OPEN

Citation: Transl Psychiatry (2016) 6, e866; doi:10.1038/tp.2016.137

www.nature.com/tp

ORIGINAL ARTICLE
Association between reduced white matter integrity in the
corpus callosum and serotonin transporter gene DNA
methylation in medication-naive patients with major
depressive disorder
E Won1,8, S Choi2,8, J Kang3, A Kim3, K-M Han1, HS Chang4, WS Tae5, KR Son6, S-H Joe7, M-S Lee1 and B-J Ham1

Previous evidence suggests that the serotonin transporter gene (SLC6A4) is associated with the structure of brain regions that are
critically involved in dysfunctional limbic-cortical network activity associated with major depressive disorder (MDD). Diffusion tensor
imaging (DTI) and tract-based spatial statistics were used to investigate changes in white matter integrity in patients with MDD
compared with healthy controls. A possible association between structural alterations in white matter tracts and DNA methylation
of the SLC6A4 promoter region was also assessed. Thirty-five medication-naive patients with MDD (mean age: 40.34, male/female:
10/25) and age, gender and education level matched 49 healthy controls (mean age: 41.12, male/female: 15/34) underwent DTI.
SLC6A4 DNA methylation was also measured at five CpG sites of the promoter region, and the cell type used was whole-blood DNA.
Patients with MDD had significantly lower fractional anisotropy (FA) values for the genu of the corpus callosum and body of the
corpus callosum than that in healthy controls (family-wise error corrected, P o0.01). Significant inverse correlations were observed
between SLC6A4 DNA methylation and FA (CpG3, Pearson's correlation: r = − 0.493, P = 0.003) and axial diffusivity (CpG3, Pearson's
correlation: r = − 0.478, P = 0.004) values of the body of the corpus callosum in patients with MDD. These results contribute to
evidence indicating an association between epigenetic gene regulation and structural brain alterations in depression. Moreover,
we believe this is the first report of a correlation between DNA methylation of the SLC6A4 promoter region and white matter
integrity in patients with MDD.

Translational Psychiatry (2016) 6, e866; doi:10.1038/tp.2016.137; published online 9 August 2016

INTRODUCTION multiple genes, although genetic linkage and association studies

The pathophysiology of major depressive disorder (MDD) involves have not identified specific strong and consistent MDD suscept-
interactions between susceptible genotypes, chronic stress and an ibility genes.6 Altered serotonergic neurotransmission has repeat-
adverse developmental environment, which lead to alterations in edly been implicated as a key contributor to the etiology of
the biochemistry, neuroplasticity and structure of the brain.1 depression;7 therefore, genes that contribute to regulation of
Neuroimaging studies on depression have consistently identified serotonin activity have been widely studied in relation to MDD.
neuroanatomical alterations in gray matter regions that partici- Since the serotonin transporter is responsible for serotonin
reuptake at the terminals and cell bodies of serotonergic neurons,
pate in affect regulation.2 Given that white matter tracts connect
and since it is the primary molecular target for selective serotonin
various gray matter areas of the brain, many studies have also
reuptake inhibitors,8 the serotonin transporter gene (SLC6A4) has
investigated possible alterations in white matter architecture and been particularly well-studied. One of the polymorphic sites in the
integrity in patients with MDD. Techniques using diffusion tensor SLC6A4 is an insertion/deletion in the 5′-flanking promoter region
imaging (DTI) and tract-based spatial statistics (TBSS) have made it (serotonin transporter-linked polymorphic region, 5-HTTLPR),
possible to detect such alterations in a more sensitive and accurate which is associated with MDD-related phenotypes, including
manner.3 Previous DTI studies have found significant correlations vulnerability to MDD9 and response to antidepressant treatment.10
between depression and altered integrity of white matter tracts that Studies suggesting an important interaction between genetic
contribute to emotional regulation,4 such as the superior long- and environmental factors in the pathophysiology of MDD are
itudinal fasciculus, corpus callosum, uncinate fasciculus, internal and accumulating,11 with the majority reporting an interaction
external capsule, cingulum and anterior corona radiata.1 between 5-HTTLPR and stress in the development of depression.12
The heritability of MDD is estimated to range from 31 to 42%5 However, the variability in transcription accounted for by 5-HTTLPR is
and the pathogenesis is thought to be significantly influenced by likely low to moderate, and epigenetic mechanisms have therefore

1
Department of Psychiatry, Korea University Anam Hospital, Korea University College of Medicine, Seoul, Korea; 2Department of Brain and Cognitive Engineering, Korea
University, Seoul, Korea; 3Department of Biomedical Science, Korea University, Seoul, Korea; 4Department of Medical Bioscience, Graduate School, Soonchunhyang University,
Bucheon, Korea; 5Brain Convergence Research Center, Anam Hospital, Korea University Medical Center, Seoul, Korea; 6Department of Radiology, Korea University Medical Center,
Korea University College of Medicine, Seoul, Korea and 7Department of Psychiatry, Korea University Guro Hospital, Korea University College of Medicine, Seoul, Korea.
Correspondence: Dr B-J Ham, Department of Psychiatry, Korea University Anam Hospital, Korea University College of Medicine, 73, Inchon-ro, Seongbuk-gu, 136-705 Seoul, Korea.
E-mail: [email protected]
8
These authors contributed equally to this work.
Received 2 June 2015; accepted 5 June 2016
 The corpus callosum and serotonin transporter gene in depression
E Won et al
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been emphasized.7 Epigenetic regulation of SLC6A4 expression by protocol was approved by the ethics review board of Korea University
DNA methylation or histone acetylation has been suggested as a College of Medicine, and signed informed consent was obtained from all
mechanism for gene–environment interactions that contribute participants according to the Declaration of Helsinki.
to MDD.13 Increased cytosine–guanine (CpG) methylation at
SLC6A4 promoter regions, has been associated with lifetime Analysis of SLC6A4 DNA methylation
history of depression,14 post-stroke depression15 and worsening Peripheral blood samples were obtained from all participants to analyze
clinical presentation of depression.16 In contrast, increased SLC6A4 DNA methylation, and the cell type used was whole-blood DNA.
second trimester maternal depressed mood was associated with The CpG-rich region of the promoter between − 34 and − 5 relative to the
decreased maternal and infant SLC6A4 promoter methylation.13 transcriptional start included 5 CpG sites (CpG1 = − 34, CpG2 = − 30,
A negative association has also been reported, with no difference CpG3 = − 16, CpG4 = − 7, and CpG5 = − 5). The region selected covers from
in SLC6A4 promoter methylation levels being observed in − 66 bp upstream to the transcription start site of SLC6A4, which is a part
unmedicated MDD patients and healthy controls.17 of the CpG island located from − 230 bp to +960 bp of the gene, and
shows the highest level of CpG methylation throughout the CpG island
Numerous studies have reported that 5-HTTLPR is associated (http://www.ncbi.nlm.nih.gov/epigenomics). This means that the screened
with brain structure, particularly regional gray matter volume or region in this study could represent the methylation levels of the CpG
thickness18 and white matter integrity,19 which may be associated island. In addition, the CpG island is the only methylated site in the whole
with the dysfunctional limbic-cortical network activity found in SLC6A4 region in the brain. Thus, we considered this region to be suitable
MDD.3 Recent studies have also reported that the status of SLC6A4 for testing the relationship between CpG methylation levels and SLC6A4
promoter region DNA methylation is associated with gray matter expression.
structure and function.7,20,21 However, only a few studies have Methylation analyses of the SLC6A4 promoter region were conducted
demonstrated a direct correlation between epigenetic gene using the bisulfite pyrosequencing method. PCR and sequencing primers
regulation and structural brain alterations associated with were designed using Pyrosequencing Assay Design Software v2.0 (Qiagen,
Hilden, Germany). PCR reactions were performed in a volume of 20 μl with
depression.22 Furthermore, previous studies have not examined ⩾ 20 ng converted DNA, 2.5 μl 10 × Taq buffer, 5 U hot-start Taq
the association between changes in white matter structure and polymerase (Enzynomics, Daejeon, Korea), 2 μl of each 2.5 mM dNTP
SLC6A4 DNA methylation in patients with MDD. mixture, 1 μl of 10 pmol per μl Primer-S and 1 μl of 10 pmol per μl
In the present study, we used DTI and TBSS to investigate biotinylated-Primer-As. The amplification was conducted according to the
changes in white matter integrity of patients with MDD compared general guidelines of pyrosequencing: denaturing at 95 °C for 10 min,
with healthy controls, and the possible association between followed by 45 cycles at 95, 55 and 72 °C, each for 30 s, and a final
structural alterations of white matter tracts and SLC6A4 promoter extension at 72 °C for 5 min. The PCR reaction (2 μl) was confirmed by
region DNA methylation status. First, we hypothesized that electrophoresis in a 2% agarose gel and visualized by ethidium bromide
patients with MDD would exhibit altered integrity in white matter staining. Single-stranded DNA templates were prepared from 16 to 18 μl of
biotinylated PCR products using streptavidin Sepharose HP beads
tracts that are related to cortico-limbic circuit alterations
(Amersham Biosciences, Uppsala, Sweden) following the PSQ 96 sample
associated with depression. Second, we hypothesized that white preparation guide for multichannel pipettes. Fifteen picomoles of the
matter tracts with decreased integrity in MDD patients would be respective sequencing primer were added for analysis. Sequencing was
associated with increased methylation of CpG sites at the SLC6A4 performed on a PyroMark ID system with the Pyro Gold reagents kit
promoter region. (Qiagen), according to the manufacturer’s instruction and without further
optimization. The percentages of individual methylation at five CpG sites
using the pyrosequencing method were calculated and analyzed.
MATERIALS AND METHODS
Participants MRI acquisition
We studied 35 medication-naive patients with MDD who had never taken Diffusion data were acquired on a 3.0 T Siemens Trio whole-body imaging
antidepressants before, and 49 healthy controls. Patients were recruited system (Siemens Medical Systems, Erlangen, Germany). DTIs were acquired
from the outpatient psychiatric clinic of Korea University Anam Hospital, using an echo-planar imaging sequence with the following parameters:
located in Seoul, Korea. Diagnosis was determined by a board-certified repetition time: 6300 ms; echo time: 84 ms; field-of-view: 230 mm;
psychiatrist, according to the Diagnostic and Statistical Manual for Mental 128 × 128 matrix; 3-mm slice thickness with no gap; voxel size
Disorders, 4th Edition, Text Revision (DSM-IV-TR), using the Korean version 1.8 × 1.8 × 3.0 mm; diffusion directions = 20; number of slices = 50; b-values:
of the Structured Clinical Interview for DSM-IV (SCID-IV). Severity of 0 and 600 s mm − 2; acceleration factor (iPAT-GRAPPA) = 2 with 38 reference
depression was measured by the 17-item Hamilton Depression Rating lines for phase encoding direction and 6/8-phase partial Fourier.
Scale (HDRS) on the day of magnetic resonance imaging (MRI) acquisition.
Patients with primary or comorbid psychiatric diagnoses other than MDD
were excluded from the study, including personality disorders of clinical MRI processing
significance. Patients with lifetime exposure to DSM-IV dependence- or We used the Functional MRI of the Brain (FMRIB) Software Library (FSL
abuse-related drugs, other than nicotine, caffeine and social drinking of version 4.1.9, Oxford, UK, http://www.fmrib.ox.ac.uk/fsl/) for preprocessing
alcohol, were also excluded. Patients with serious or unstable medical of raw DTI data, and performed voxel-wise statistical analyses of the
illnesses or primary neurological illnesses, such as cerebrovascular disease, fractional anisotropy (FA) data using TBSS version 4.1.9.23 First, the raw
Parkinson's disease and epilepsy were also excluded. Two patients had a data for each participant were software-corrected for Eddy current
history of suicide attempt in the present depressive episode, one being distortions and head movements. Using the Brain Extraction Tool (BET;
drug intoxication and the other self-strangulation, but both attempts were Oxford Centre for Functional Magnetic Resonance Imaging of the Brain
not performed to an injurious degree. The other 33 patients did not have a (FMRIB), Oxford, United Kingdom), a single binarized brain mask was
history of suicide attempt in the present depressive episode. Also 18 generated in diffusion space, using ‘1’ to represent inside the brain and ‘0’
patients confirmed to have a psychiatric family history, while the other 17 to represent outside the brain (b = 0). Last, the diffusion tensor models
patients denied such a history. However, the absence of psychiatric family were fitted to the corrected data of the FA images.
history in these patients could not be ascertained, as the family members We checked the quality of the FA images to filter out any distorted data
were not assessed directly. Forty-nine healthy controls matched for age, before performing TBSS analysis. However, there were no distorted data
gender and education level were recruited by advertisements in the due to participant motion or scanner noise. FA images from each
community. Healthy controls were screened for major psychiatric histories, participant were aligned to the FMRIB58_FA template image and
and no healthy controls had psychiatric disorders. Healthy participants transformed into a 1 × 1 × 1 mm3 standard space (Montreal Neurological
who consumed alcohol more than to a social degree were also excluded. Institute 152 standard) using the FMRIB Non-linear Image Registration Tool
The age of participants in both groups ranged from 21 to 64 years. All (FNIRT). Non-linear transformed FA images were applied to the original FA
participants were right-handed, as revealed by the Edinburgh Handedness image for each participant. An averaged FA image was then generated
Test, and were self-identified Koreans with ethnic origin ascertained by using the transformed FA images and extracted to create a mean FA
confirming the ethnicity of three generations of the patients’ families. The skeleton image representing the centers of all white matter tracts. The FA

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Figure 1. Results of tract-based spatial statistics (TBSS) analyses demonstrating significantly reduced fractional anisotropy (FA) values for the
body of the corpus callosum (bCC) and genu of the corpus callosum (gCC) in medication-naive patients with major depressive disorder (MDD)
(family-wise error corrected, P o0.01). Background images are the mean FA maps across all participants, with green voxels representing the
mean FA skeleton images. Red and yellow voxels represent the white matter regions in which FA values were significantly lower in patients
with MDD than in healthy controls. Enlarged images show the hand-drawn region-of-interest masks. Red areas represent the body of the
corpus callosum and blue areas represent the genu of the corpus callosum.

skeleton image was set to a threshold of FA40.2, to include the major continuous variables (age, years of education and HDRS scores) and a χ2-
white matter pathways. test for gender. Analysis of covariance (ANCOVA) was performed to
Voxel-wise cross-subject statistics were applied to the skeletonized data calculate differences in SLC6A4 promoter region methylation between
for each participant using the FSL randomize program (University of patients with MDD and healthy controls, including age as a covariate, as an
Oxford, Oxford, United Kingdom). We performed a two-sample t-test for FA association between aging and DNA methylation has previously been
comparisons between patients with MDD and healthy controls. Permuta- reported.31 The alpha level was set at Po0.05/5 = 0.01 after a Bonferroni's
tion testing was performed with 5000 permutations using threshold-free correction for number of CpG sites (CpG1, CpG2, CpG3, CpG4 and CpG5).
cluster enhancement.24 The cluster size was always above 100 voxels and The differences in AD and RD values of the ROIs between patients with
the level of significance was set at a family-wise error (FWE) corrected MDD and healthy controls were analyzed using ANCOVA with age as a
Po0.01 for multiple comparisons. The anatomical location of white matter covariate, as age-related alterations in white matter microstructure have
tracts with significantly different FA values between patients with MDD previously been reported.32 The ROIs were tested at P o0.05/2 = 0.025,
and healthy controls were identified with the John Hopkins University after Bonferroni's corrections for number of ROIs.
(JHU) ICBM-DTI-81 white matter labels in FSL. Binary mask images were A two-tailed Pearson's correlation was performed to analyze the
drawn from the identified regions using FSL, and were defined as the correlations between the extracted FA, AD and RD values and SLC6A4
regions-of-interests (ROIs). Individual means of FA, axial diffusivity (AD) and methylation, controlling for age, as an association between aging and DNA
radial diffusivity (RD) values were extracted from the ROIs (see Figure 1). methylation,31 and alterations in white matter microstructure have previously
DTI expresses the character of the axon fiber using FA and other been reported.32 Each ROI was separately tested at Po0.05/5 = 0.01 after
diffusivity values such as AD and RD. The FA index is sensitive to the Bonferroni's corrections for the five CpG sites. All statistical analyses were
presence and integrity of white matter fibers,25 and higher FA values
performed using SPSS version 12.0 (SPSS, Chicago, IL, USA). In addition, we
represent either an increase in number and size of axon fibers, or a
analyzed the difference between the correlation coefficients of each
decrease in density of crossing fibers.26 Previous studies have suggested
diagnostic group, using Fisher's r-to-z transformation (two-tailed).
AD values to be sensitive to axonal pathologies and RD values to be
Post hoc statistical power calculations for the ANCOVA and comparison
sensitive to myelination,27–29 which may reflect white matter tissue
of correlation coefficients, were performed using the G*Power analysis
microstructure that in turn affects functional connectivity within a neural
circuit. Therefore, AD and RD values may complement FA values, and help program.33 The power calculations were based on the effect sizes which
to interpret potential underlying tissue microstructure alterations.30 came from Cohen’s f for ANCOVA and Cohen’s q for correlation
difference.34 Effect sizes were defined as small, medium or large according
to the following criteria. For Cohen’s f, 0.01 ⩽ small effect sizeo0.25,
Statistical analyses 0.25 ⩽ medium effect sizeo0.40, 0.40 ⩽ large effect size. For Cohen's q,
Differences in demographics between medication-naive patients with 0.01 ⩽ small effect sizeo0.30, 0.30 ⩽ medium effect sizeo0.50,
MDD and healthy controls were analyzed using two-sample t-tests for 0.50 ⩽ large effect size.35

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Table 1. Demographic and clinical characteristics of drug naive patients with MDD and healthy controls

MDD patients (n = 35) Healthy controls (n = 49) t/χ2/F df P-value

Age (years) 40.34 (11.08) 41.12 (14.11) t = − 0.283 81.2 0.786

Gender (male/female) 10/25 15/34 χ2 = 0.041 1 0.84
Education (years) 13 (3.02) 14.14 (3.06) t = − 1.696 82 0.094
HDRS-17a 20.2 (4.95) 2.18 (2.14) t = 20.222 43.11 o0.001a
Illness duration (months) 12.03 (24.55) — — — —

SLC6A4 methylation (%)

CpG1 4.25 (1.76) 5.38 (2.14) F = 6.397 81 0.013
CpG2 2.45 (1.43) 1.61 (1.21) F = 8.365 81 0.005b
CpG3 6.90 (2.00) 6.10 (1.53) F = 4.702 81 0.033
CpG4 5.69 (1.96) 5.83 (2.24) F = 0.085 81 0.772
CpG5 1.75 (1.50) 1.59 (1.16) F = 0.31 81 0.579
Abbreviations: df, degree of freedom; HDRS-17, 17-item Hamilton Depression Rating Scale; MDD, major depressive disorder; SLC6A4, serotonin
transporter gene. Data are represented as mean (s.d.), unless otherwise indicated. aSignificance level for demographic and clinical characteristics. Po 0.05.
b
Significance level for SLC6A4 methylation was corrected for multiple comparisons using Bonferroni's correction; Po0.05/5 (CpG1, CpG2, CpG3, CpG4 and
CpG5) = 0.01.

RESULTS
Demographic and clinical characteristics and SLC6A4 DNA Table 2. Regions showing significantly decreased FA values in drug
methylation status naive patients with MDD compared with healthy controls
There were no significant differences in the demographic variables Cluster size P-valuea MNI peak Anatomical region
tested between patients with MDD and the healthy controls, with coordinates (mm)
the predicted exception of HDRS scores (Table 1). The level of
SLC6A4 DNA methylation was significantly higher at CpG2 in X Y Z
patients with MDD than in healthy controls (F(1,81) = 8.365,
P = 0.005; Table 1), with medium effect size (Cohen’s f = 0.322, 1872 0.004 −2 23 13 Genu of corpus callosum
partial η2 = 0.094) as defined by Cohen.35 Body of corpus callosum
Abbreviations: FA, fractional anisotropy; MDD, major depressive disorder;
TBSS analysis, AD and RD values MNI, Montreal Neurological Institute coordinates. aThreshold-free cluster
Medication-naive patients with MDD showed significantly lower enhancement corrected P-value (family-wise comparisons, error corrected,
FA values for the genu of the corpus callosum and body of the Po0.01).
corpus callosum compared with healthy controls (FWE corrected,
P = 0.004; Table 2 and Figure 1). Therefore, the genu of the corpus
callosum and body of the corpus callosum were defined as correlations between any DTI measures of the body of the corpus
the ROIs.
callosum and SLC6A4 DNA methylation status.
AD values of the body of the corpus callosum were significantly
For the genu of the corpus callosum, no significant correlations
different between the two groups. The MDD group demonstrated
occurred in patients with MDD. However, a significant inverse
lower AD values than the healthy control group (F(1,81) = 11.577,
correlation between the FA values of the genu of the corpus
P = 0.001), with medium effect size (Cohen’s f = 0.322, η2 = 0.125).
callosum and SLC6A4 DNA methylation at CpG4 was observed in
RD values of the body of the corpus callosum were also
healthy controls (Pearson's correlation: r = − 0.372, P = 0.009;
significantly different between the two groups. The MDD group
Table 4 and Figure 3). In addition, a significant positive correlation
demonstrated higher RD values than the healthy control group
between the RD values of the genu of the corpus callosum and
(F(1,81) = 23.084, P o 0.001), with large effect size (Cohen’s f = 0.53,
SLC6A4 DNA methylation status at CpG4 occurred in the healthy
η2 = 0.222). In addition, RD values of the genu of the corpus
control group (Pearson's correlation: r = 0.388, P = 0.006; Table 4
callosum were significantly different between the two groups. The
and Figure 3).
MDD group demonstrated higher RD values compared with the
Patients with MDD showed significantly higher correlations
healthy control group (F(1,81) = 27.793, P o0.001), with large effect
between FA values of the body of the corpus callosum and
size (Cohen’s f = 0.59, η2 = 0.255). These results are summarized in
SLC6A4 DNA methylation at CpG3 compared with healthy controls
Table 3 and Figure 2.
(FA: z = − 2.16, P o 0.05), with large effect size (Cohen’s q = 0.5).
Also, patients with MDD showed significantly higher correlations
Correlations between FA, AD and RD values, and SLC6A4 DNA between AD values of the body of the corpus callosum and
methylation status SLC6A4 DNA methylation at CpG3 compared with healthy controls
A significant inverse correlation between the FA values of the (z = − 2.56, P o 0.05), with medium effect size (Cohen’s q = 0.45).
body of the corpus callosum and SLC6A4 DNA methylation at However, the correlations between FA and RD values of the genu
CpG3 was observed for patients with MDD (Pearson's correlation: of the corpus callosum and SLC6A4 DNA methylation at CpG4 in
r = − 0.493, P = 0.003; Table 4 and Figure 3), in conjunction with a healthy controls were not significantly higher compared with
marginally significant inverse correlation at CpG4 (Pearson's patients with MDD (FA: z = 0.57, P40.1; RD: z = − 1.04, P40.1).
correlation: r = − 0.438, P = 0.01; Table 4). A significant inverse
correlation between the AD values of the body of the corpus
callosum and SLC6A4 DNA methylation at CpG3 was also found in DISCUSSION
the MDD group (Pearson's correlation: r = − 0.478, P = 0.004; The present study found significantly lower FA and AD values and
Table 4, Figure 3). In healthy controls, there were no significant higher RD values of the body of the corpus callosum, as well as

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Table 3. AD and RD values of the bCC and gCC in drug naive MDD patients and healthy controls

MDD patients (n = 35) Healthy controls (n = 49) F P-value

Axial diffusivity
gCC 0.001709 (1.08E − 05) 0.001681 (1.09E − 05) 3.204 0.077
bCC 0.001739 (1.32E − 05) 0.001793 (9.43E − 06) 11.577 0.001a

Radial diffusivity
gCC 0.000345 (7.44E − 06) 0.000283 (8.71E − 06) 27.793 o 0.001a
bCC 0.000454 (7.67E − 05) 0.000404 (6.86E − 06) 23.084 o 0.001a
Abbreviations: AD, axial diffusivity; bCC, body of corpus callosum; gCC, genu of corpus callosum; MDD, major depressive disorder; RD, radial diffusivity. Data
are represented as mean (s.e.), unless otherwise indicated. aSignificance level was corrected for multiple comparisons using Bonferroni's correction; Po0.05/2
(BCC, GCC) = 0.025.

Figure 2. The mean axial diffusivity (AD) values for the body of the corpus callosum were significantly different between medication-naive
patients with major depressive disorder (MDD) and healthy controls, with significantly decreased AD values of the body of the corpus
callosum in patients with MDD. The mean radial diffusivity (RD) values for the body of the corpus callosum and genu of the corpus callosum
were significantly different between patients with MDD and healthy controls, with patients showing significantly increased RD values. Bars
represent s.e. '*' indicates significant P-values corrected for multiple comparisons using the Bonferroni's correction; P o0.05/2 (body of the
corpus callosum, genu of the corpus callosum) = 0.025.

significantly lower FA values and higher RD values of the genu of maturation process is axonal myelination,39 and disruption in the
the corpus callosum in patients with MDD. These findings indicate myelination trajectory can induce deficits in executive and daily
decreased integrity of the body of the corpus callosum and genu functioning.38 Critical functions such as working memory, cogni-
of the corpus callosum in these patients. Significantly elevated tion and emotional processing can be negatively influenced by
SLC6A4 DNA methylation was also observed in patients with MDD deficits of the corpus callosum40 which may predispose indivi-
compared with that in healthy controls. Furthermore, we duals to more severe depressive symptoms.41 Structural changes
identified significant inverse correlations between DNA methyla- in the corpus callosum have been shown to induce altered
tion status of the SLC6A4 promoter region and both FA and AD hemispheric connectivity and impaired emotional and cognitive
values of the body of the corpus callosum in patients with MDD. control, which are reported in patients with MDD.42 Treatment-
To the best of our knowledge, the present study is the first report naive young women with early-onset dysthymia,43 as well as
of an association between brain white matter structural alterations
adolescents and adults with early-onset MDD were reported to
and SLC6A4 DNA methylation status in patients with MDD.
have a smaller corpus callosum compared with healthy
The corpus callosum is the largest commissural white matter
pathway connecting the hemispheres of the human brain, and is controls.38,44 DTI studies have reported lower FA values of the
an essential component in brain lateralization and inter- corpus callosum in treatment-naive young adults with a first
hemispheric communication.36 The genu is the most rostral episode of MDD,45 in middle-aged patients with MDD4 and in a
region of the corpus callosum and forms connections between group of MDD patients who were either treatment-naive or taking
prefrontal brain regions.37 The most caudal region of the corpus medication.3 FA deficits associated with lower AD and higher RD
callosum is the splenium, and the body is the midsection between values in the corpus callosum have also been identified in patients
the genu and splenium. In general, the overall area of the corpus with MDD.46 Also, a recent study has reported increased
callosum increases during childhood and adolescence,36 and depression severity to be significantly related to reduced FA of
maturation of the corpus callosum corresponds to maturation of the corpus callosum in patients with mild Alzheimer's disease.47
cognitive processes.38 The core physiological mechanism in this Our present findings are consistent with these previous studies

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Table 4. Results for Pearson's correlation analysis between FA, AD and RD values of the gCC and bCC and SLC6A4 methylation (CpG1, CpG2, CpG3,
CpG4 and CpG5) in drug naive MDD patients and healthy controls

SLC6A4 methylation

CpG1 CpG2 CpG3 CpG4 CpG5

FA
MDD patients
gCC − 0.068 (0.702) − 0.195 (0.27) − 0.28 (0.108) − 0.253 (0.15) − 0.132 (0.458)
bCC − 0.301 (0.084) − 0.255 (0.15) − 0.493 (0.003)a − 0.438 (0.01)b − 0.142 (0.422)
Healthy controls
gCC − 0.191 (0.192) − 0.17 (0.249) − 0.203 (0.167) − 0.372 (0.009)a − 0.095 (0.519)
bCC − 0.022 (0.88) − 0.03 (0.840) − 0.042 (0.778) − 0.117 (0.428) 0.013 (0.931)

AD
MDD patients
gCC − 0.188 (0.288) − 0.315 (0.07) − 0.419 (0.14) − 0.361 (0.036) − 0.301 (0.084)
bCC − 0.225 (0.2) − 0.252 (0.151) − 0.478 (0.004)a − 0.341 (0.049) − 0.293 (0.092)
Healthy controls
gCC 0.215 (0.143) − 0.139 (0.345) − 0.101 (0.493) 0.201 (0.170) − 0.042 (0.779)
bCC 0.024 (0.872) 0.012 (0.938) 0.068 (0.647) 0.082 (0.577) − 0.080 (0.587)

RD
MDD patients
gCC 0.034 (0.847) 0.124 (0.486) 0.190 (0.282) 0.168 (0.341) 0.042 (0.815)
bCC 0.237 (0.178) 0.156 (0.378) 0.307 (0.077) 0.294 (0.092) 0.021 (0.907)
Healthy controls
gCC 0.200 (0.173) 0.153 (0.298) 0.175 (0.234) 0.388 (0.006)a 0.064 (0.664)
bCC 0.028 (0.850) 0.041 (0.780) 0.022 (0.883) 0.107 (0.470) −0.041 (0.783)
Abbreviations: AD, axial diffusivity; bCC, body of corpus callosum; FA, fractional anisotropy; gCC, genu of corpus callosum; MDD, major depressive disorder; RD,
radial diffusivity; SLC6A4, serotonin transporter gene. All data are given as coefficient of Pearson's correlation controlling for age (P-value). aSignificance level
for SLC6A4 methylation was corrected for multiple comparisons using Bonferroni's correction; P o0.05/5 (CpG1, CpG2, CpG3, CpG4 and CpG5) = 0.01.
b
Statistical significance: P = 0.01.

reporting significant alterations in the corpus callosum of patients occur not only in the developing fetus, but also in individuals
with depression. throughout the human life-span.53 High methylation density at
We also observed a significant difference in DNA methylation at the gene promoter region typically silences gene expression, so
CpG2 of the SLC6A4 promoter region, with increased methylation increased methylation of the SLC6A4 promoter region may result
in patients with MDD. This finding is consistent with a previous in a loss of gene functioning.54 The serotonin transporter is
study that reported an association between increased SLC6A4 essential to determining the duration and intensity of serotonin
promoter methylation and susceptibility to major depression.14 communication with pre- and post-synaptic receptors,55 including
Furthermore, we identified significant inverse correlations the serotonin 1A receptor, which is implicated in synaptic function
between FA and AD values of the body of the corpus callosum and plasticity.48 Therefore, decreased expression of SLC6A4 due to
and SLC6A4 methylation at CpG3 in patients with MDD. Our increased DNA methylation may decrease serotonin uptake and
results indicate a decrease in integrity of the body of the corpus produce a deficiency in serotonin activity.54 Accordingly, SLC6A4
callosum, which is associated with an increase in SLC6A4 promoter promoter methylation has been associated with lower in vivo
region methylation. This is similar to the results of a recent study measures of serotonin synthesis in specific brain areas.56 Given
which had reported increased SLC6A4 promoter methylation to be that alterations in serotonergic modulation can have unexpected
associated with brain structure, with smaller hippocampal
effects on neuronal growth,57 brain regions with increased
volumes being associated with higher levels of methylation.21
methylation of the SLC6A4 promoter region may exhibit dysfunc-
The authors of this study stated that one of the underlying
tional brain morphology and physiology.48 As stress has repeatedly
physiological mechanisms of how SLC6A4 methylation is asso-
ciated with brain structure, may be the trophic actions of been associated with MDD, known to precipitate depressive
serotonin.48 We speculate the inverse correlation between episodes and influence the severity, duration and natural course
methylation status and white matter integrity observed in our of the disorder,58 patients with MDD are more likely to have been
results, may also be explained by the role of SLC6A4 DNA exposed to stress. Therefore, our results may represent an
methylation in regulating neuronal plasticity of the body of the association between alterations in serotonin activity influenced by
corpus callosum, through alterations in serotonin activity. increased SLC6A4 promoter methylation levels due to stress
Not only is serotonin involved in basic morphogenetic activities exposure, and changes in white matter integrity in MDD.
during brain development, such as modulation of neural cell We also observed a significant negative correlation between FA
proliferation, migration and differentiation, axonal guidance and values of the genu of the corpus callosum and SLC6A4
synaptogenesis,49 but is also involved in adult neurogenesis.50 methylation at CpG4, as well as a significant positive correlation
Chronic exposure to several forms of stress have been shown to between RD values of the genu of the corpus callosum and
alter chromatin structure in the brain, such as increases in DNA SLC6A4 methylation at CpG4 in healthy controls. These correla-
methylation or histone acetylation,51 and several studies have tions were not significantly higher compared with patients with
found associations between stress and altered promoter methyla- MDD, when the difference between correlation coefficients of
tion levels of the SLC6A4 in particular.52 Also, epigenetic changes each diagnostic group was analyzed.

Translational Psychiatry (2016), 1 – 9

 The corpus callosum and serotonin transporter gene in depression
E Won et al
7

Figure 3. (a) Significant inverse correlations between fractional anisotropy (FA) values and axial diffusivity (AD) values for the body of the
corpus callosum and serotonin transporter gene (SLC6A4) methylation at CpG3 were observed in medication-naive patients with MDD. (b) A
significant inverse correlation between the FA values for the genu of the corpus callosum and SLC6A4 methylation at CpG4 was observed in
healthy controls. In addition, a significant positive correlation between the RD values for the genu of the corpus callosum and SLC6A4
methylation at CpG4 was observed in healthy controls. MDD, major depressive disorder; RD, radial diffusivity.

To our knowledge, this is the first report on brain white matter Although the majority of studies investigating DNA methylation
structural changes and SLC6A4 promoter methylation in patients patterns in humans have been performed on peripheral blood
with MDD. Neurotrophic effects of antidepressants have been cells, such studies are considered to be constrained by access to
reported;59 therefore, only psychotropic medication-naive patients tissue.61 As DNA methylation changes are tissue specific, it is
with MDD were included in the present study. Although our study suggested that the results of studies on epigenetic mechanisms
has multiple strengths, there are also limitations to consider. There need to be considered in the context of the cells that are assessed.
is no clear consensus regarding the appropriate number of CpG Nevertheless, there is emerging evidence for the relevance of
loci of the SLC6A4 promoter region for DTI analyses on SLC6A4 peripheral SLC6A4 methylation for brain processes,21 and DNA
DNA methylation. However, the number of CpG loci in our study is methylation at the SLC6A4 promoter is suggested not to be
relatively fewer compared with other recent studies,13 and may limited to peripheral tissues, but paralleled in the brain.52 Fourth,
therefore affect the ability to detect a correlation between SLC6A4 the age of participants in both groups ranged from 21 to 64 years,
DNA methylation status and white matter tract alterations in which can be considered high. However, our mean age (s.d.) for
patients with MDD. Second, we did not assess psychosocial MDD patients was 40.34 (11.08), which is similar to previous DTI
stressors such as childhood adversity and stressful life events. studies on major depression.1 Therefore, we considered the age
Stress has been shown to alter SLC6A4 promoter methylation range of our sample to be capable of representing MDD patients
levels,52 and white matter integrity has been reported to be in general. Also, we considered concentrating on a certain age
sensitive to adverse experiences.60 Therefore, the influence of group would overly emphasize sample homogeneity, which may
such environmental factors on our results is uncertain. However, hinder generalizing our results. Furthermore, heavy alcohol
as stated above, stress is known to precipitate depressive episodes consumption and smoking have been reported to affect white
and influence the natural course of the disorder.58 Therefore we matter microstructure.62,63 Although we had excluded participants
considered patients with MDD more likely to have been exposed who consumed alcohol more than to a social degree, the exact
to stress compared with healthy controls. Third, the sample used amount of alcohol consumption and proportion of smokers were
to analyze SLC6A4 DNA methylation, was peripheral blood DNA. not assessed, which we consider to be a limitation as the influence

Translational Psychiatry (2016), 1 – 9

 The corpus callosum and serotonin transporter gene in depression
E Won et al
8
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