Prenatal Exposure to Maternal Depressed Mood and the
MTHFR C677T Variant Affect SLC6A4 Methylation in
Infants at Birth
Angela M. Devlin1*, Ursula Brain2, Jehannine Austin3, Tim F. Oberlander1,2
1Department of Pediatrics, Child & Family Research Institute, University of British Columbia, Vancouver, Canada, 2Early Human Experience Unit, Child & Family Research
Institute, University of British Columbia, Vancouver, Canada, 3Department of Psychiatry, Child & Family Research Institute, University of British Columbia, Vancouver,
Background: Prenatal and early postnatal exposure to maternal depression may ‘‘program’’ childhood behavior via
epigenetic processes such as DNA methylation. Methylenetetrahydro-folate reductase (MTHFR) is an important enzyme in
the generation of methyl groups for DNA methylation. The common MTHFR C677T variant is associated with depression in
men and non-pregnant women, and with global changes in DNA methylation. This study investigated the effect of maternal
MTHFR C677T genotype on antenatal maternal mood, and their impact on the gene-specific methylation in pregnant
women and their newborn infants. The methylation status of SLC6A4, which encodes the transmembrane serotonin
transporter, and BDNF, which encodes brain derived neurotrophic factor, were assessed because of their potential role in
Methods/Principal Findings: Depressed mood was assessed by the Edinburgh Postnatal Depression Scale (EPDS) and the
Hamilton Rating Scale for Depression (HAM-D) in women (n=82, all taking folate) during the 2ndand 3rdtrimesters of
pregnancy. The methylation status of SLC6A4 and BDNF were assessed in 3rd trimester maternal peripheral leukocytes and
in umbilical cord leukocytes collected from their infants at birth. Women with the MTHFR 677TT genotype had greater 2nd
trimester depressed mood (p,0.05). Increased 2ndtrimester maternal depressed mood (EPDS scores) was associated with
decreased maternal and infant SLC6A4 promoter methylation (p,0.05), but had no effect on BDNF promoter methylation.
Conclusions: These findings show that the MTHFR C677T variant is associated with greater depressed mood during
pregnancy. We further showed that prenatal exposure to maternal depressed mood affects gene-specific DNA methylation
patterns. These findings support the concept that alterations in epigenetic processes may contribute to developmental
programming of behaviour by maternal depression.
Citation: Devlin AM, Brain U, Austin J, Oberlander TF (2010) Prenatal Exposure to Maternal Depressed Mood and the MTHFR C677T Variant Affect SLC6A4
Methylation in Infants at Birth. PLoS ONE 5(8): e12201. doi:10.1371/journal.pone.0012201
Editor: Robert Feil, CNRS, France
Received April 6, 2010; Accepted July 15, 2010; Published August 16, 2010
Copyright: ? 2010 Devlin et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by grants from the Canadian Institutes for Health Research (T.F.O.). A.M.D. holds a New Investigator Salary Award from the
Heart and Stroke Foundation of Canada. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
Approximately, 15% of mothers experience mood disturbances
during pregnancy and up to one third are treated with a serotonin
reuptake inhibitor antidepressant (SRI) medication . These two
environmental factors may be among the earliest adverse life
experiences that ‘‘programs’’ or (re-programs) the physiological,
neuroendocrine and metabolic adaptations that underlie early
human brain development, setting a course of health or illness that
may last a life time. Increasing evidence points to the links between
antenatal maternal depressed and anxious mood and risk for
neurobehavioral disturbances during childhood [2,3].
The molecular mechanisms underlying developmental pro-
gramming are poorly understood but may involve the interplay
between genetic and epigenetic processes, and prenatal environ-
mental factors such as maternal mood. Epigenetic processes
include DNA methylation and chromatin modifications (histone
methylation, acetylation), patterns of which are inherited [4,5] but
are responsive to environmental shifts, such as stress, and are
especially vulnerable during development [6–8]. For example,
studies in a rodent model have shown that variations in early life
experience (maternal care over the first week of life) is associated
with decreased HPA stress responsivity in early infancy, and
involves changes in the methylation status of the hippocampal
glucocorticoid receptor (GR) gene (Nr3c1) and Nr3c1 expression
. This phenomena has also been demonstrated in humans. We
recently reported an association between exposure to increased 3rd
trimester maternal depressed mood and NR3C1 promoter
methylation in newborn infants, and HPA stress reactivity at 3
months , even when mothers had been treated with a selective
serotonin reuptake inhibitor antidepressant. Furthermore, recent
studies have shown methylation-silencing of rRNA and NR3C1
PLoS ONE | www.plosone.org1August 2010 | Volume 5 | Issue 8 | e12201
expression in hippocampus from suicide victims with a history of
child abuse [9,10].
The neurochemical serotonin (5-HT) plays a critical link
between early life experience and an increased risk for emotional
disturbances in childhood . Reduced 5-HT levels may increase
a susceptibility for life time risk for depression, reflecting a
‘‘serotonergic vulnerability’’ . A key regulator of 5-HT levels is
the transmembrane serotonin transporter (5-HTT) that governs
the reuptake of 5-HT and as such, determines the magnitude and
duration of the 5-HT action. A 44 base pair insertion/deletion
variant (referred to as 5-HTTLPR), in the promoter of the gene
that encodes 5-HTT (SLC6A4), is believed to contribute to
variations in 5-HTT expression, and as such, variations in 5-
HTT-dependent 5-HT reuptake efficiency [13–15]. The 5-
HTTLPR variant has been shown to influence vulnerability to
the impact of early stressful life events [16,17]. Furthermore, 5-
HTT expression may also be regulated by epigenetic mechanisms.
The methylation status of the SLC6A4 promoter was shown to play
a role in governing SLC6A4 mRNA levels, however, this was
dependent on the 5-HTTLPR genotype .
The objective of this study was to assess the effect of antenatal
maternal depressed mood on the methylation status of SLC6A4
and brain derived neurotrophic factor (BDNF) in pregnant women
and their infants at birth. SLC6A4 and BDNF were chosen as target
genes because methylation plays a role in governing SLC6A4
expression  and expression of Bdnf in a rat model was shown to
be regulated by methylation and sensitive to early adverse life
experience . Several population studies have shown an
association of the the gene for methylenetetrahydrofolate reduc-
tase (MTHFR), an enzyme required for folate metabolism and the
generation of methyl groups [19,20], with global changes in DNA
methylation [21–23] and depressed mood and depressive disorders
in non-pregnant populations [24–28]. As such, we further assessed
the relationship of the MTHFR C677T variant with antenatal
mood and SLC6A4 and BDNF methylation status.
Maternal and neonatal demographic characteristics did not
vary significantly with maternal MTHFR C677T genotype
(Table 1). Genotype frequencies for the MTHFR C677T variant
were 15.1% TT, 41.9% CT, and 43.0% CC (Table 2), similar to
previous reports in non-pregnant women and men [19,20,28]. At
26 weeks gestation, women with the MTHFR 677TT genotype
had significantly higher EPDS scores (F=4.99; p=0.009;
g ´2=.11) compared to women with the MTHFR 677CT and
677CC genotypes (Table 2), controlling for serotonin reuptake
inhibitor (SRI)-treatment. No association between MTHFR
C677T genotype and maternal mood at 33 weeks was observed.
As shown in Figure 1, we assessed the methylation status of 10
CpGs in the SLC6A4 promoter and 12 CpGs in the BDNF
promoter. The methylation status of the SLC6A4 promoter was
significantly lower in mothers with increased depressed mood
symptoms at 26 weeks gestation (p,0.05) (Table 3). Using a
multivariate model, controlling for SRI exposure and MTHFR
C677T genotype, the relationship between maternal depressed
mood and lower methylation status was most evident for CpG sites
1,4,5,6,7 and 9 (sites 6 and 9 illustrated in Figure 2A), (F=5.23,
p=0.024, g ´2=0.065; F=4.6, p=0.034, g ´2=0.058; F=4.0,
p=0.050, g ´2=0.050; F=8.89, p=0.004, g ´2=0.104; F=6.5,
p=0.013, g ´2=0.078; F=6.1, p=0.015, g ´2=0.075, respectively).
Importantly, the methylation status of the SLC6A4 promoter was
unaffected by maternal MTHFR C677T genotype, SRI exposure,
Table 1. Maternal MTHFR C677T genotype and demographic data of the pregnant women and their infants.
Maternal MTHFR C677T Genotype
CC (n=40) CT (n=36)TT (n=14)
Maternal age at birth, years (SD)32.4 (4.5)33.4 (4.9)31.4 (5.6)
Maternal education, years (SD) 16.8 (2.5)16.5 (3.3)15.6 (2.9)
Delivery type, % caesarian-section363136
SRI treated during pregnancy, %384857
Alcohol use - drinks during pregnancy, %
Tobacco Use, %007
Newborn Infant Characteristics
Prenatal SRI exposure, days (SD)239 (52)235 (71)178 (85.6)
Birth weight, g (SD)3469 (613)3492 (476)3523 (458)
Head Circumference, cm (SD)34.8 (1.4)34. 6 (1.3)35.1 (1.3)
Length, cm (SD)51.0 (3.15)51.6 (2.6)52.1 (2.5)
Gestational age at birth, weeks (SD)40.0 (1.4)39.3 (1.6)39.8 (1.4)
Gender, % M / F38/6244/5664/36
Apgar score at 1 minute (SD)8.1 (1.5)7.6 (1.5)7.6 (1.5)
Apgar score at 5 minute (SD)9.0 (0.46)8.8 (0.78)8.7(0.9)
PLoS ONE | www.plosone.org2August 2010 | Volume 5 | Issue 8 | e12201
or mood at 33 weeks gestation. Maternal BDNF promoter
methylation status was unaffected by maternal MTHFR C677T
genotype, antenatal mood scores at 26 weeks and 33 weeks, or
SRI exposure (Table 4).
Similar to what we observed for maternal SLC6A4 promoter
methylation status, decreased SLC6A4 promoter methylation status
at CpG site 6 and 9 in newborns was associated with increased
levels of maternal depressed mood symptoms during the second
trimester (F=5.0, p=0.029, g ´2=0.070; F=4.410, p=0.039,
g ´2=0.06, respectively) (Table 3 and Figure 2B). To address the
potential for a heritable SLC6A4 epigenotype in infants we assessed
the relationship of maternal SLC6A4 promoter methylation status
to infant SLC6A4 methylation status and found no significant
relationship. Methylation status was not associated with maternal
and infant MTHFR C677T genotype or prenatal SRI exposure.
Methylation status of the infant SLC6A4 promoter was unaffected
by maternal mood score at 33 weeks gestation. Neonatal BDNF
promoter methylation status was not associated with either
Table 2. Influence of maternal MTHFR C677T and BDNF V66M
genotypes on mood scores in the 2nd trimester of
MTHFR C677T Genotype (rs1801133)
Mood Scores CC (43.0%) CT (41.9%)TT (15.1%)
HAM-D total score 8.7561.48.2161.2 13.4662.2
EPDS score8.0061.2 5.8360.912.0061.6b
Note: Values shown are means 6 SE (standard error), n=86 for MTHFR C677T.
aMaternal mood was assessed by the Edinburgh Postnatal Depression Scale
(EPDS)  and the Hamilton Rating for Depression Scale (HAM-D) . Effects
of the MTHFR C677T variant on maternal mood scores was assessed by analysis
of covariance (ANCOVA), with SRI-treatment as a covariate in the analysis.
bp,0.05 compared to MTHFR 677CC and 677CT genotype groups.
Figure 1. Schematic representation of the SLC6A4 and BDNF promoters analyzed for methylation status. The portion analyzed by
bisulfite pyrosequencing is shown in bold. The CpGs are underlined and numbered. Numbering of the gene sequence is relative to the transcriptional
PLoS ONE | www.plosone.org3 August 2010 | Volume 5 | Issue 8 | e12201
maternal or neonatal MTHFR C677T genotype, antenatal
maternal mood scores, or prenatal SRI exposure (Table 4).
As a first step towards delineating a role for epigenetic
mechanisms in the programming of childhood behaviour by
prenatal exposure to maternal depressed mood we assessed the
effect of antenatal maternal depressed mood on the methylation
status of SLC6A4 and BDNF in pregnant women and their infants
at birth. There are three main findings of this study. First we found
that that 2ndtrimester maternal depressed mood symptoms scores
are associated with maternal MTHFR C677T genotype, such that
women with the MTHFR 677TT genotype have the greatest
depressed mood symptoms. Second, we found that antenatal
maternal mood is associated with maternal and neonatal SLC6A4
promoter methylation status. In particular, increased maternal
depressed mood symptoms in the 2ndtrimester are associated with
lower maternal SLC6A4 promoter methylation status, but not the
BDNF promoter. Interestingly, these associations were not
observed during the 3rdtrimester and maternal SRI treatment
did not play a role in any of these relationships. Third, similar to
what we observed in the pregnant women, SLC6A4 promoter
methylation status was also lower in newborn infants from mothers
who reported higher depressed mood symptoms during the 2nd
trimester. This relationship was unrelated to maternal SLC6A4
methylation status, and unaffected by maternal and infant
MTHFR C677T genotype, or prenatal exposure to maternal
SRI medication. Given the role for MTHFR in methyl
metabolism, these findings suggest that disturbances in methyl
metabolism, such as those associated with the MTHFR 677TT
genotype [19,21], may contribute to the pathology of depression
during pregnancy. These findings further suggest that prenatal
exposure to maternal depressed mood during the second trimester
of pregnancy can alter gene-specific DNA methylation patterns in
newborns, and thereby set-up, via epigenetic mechanisms,
processes that alter SLC6A4 expression that may have long-term
consequences. Given we found the MTHFR C677T variant is
associated with greater antenatal depressed mood symptoms in
women and that the SLC6A4 promoter methylation status in
women and infants was affected by maternal mood our finding of
no direct effect of the MTHFR C677T variant on SLC6A4
promoter methylation status was unexpected. The reason behind
this finding is unknown but may simply be the consequence of
insufficient power to detect such an effect, given the small number
of women with the MTHFR 677TT genotype (n=14).
In this study we report that increased maternal depressed mood
during the 2ndtrimester of pregnancy was associated with reduced
methylation of the maternal and neonatal SLC6A4 promoter
region. Conceivably, such reduced methylation may lead to
increased SLC6A4 expression and availability of 5-HTT, and as
such, result in increased 5-HT reuptake and lower intrasynaptic 5-
HT. In the mature brain this might not have a noticeable impact,
but in the developing brain such altered serotonergic tone may
have long term effects on behavior . Prior to the neurotrans-
mitter role of 5-HT, it plays critical roles as a trophic factor
modulating neuronal differentiation and growth, therefore it is
conceivable that changes in 5-HT via altered levels of the
serotonin transporter during critical periods of development alters
brain function and increases vulnerability to affective disorders
later in life . Altered central 5-HT, possibly via changes in
methylation of regulatory regions of SLC6A4 affects 5-HT levels
during fetal development and may have a long term impact on the
developing brain that ‘‘programs’’ subsequent child emotional
development . This has been demonstrated in Slc6a42/2
mice, which have no 5-HTT, increased intrasynaptic 5-HT
(analogous to the pharmacological effect of an SRI), and increased
depressed and anxious behaviors in adulthood, suggesting long-
term consequences associated with early altered 5-HT levels .
The association between maternal mood and SLC6A4 methylation
status may offer an insight into processes, beyond genetic
variations in SLC6A4 that alters serotonergic tone during
development. Demonstrating an effect of altered neonatal SLC6A4
methylation status on developmental outcomes will provide
evidence of a functional relationship and long-term consequences
of such a relationship. These studies remain to be determined.
The methylation status of the SLC6A4 promoter in whole blood
from pregnant women and newborn infants observed in this study
were within the same range previously reported by others for the
mean methylation status of the SLC6A4 promoter in lymphoblast
Table 3. Maternal and infant SLC6A4 promoter methylation status according to maternal MTHFR C677T genotype.
(% methylation)Maternal MTHFR C677T Genotype
(% methylation)Maternal MTHFR C677T Genotype
CC (n=35)CT (n=36)TT (n=12)CC (n=35)CT (n=36)TT (n=12)
10.264.19.6062.5 8.7062.9CpG 19.8962.99.6963.49.2363.5
CpG 24.4261.84.3061.83.6361.0 CpG 24.7662.44.3262.0 4.1061.6
7.3862.17.2262.1 6.1661.5 CpG 37.8062.67.4062.6 7.9464.2
4.3261.84.2061.63.5661.5 CpG 44.7162.44.1761.93.9161.4
7.3561.97.5861.96.4461.4CpG 57.6262.2 7.2661.97.5463.6
4.2861.5 4.0861.33.5161.2CpG 6a
7.4162.2 7.6361.8 6.6162.0 CpG 77.6162.47.2662.27.0362.7
5.4461.35.4561.04.8761.0 CpG 85.7761.5 5.6961.44.9961.6
7.9662.1 7.9362.06.8262.0CpG 9a
8.3163.2 7.6662.2 6.8262.0
CpG 108.3961.78.5061.3 8.3661.2CpG 1010.1862.010.0061.69.4062.0
Note: Values shown are means 6 SE (standard error).
ap,0.05, significant effect of maternal Edinburgh Postnatal Depression Scale (EPDS) score (maternal mood) at 26 weeks gestation, as determined by analysis of
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Figure 2. Relationship between SLC6A4 promoter methylation status, maternal MTHFR C677T genotype, and pregnancy mood
scores. The relationship between (A) maternal and (B) infant SLC6A4 CpG 6 and CpG 9 methylation status and maternal EPDS scores (depressed
mood scores) at 26 weeks gestation are shown. Points are plotted according to maternal MTHFR C677T genotype: solid triangle, CC; solid circle, CT;
solid square, TT.
Table 4. Maternal and infant BDNF promoter methylation status according to maternal MTHFR C677T genotype.
Maternal MTHFR C677T Genotype
Maternal MTHFR C677T Genotype
CC (n=35)CT (n=36) TT (n=12)CC (n=35)CT (n=36)TT (n=12)
CpG 12.7860.92.9260.82.6960.8CpG 1 2.6560.92.5560.6 2.4860.4
CpG 2 2.5661.02.6460.9 2.2960.3CpG 2 2.1860.92.0860.71.8060.2
CpG 35.9563.65.7462.74.760.6CpG 35.3962.85.1362.84.4460.9
CpG 4 7.2463.97.6163.7 6.0960.9 CpG 46.8263.9 6.3763.65.4060.9
CpG 54.8261.64.8661.34.1360.5CpG 5 4.2061.64.0761.2 3.6460.5
CpG 65.2061.65.5661.44.6360.6CpG 64.6761.64.3561.33.8560.2
CpG 72.4560.82.6260.72.2060.4CpG 7 2.1960.72.0260.41.8760.2
CpG 88.1262.97.6961.56.9260.7CpG 87.2562.16.7961.26.2960.6
CpG 94.3261.24.1661.03.5560.6 CpG 93.8161.23.6360.83.3360.4
CpG 103.6361.23.7961.1 3.2360.7CpG 10 3.5961.53.1361.02.960.5
CpG 114.8361.84.9361.4 4.3160.9CpG 11 4.7361.94.3761.1 4.0560.6
CpG 127.8362.97.6461.9 6.7061.0CpG 12 7.4963.7 6.8861.96.2360.81
Note: Values shown are means 6 SE (standard error).
PLoS ONE | www.plosone.org5 August 2010 | Volume 5 | Issue 8 | e12201
cell lines . In the current study we used bisulfite pyrosequenc-
ing to analyze a 130 bp region of the SLC6A4 promoter adjacent
to exon 1a, and quantified the methylation status of 10 CpG sites.
This region corresponds to a portion of the much larger region of
the SLC6A4 promoter analyzed in lympophoblast cell lines that
quantified 81 CpG sites by traditional bisulfite sequencing . In
this prior study it was also shown that the methylation status of 4 of
the 81 CpG sites correlated with SLC6A4 mRNA levels. In the
region we analyzed CpG 8 corresponds to one of these sites at bp
872. We do recognize that our analysis was conducted in a
heterogeneous mixture of cell types (whole blood), which may
confound our findings. SLC6A4 is expressed predominantly by
platelets, lymphocytes, and monocytes in blood but in the current
study we were unable to assess blood cell-specific differences in
SLC6A4 methylation status.
Interestingly BDNF methylation status was not affected by
antenatal maternal mood, MTHFR C677T genotype or SRI
exposure. One study did show differential methylation of 4 CpG
sites in the coding sequence around the BDNF V66M variant, with
the M allele associated with less methylation in human frontal
cortex postmortem brain tissue . The BDNF 66M allele has
been associated with depression in elderly subjects  and is
associated with reduced hippocampal volume . In the current
study we found no effect of the V66M variant on maternal
depressed mood scores and no effect of maternal BDNF V66M
genotype on maternal or infant BDNF promoter methylation status
(results not shown). Studies in a rat model have shown that
exposure to adverse maternal care giving in the first postnatal
week following birth is associated with differential methylation of
the 59 region of the Bdnf gene and changes in Bdnf mRNA
expression in prefrontal cortex from adult rats and that this is
transferred to the next generation . It remains to be determined
how well gene-specific DNA methylation patterns in blood cells
correlate with gene-specific DNA methylation patterns in brain
regions, such as the hippocampus and prefrontal cortex.
Several studies have reported an association between the
MTHFR C677T variant and depression [24–28] but the role of
MTHFR in the pathology of depression remains to be determined.
One would expect that the metabolic changes associated with the
MTHFR 677TT genotype, such as elevated plasma total
homocysteine [19,20], or changes in global DNA methylation
[21–23], are contributing factors. However, the degree of these
changes are most pronounced in the presence of low folate status
[23,35]. The folate status of the women in our current study was
not evaluated, but given all the women were taking folic acid
supplements and living in an environment with mandatory folic
acid fortification of the food supply it is unlikely that any of the
women in our study had poor folate status.
Taken together, our findings suggest there may be a three-way
interaction between maternal MTHFR C677T genotype, maternal
depressed mood during pregnancy, and gene-specific changes in
DNA methylation patterns such that maternal MTHFR 677TT
genotype may predispose women to mood disturbances during
pregnancy, which in turn influences gene-specific DNA methyl-
ation patterns, such as that observed for SLC6A4. Decreased
methylation of the SLC6A4 promoter may result in increased
SLC6A4 expression and changes in central serotonergic tone that
might contribute to ‘‘programming’’ infant and childhood
behaviour. This association between antenatal maternal depressed
mood and SLC6A4 methylation status is a first step towards a more
complete understanding of how early life experience, genotype,
and epigenetic processes contribute to development. Further
studies are required to to assess the effect of MTHFR C677T
variant, maternal mood and changes in SLC6A4 promoter
methylation status on SLC6A4 expression and its impact on infant
Materials and Methods
With approval from the University of British Columbia
Research Ethics Board, Children’s and Women’s Health Centre
of British Columbia Research Review Committee, and written
informed consent, a cohort (n=98) of mothers was recruited in
their early second trimester as part of a study of the impact of
prenatal SRI exposure on neonatal health [36,37]. Of the original
98 mothers who completed a second trimester data collection,
samples from 16 mothers and infants at delivery were not available
for analysis (i.e. mothers withdrew for personal reasons prior to
delivery, inadequate DNA yield, infant cord blood sample was not
obtained at birth) leaving 49 maternal and infant samples that
were not treated with SRI medications, and 33 samples that were
treated with SRI medications. Mothers were only included in the
study if they took no other serotoninergic medications or other
psychotropic medications during their pregnancy. All mothers
were taking folic acid (1 mg folate/day) during their pregnancies,
either as a component of a prenatal vitamin supplement or on its
own. Maternal blood (mid 3rdtrimester) and neonatal cord
(venous) blood samples were obtained for genotyping and DNA
Maternal Mood Assessment
Prenatal maternal mood was assessed using clinician- (blinded
to SRI-treatment group status) and self-rated measures at the time
of study enrollment (approximately 26 weeks) and at 33 weeks
gestation. Measures included the Hamilton Rating Scale for Depression
(HAM-D), a 21-item clinician administered scale designed to assess
the severity of depression . The Edinburgh Postnatal Depression
Scale (EPDS) is a 10 item, self-rated instrument used to assess
symptoms of depressed mood in both pre and postnatal settings
. Higher scores on these scales indicate higher levels of
depression in the patient.
Genomic DNA was extracted from maternal and newborn
leukocytes using the Flexigene DNA Blood Kit (Qiagen, Valencia,
CA). The MTHFR C677T (rs1801133), and BDNF V66M (rs6265)
variants were genotyped using TaqMan SNP genotyping assay
reagents and a 7500 Real Time PCR System (Applied Biosystems)
following the manufacturer’s suggested protocol.
Quantitative Analysis of Gene-Specific DNA methylation
The methylation status of CpG-rich regions in the SLC6A4 and
BDNF gene promoters (Figure 1) were quantified by bisulfite
Pyrosequencing . The region of SLC6A4 analyzed was within
the same region shown to be differentially methylated and
associated with changes in SLC6A4 mRNA expression [18,41].
We analyzed a region of the SLC6A4 promoter between 2479 and
2350, relative to the transcriptional start site, which contains 10
CpG sites and is adjacent to exon 1a . For BDNF we analyzed
a CpG-rich region of the promoter between 2694 and 2577,
relative to the transcriptional start, which contains 12 CpG sites.
The region of BDNF we analyzed for methylation status
corresponds to an analogous region in rat Bdnf, which was shown
to be differentially methylated and associated with Bdnf mRNA
Genomic DNA from leukocytes (1 mg) was bisulfite-treated
using the EpiTect Bisulfite Kit (Qiagen) following the manufac-
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turer’s suggested protocol, and stored at 220uC until further
analysis. A 130 bp fragment of the SLC6A4 promoter (Fig. 1) and a
118 bp fragment of the BDNF promoter were amplified by PCR
from bisulfite-treated DNA using HotStar Taq DNA Polymerase
(Qiagen) and the following primers for SLC6A4: PMHSERTF, 59-
PMHSERTR, 59-AAAAATCCTAACTTTCCTACTCT TTAA-
CTT-39; and for BDNF: PMHBDNFF, 59-GTGGGGTAG-
GAGGGGAGTAGTAT-39 and PMHBDNFR, 59-AAATCCCC-
CAATCAACTCTCT-39 (IDT Inc, Coralville, IA), with the
reverse primer containing a biotin at the 59 end. Cycling
conditions were 94uC for 15 minutes followed by 50 cycles of
94uC for 1 minute, 60uC for 1 minute, and 72uC for 1 minute
with a final extension of 10 minutes at 72uC. PCR products were
purified and sequenced using a PyroMark MD System (Biotage,
Foxboro, MA) following the manufacturer’s suggested protocol
and the following sequencing primers for SLC6A4 and BDNF,
CAAAT-39 and PMHBDNFS, 59-GGTAGGAGGGGAGTAG-
TA-39 (IDT). The percent methylation at each CpG site was
quantified using the Pyro Q-CpG software, version 1.0.9 (Biotage).
The effects of the MTHFR C677T variant on maternal
depressed mood scores was assessed by analysis of covariance
(ANCOVA), with genotype as the independent variable and SRI-
treatment as a covariate in the analysis. The effect of maternal and
infant MTHFR C677T genotype on maternal and infant SLC6A4
and BDNF promoter methylation status at specific CpG sites was
assessed using multiple analyses of covariance (MANCOVA)
models with maternal EPDS score (depressed mood score) and
SRI-treatment as covariates. Effect sizes (eta squared) were also
calculated. All analysis was conducted using SPSS, version 16.0
(SPSS Inc, Chicago, IL).
We are grateful to Rachel Wade for her technical assistance with the
methylation and genotyping assays.
Conceived and designed the experiments: AD JA TFO. Performed the
experiments: AD UB. Analyzed the data: AD TFO. Contributed reagents/
materials/analysis tools: AD TFO. Wrote the paper: AD TFO.
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