Molecular Genetics and Metabolism 98 (2009) 166–172

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Molecular Genetics and Metabolism

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Vitamin B12 and birth defects

Fei Li a, David Watkins a, David S. Rosenblatt a,b,*
a
Department of Human Genetics, McGill University, 1205 Avenue Docteur Penfield, N5/13, Montreal, Que., Canada H3A 1B1
b
Division of Medical Genetics, Department of Medicine and Department of Medical Genetics, McGill University Health Centre, 1650 Cedar Avenue, Room L3-319,
Montreal, Que., Canada H3G 1A4

a r t i c l e i n f o a b s t r a c t

Article history: We have reviewed the literature on the effect of vitamin B12 (cobalamin) on development and birth
Received 5 June 2009 defects. In rodents, administration of antibodies to cubilin, a component of the intestinal receptor respon-
Accepted 5 June 2009 sible for cobalamin absorption, results in a variety of defects including neural tube defects. There is no
Available online 11 June 2009
direct evidence that this is mediated through a direct effect on cobalamin metabolism. Homozygosity
for inactive versions of the genes for CUBN coding for cubilin, AMN, coding for amnionless, the MTR gene
Keywords: coding for methionine synthase, or MTRR coding for methionine synthase reductase, is embryonic lethal
Birth defects
in mice. Homozygosity for a hypomorphic form of the MTRR gene is associated with increased occurrence
Development
Neural tube defects
of defects. In man, the following have been associated with neural tube defects: decreased maternal
Vitamin B12 serum and amniotic fluid levels of vitamin B12; decreased serum levels of cobalamin bound to the serum
Fortification transport protein transcobalamin; increased levels of homocysteine and methylmalonic acid; and the G
Folate allele in mothers and embryos at the 66A>G polymorphism in the MTRR gene. A prospective study to
determine whether fortification of food with vitamin B12 in addition to folic acid might decrease the inci-
dence of birth defects to a greater extent than does fortification with folic acid alone is warranted.
Ó 2009 Elsevier Inc. All rights reserved.

Introduction
Folate supplementation in the periconceptional period can pre-
vent occurrence and recurrence of neural tube defects [1,2]. This
finding led to fortification of cereal grains in the United States, Can-
ada and other countries with folic acid [3]. Fortification has been
associated with significantly decreased prevalence of neural tube
defects [4–6] and smaller reductions in the prevalence of other
birth defects, including transposition of the great arteries, cleft pal-
ate, pyloric stenosis, upper limb reduction defects and omphalo-
cele [7]. However, even in the presence of folic acid fortification
neural tube defects continue to occur [8], and there is thus interest
in other interventions that could further reduce the prevalence of
these disorders. The mechanism by which folate deficiency affects
neural tube closure remains unknown, although elevation of
homocysteine levels and impairment of S-adenosylmethionine-
dependent transmethylation may be implicated [9,10]. Because
deficiency of either folate or cobalamin results in similar biochem-
ical effects, including elevated homocysteine levels and impaired
synthesis of S-adenosylmethionine, cobalamin deficiency may also
contribute to development of neural tube defects and other birth
defects, and fortification of food with cobalamin together with folic
* Corresponding author. Address: McGill University Health Centre-MGH Site,
1650 Cedar Avenue L3-319, Montreal, Que., Canada H3G 1A4. Fax: +1 514 934 8536.
E-mail address: david.rosenblatt@mcgill.ca (D.S. Rosenblatt).
acid might be an effective way to further reduce birth defect prev-
alence. A role for cobalamin insufficiency in development of neural
tube defects was suggested as early as 1980 [11]. We have
reviewed the literature on animal models as well as some of the
studies that associate decreased levels of vitamin B12 or polymor-
phisms in genes that play a role in metabolism of vitamin B12, with
developmental abnormalities and birth defects in man.
Cobalamin metabolism
Vitamin B12 (cobalamin) is a water-soluble vitamin that is
required for normal cellular intermediary metabolism. There are
two reactions in mammalian cells that require derivatives of cobal-
amin for activity: the cytoplasmic enzyme methionine synthase,
which catalyzes methylation of homocysteine to form methionine,
requires methylcobalamin for activity; and the mitochondrial
enzyme methylmalonylCoA mutase, which converts methylmalo-
nylCoA generated during catabolism of branched-chain amino
acids and odd-chain fatty acids, to succinylCoA, requires adeno-
sylcobalamin for activity [12]. Deficiency of cobalamin or defects
in cobalamin metabolism results in accumulation of the substrates
methylmalonic acid and homocysteine in blood and urine.
Uptake of dietary cobalamin and its delivery to cells is a com-
plex process [12], requiring three cobalamin-binding proteins,
intrinsic factor, haptocorrin and transcobalamin, and their cell
membrane receptors (Fig. 1). Cobalamin in food is released from

1096-7192/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.ymgme.2009.06.004
 F. Li et al. / Molecular Genetics and Metabolism 98 (2009) 166–172 167

Fig. 1. Uptake of cobalamin in the gut. Cobalamin (Cbl) binds to haptocorrin (HC) in the stomach. After digestion of HC in the jejunum, cobalamin becomes bound to intrinsic
factor (IF). The IF-cobalamin complex binds to the cubam receptor (composed of cubilin and amnionless) in the distal ileum, where it is taken up by receptor-mediated
endocytosis. Following breakdown of IF, cobalamin is released into blood attached to transcobalamin (TC).

proteins in the acid environment of the stomach and becomes
bound to haptocorrin secreted in the saliva and gastric secretions.
Cobalamin is released from haptocorrin by pancreatic proteases in
the jejunum, where it becomes associated with intrinsic factor
secreted by gastric parietal cells. The intrinsic factor-cobalamin
complex is recognized by the cubam receptor, formed by two pro-
teins, cubilin and amnionless, on the epithelial cells of the distal
ileum and is taken up by endocytosis. Cobalamin taken up by intes-
tinal epithelial cells subsequently appears in the circulation bound
to transcobalamin.
Uptake of cobalamin bound to transcobalamin (holo-TC) by
cells throughout the body occurs by carrier mediated endocytosis
following binding to a cell surface receptor [13], release of cobala-
min from transcobalamin in lysosomes and transfer of free cobal-
amin to the cytoplasm by a poorly understood process that
involves the product of the LMBRD1 gene [14]. Cobalamin is subse-
quently partitioned between the mitochondrial and cytoplasmic
compartments, where it is reduced and converted to AdoCbl or
MeCbl (Fig. 2).
Methionine synthase catalyzes the methylation of homocyste-
ine to form methionine, using 5-methyltetrahydrofolate as methyl
group donor with formation of MeCbl as an intermediate. Activity
of this enzyme requires the presence of a second protein, methio-
nine synthase reductase, which maintains the enzyme-bound
cobalamin prosthetic group in its active reduced state. Because
the reaction catalyzed by methionine synthase represents the only
cellular reaction that utilizes methyltetrahydrofolate, converting it
to tetrahydrofolate, decreased activity of methionine synthase
results in trapping of folate as methyltetrahydrofolate that is
unavailable for other folate dependent reactions, including conver-
sion of deoxyuridine to thymidine and two separate steps in de
novo purine biosynthesis. Decreased conversion of homocysteine
to methionine, as the result of decreased activity of methionine
synthase, methionine synthase reductase, or methyltetrahydrofo-
late reductase (MTHFR) which synthesizes methyltetrahydrofolate,
results in accumulation of homocysteine. It also results in
decreased function of a number of methyltransferase enzymes that
utilize S-adenosylmethionine as methyl group donor.
Animal models
The first evidence of a relationship of developmental defects to
proteins involved in cobalamin transport or metabolism was only
recognized many years after the original experiments were per-
formed. Injection into rodents of antibodies raised against kidney,
placenta, or visceral yolk sac had been shown to result in decreased
fetal weight, increased resorption rates and increased incidence of
a variety of fetal defects including anophthalmia, hydrocephalus,
exencephaly, anencephaly, omphalocele, orofacial clefts, cardio-
vascular defects and urogenital malformations [15–19]. The target
antigen was a 280 kDa glycoprotein, initially named gp280, which
was concentrated at the clathrin-coated intermicrovillar regions of
kidney proximal tubules and the polarized epithelial cells of the
yolk sac [20–22]. Interaction of antibodies with gp280 led to inhi-
bition of endocytic uptake and altered processing of internalized
gp280, allowing only a small amount of the antigen to reach the
lysosomal compartment [18,23–27]. There was decreased number,
reduced size and altered shape of lysosomal vesicles [27], suggest-
ing generalized dysfunction of endocytosis. Subsequently gp280
was shown to be identical to cubilin [28] which, together with
amnionless forms cubam, the distal ileum receptor for the intrinsic
factor-cobalamin complex [29–32]. Cubilin is a large peripheral
membrane protein containing eight EGF-like and 27 CUB domains
[33,34], and plays a role in binding and endocytosis of a number of
ligands in addition to the intrinsic factor-cobalamin complex [35].
It is not clear that interaction of antibodies with cubilin exerts its
168 F. Li et al. / Molecular Genetics and Metabolism 98 (2009) 166–172
Cbl
TC
Cbl
TC
TCblR
Cytoplasm
Cbl
TC
Lysosome
LMBRD1
Cbl Cbl
Homocysteine
Fig. 2. Cellular cobalamin metabolism. Cobalamin (Cbl) bound to transcobalamin (TC) is taken up by endocytosis and converted to adenosylcobalamin (AdoCbl), which is
required for activity of the mitochondrial enzyme methylmalonylCoA mutase (MCM) or methylcobalamin (MeCbl), which is required for activity of the cytoplasmic enzyme
methionine synthase (MS). TCblR, TC receptor; MSR, methionine synthase reductase.
teratogenic effect by interference with cobalamin metabolism. It is
possible that the teratogenic effect is the result of interference with
one of the other ligands recognized by this molecule, or follows
from generalized disruption of the endocytic uptake mechanism
in the presence of antibodies.
Targeted disruption of either the CUBN gene or the AMN gene in
mice results in embryonic lethality. Homozygous CUBN null mice
have developmental retardation with abnormal endodermal func-
tion and absence of somite formation [36]. Homozygous AMN null
mice show the amnionless phenotype characterized by a poorly
developed amnion and the absence of the trunk mesoderm [29].
In man, mutations in either CUBN or AMN cause Imerslund-Gräs-
beck syndrome (MGA1; OMIM 261100), which is characterized
by decreased ability to take up cobalamin from the intestine and
mild proteinuria. Congenital urinary tract anomalies have been
seen in Imerslund-Gräsbeck syndrome patients with AMN muta-
tions [37].
Homozygosity for knockouts of either the MTR or MTRR gene is
embryonic lethal in mice [38,39]. Mice homozygous or heterozy-
gous for a hypomorphic MTRR allele had an elevated incidence of
ventricular septal defects among their offspring, and homozygous
and heterozygous offspring had a higher risk of ventricular septal
defects [40].
Exposure of pregnant mice to ethanol results in high frequen-
cies of a number of malformations, including neural tube defects,
branchial arch abnormalities and cardiac defects. The rates of these
disorders were reduced by administration of folinic acid and cobal-
amin together during ethanol exposure; cobalamin alone did not
have any significant protective effect, and while folinic acid alone
did have a protective effect, the effect of the two vitamins together
was significantly greater [41]. The ability of dexamethasone to im-
MCM
MethylmalonylCoA SuccinylCoA
AdoCbl
MMAA
AdoCbl
MMAB
Mitrochondrion
Cbl
MMACHC
MMADHC
Cbl
MMADHC
Cbl
MSR
MeCbl
MS
Methionine
pair fusion of excised murine palatal shelves in organ culture is an
in vitro model of dexamethasone-induced cleft palate. Cobalamin
was shown to support shelf fusion in this system in the presence
of concentrations of dexamethasone that would normally prevent
fusion [42].
A large number of inbred mouse strains have been developed
with increased susceptibility to various birth defects. Several of
these have been tested for the ability of folate to protect against
development of birth defects, and in some cases a protective effect
of folate has been demonstrated [43]. For the most part, similar
experiments with cobalamin have not been carried out. One excep-
tion is the Axd (axial defects) mutation. It has been shown that
supplementation on days 8 and 9 of pregnancy with methionine
can decrease the frequency of neural tube defects in the products
of heterozygous Axd/+ matings, while neither folinic acid nor
cobalamin had any effect [44].
Thus, while it is tempting to speculate that cobalamin treat-
ment might correct the defects seem in some of these animal mod-
els, data are either incomplete or contradictory. Nonetheless, the
fact that disruptions of so many genes involved with vitamin B12
metabolism result in developmental abnormalities indicates that
further study is warranted.
Human studies
Metabolites
Human studies are numerous and the results are interesting but
still not conclusive. Cobalamin derived from the maternal diet is
delivered to the embryo by active transport across the placenta
and subsequently concentrated in the fetus during pregnancy
 F. Li et al. / Molecular Genetics and Metabolism 98 (2009) 166–172
[45]. Several studies have investigated serum cobalamin levels in
maternal blood, either in women pregnant with a neural tube
defect fetus or in women who have previously had a child with a
neural tube defect [11,46–64]. Some but not all studies have shown
decreased serum cobalamin levels in mothers of affected fetuses
compared to controls. A systematic review of these studies identi-
fied a moderate association between low maternal serum cobala-
min levels and risk of neural tube defects, but recognizing design
limitations and small number of participants in the studies ana-
lyzed, argued for a large observational study in early pregnancy
to definitively answer the question [65]. It should be noted that
the serum cobalamin levels associated with neural tube defects
in these studies, while lower than levels in controls, were not in
the range usually considered deficient and associated with mega-
loblastic anemia and neurological problems. This suggests that
the concept of cobalamin sufficiency may need to be reconsidered,
especially in the context of pregnancy. No correlation between the
presence of a neural tube defect and serum cobalamin level was
detected in children with neural tube defects [55,58,60].
Cobalamin levels in amniotic fluid from neural tube defect preg-
nancies have been measured in a small number of studies. In most
cases cobalamin levels have been lower in amniotic fluid from af-
fected pregnancies compared to controls, although the difference
has not reached statistical significance in all studies [48,53,66–
70], and the number of subjects has been small in most cases. In
some cases the maternal serum cobalamin level was in the normal
range in the presence of decreased amniotic fluid cobalamin, sug-
gesting that insufficient dietary intake was not the cause of cobal-
amin insufficiency in the fetus [48]. No difference in amniotic fluid
cobalamin compared to controls was detected in a study of 170
unaffected pregnancies in mothers who had previously had a child
with a neural tube defect [66].
Since elevated serum methylmalonic acid (MMA) is a marker of
functional cobalamin insufficiency, MMA levels have been studied
in neural tube defect pregnancies. In one study of 32 neural tube
defect pregnancies, maternal serum MMA was elevated compared
to control pregnant women, with a 13-fold increase in risk of hav-
ing a child with a neural tube defect in women in the ninetieth per-
centile. There was no increase in serum MMA in nonpregnant
women who had previously had a baby with a neural tube defect
compared to controls [71]. A study in a region of China with a high
frequency of neural tube defects found no difference in serum
MMA levels between women with neural tube defect pregnancies
and those with unaffected pregnancies, although levels in both af-
fected and unaffected women were elevated compared to controls
from elsewhere in the country [61]. However, the number of sub-
jects that have been studied is small, and it is not possible to draw
firm conclusions at this time.
Cobalamin circulating bound to transcobalamin (holo-TC) rep-
resents the pool of cobalamin that is available for uptake by cells,
and may represent a better measure of physiologically available
cobalamin than serum cobalamin level. Decreased levels of holo-
TC were observed in the serum of mothers who were pregnant
with a neural tube defect fetus [72] or who had previously had a
child with a neural tube defect [47,73]. In both cases, there was a
2- to 3-fold increase in risk of having a child with a neural tube
defect in the lowest quartile compared to the highest quartile.
Elevated levels of both transcobalamin and a second cobalamin-
binding protein, haptocorrin, and increased levels of apo-transco-
balamin (transcobalamin without bound cobalamin) and
apo-haptocorrin have been detected in amniotic fluid of mothers
pregnant with a neural tube defect fetus as well as in the amniotic
fluid of mothers who had previously had a child with a neural tube
defect or other type of birth defect [66,70].
The effect of cobalamin status in patients with orofacial clefts
has not been studied as extensively as in neural tube defects. There
169
is an association between maternal hyperhomocysteinemia and
risk of having an infant with cleft lip or palate [74], and some,
but not all, studies of periconceptional supplementation with high
dose folic acid or multivitamins have shown reduced risk of cleft
lip with or without cleft palate [75–77]. Results of studies of serum
cobalamin in mothers are inconsistent. One study of serum cobal-
amin in mothers during pregnancy showed no difference between
mothers of affected fetuses and controls [56], while a study of
mothers who had previously delivered an affected child found a
significant decrease in cobalamin compared to controls [78]. Stud-
ies of estimated cobalamin intake found no differences between
mothers of children with orofacial clefting and controls [79,80].
At present there is insufficient evidence to demonstrate any effect
of cobalamin status on risk of having a child with cleft lip and/or
cleft palate.
Polymorphisms in genes affecting cobalamin transport and
metabolism
The 677C>T polymorphism in the MTHFR gene, which encodes
the enzyme methylenetetrahydrofolate reductase that catalyzes
synthesis of the methyltetrahydrofolate substrate for the reaction
catalyzed by methionine synthase, represents a model for the
effects of a genetic polymorphism in development of neural tube
defects. The T allele at this polymorphism results in a thermolabile
enzyme with approximately 40–50% of control activity, and homo-
zygosity for the T allele is associated with increased levels of
homocysteine in the presence of low serum folate [81]. Homozy-
gosity for the T allele is a risk factor for neural tube defects and
other birth defects [82].
Since cobalamin deficiency also results in elevated plasma
homocysteine, mutations affecting cobalamin metabolism have
been investigated for a role in neural tube defect development.
The most frequently studied genes and polymorphisms are listed
in Table 1. A number of studies have investigated the role of the
c.66A>G (p.I22M) polymorphism in the MTRR gene in modifying
the risk of developing birth defects. This polymorphism results in
a protein that appears to interact less effectively with methionine
synthase [83]. Homozygosity for the G allele at this locus is associ-
ated with increased plasma total homocysteine in the presence of
low levels of serum cobalamin in pregnant women [84]. Several
studies have identified an increased risk of developing a neural
tube defect in mothers [85–89] or offspring [85–88] with the GG
or AG genotype at this polymorphism, but other studies have not
shown any effect on neural tube defect risk [62,70,90,91] or risk
of other birth defects [70,92,93], and two studies appeared to dem-
onstrate a protective effect of the G allele [94,95]. All of these stud-
ies have been limited by the size of the study population. A meta-
analysis performed using the results of several of these studies
found that the maternal GG genotype at MTRR was associated with
a small increase in risk of having a child with a neural tube defect
(odds ratio of 1.55, 95% confidence interval 1.04–2.30). There was
no effect of the GG allele in offspring on neural tube defect risk
[89]. The effect of the c.66A>G polymorphism may be increased
in the presence of low serum cobalamin levels or elevated serum
methylmalonic acid [85,89,93] or in the presence of predisposing
alleles at other genes [86,95].
Association of the G allele at the c.2756A>G polymorphism in
the MTR gene with an increased risk of neural tube defects or cleft
lip and palate when present in the mother or the offspring has been
reported [86,87,94,96], but most studies have not identified any
effect of this polymorphism on risk for birth defects
[62,70,84,97–103]. Even in the absence of an independent effect
on neural tube defect risk, presence of the MTR polymorphism
together with the MTRR polymorphism may result in an increased
NTD risk [85].
170 F. Li et al. / Molecular Genetics and Metabolism 98 (2009) 166–172
Table 1
Genes involved in cobalamin metabolism, and polymorphisms that have been studied for their effects on birth defects.
Gene Location Protein
AMN 14q32 Amnionless
CUBN 10p12.1 Cubilin
MTHFR 1p36.3 Methylenetetrahydrofolate reductase
MTR 1q43 Methionine synthase
MTRR 5p15.3-p15.2 Methionine synthase reductase
TCN2 22q11.2-qter Transcobalamin (TC)
The c.776C>G polymorphism in the TCN2 gene, which encodes
transcobalamin, results in decreased holo-TC levels in homozygotes,
with increased plasma total homocysteine and methylmalonic acid
[104,105] reflecting decreased function of cobalamin-dependent
enzymes. One study has suggested that homozygosity for the G
allele at this locus is associated with increased risk of spina bifida
[106], while another described significant overtransmission of the
C allele in offspring with cleft lip with or without cleft palate [107].
In contrast with these conflicting positive results, most studies
have found no effect of the TCN2 776C>G polymorphism on neural
tube defect [62,70,101,108,109] or cleft lip and cleft palate risk
[110].
In a recent study, a SNP (rs1907362) in the twenty-seventh
intron of the CUBN gene was associated with decreased risk of
spina bifida, and was related to increased cobalamin level [111].
No sequence change in the coding region of the gene that is linked
to this SNP has been identified.
Thus, as for the metabolic studies, although it is possible to find
data in support of an effect for genes involved in cobalamin metab-
olism or transport on birth defects, proper interpretation of these
results await large prospective studies.
Discussion
Birth defects are caused by the confluence of a number of genet-
ic and environmental factors. There are now several lines of evi-
dence that deficiency of cobalamin and alterations of cobalamin
metabolism might play a role in the development of neural tube
defects, and possibly other birth defects as well. As described
above, decreased maternal serum cobalamin and decreased amni-
otic fluid cobalamin levels have been associated with increased
risk of neural tube defects. A systematic review of several studies
identified a moderate association between low maternal serum
cobalamin levels and risk of neural tube defects [65]. Smaller num-
bers of studies have implicated decreased maternal serum holo-TC
levels, elevated levels of apo-transcobalamin and apo-haptocorrin,
and increased levels of maternal serum methylmalonic acid with
increased neural tube defect risk. The presence of the G allele at
the c.66A>G polymorphism in the MTRR gene, which encodes
methionine synthase reductase, has also been associated with
increased neural tube defect risk. A meta-analysis indicates a 1.5-
fold increase in risk for neural tube defects in subjects with the
GG genotype at this polymorphism [89]. Some studies suggest that
the risk caused by this genotype is increased in the presence of low
serum cobalamin [85]. Transgenic mice heterozygous or homozy-
gous for a hypomorphic mutant of the MTRR gene are at increased
risk of developing birth defects or bearing offspring with neural
tube defects [40]. Administration of cobalamin protects against
development of birth defects in ethanol-treated mice and
promotes palatal fusion caused by dexamethasone in an in vitro
system [42].
The finding that cobalamin deficiency represents an indepen-
dent risk factor for birth defect [51] has led to suggestions that
OMIM Polymorphism
cDNA Protein
605799
602997 rs1907362
607093 677C ? T Ala222Val
156570 2756A ? G Asp919Gly
602568 66A ? G Ile22Met
275350 776C ? G Pro259Arg
supplementation of food with both folic acid and cobalamin should
result in further decrease in the occurrence of birth defects beyond
that mediated by folic acid supplementation alone [112]. The origi-
nal case for folic acid supplementation was strengthened by data
from a large prospective study [1]. The studies described in the
present review provide sufficient information to support carrying
out a similar prospective study using cobalamin.
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