) as
indicative of marginal or deficient stores (values o180 pmol/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 socioeco-
nomic conditions and nutritional deficiency) and genetic
factors (presence of one polymorphism or combinations of
several polymorphisms in genes of key enzymes of homo-
cysteine 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,

European Journal of Clinical Nutrition

 Association between vitamin deficiency and polymorphisms
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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) 0.06 (0.07) 0.0028 0.407
Serum folate (nmol/l) 0.34 (0.06) 0.1548 o0.001
Creatinine (mg/dl) 0.19 (0.12) 0.0081 0.102
Model 1 SAM/SAH 0.01 (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) 0.20 (0.17) 0.0061 0.239
Model 2 tHcy (mmol/l) 0.01 (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) 0.01 (0.03) 0.0001 0.900

Intercept 3.27 (0.26) — o0.001

Cbl (pmol/l) 0.11 (0.10) 0.0099 0.267
Model 3 Serum folate (nmol/l) 0.05 (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) 0.05 (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.
a
Log-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
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), Po0.05. How-
ever, 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 ¼ 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

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

a
MTHFR C677T
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.
a
Student’s t-test performed on the log-transformed variables adjusted by covariates age, ethnic group, parity and per capita income.
b
Student’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
that the result found in this study may be due to the effect
of interactions between MTHFR C677T and A1298C poly-
morphisms 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 investi-
gation, 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 con-
centrations 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 com-
pared with all combinations of genotypes for these poly-
morphisms, 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 decreas-
ing 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.

European Journal of Clinical Nutrition


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 concen-
trations 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 sub-
stitution 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 polymor-
phisms in pregnant women. On the other hand, the MTHFR
677TT genotype in combination with MTR 2756AG geno-
type 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,
Association between vitamin deficiency and polymorphisms
PR Barbosa et al
1019
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 (Cblp102 pmol/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 polymor-
phisms 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 consider-
ing 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.
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.
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
CNPq, Brasilia, DF, Brazil. We thank the Hospital Regional of
Conjunto Hospitalar de Sorocaba and Hospital Santa Lucin-
da and the pregnant women who participated in the study.
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