Introduction

Stress is believed to play an important role in the pathogenesis of Major Depressive Disorder (MDD). There is much debate about the effect of childhood adversity on later psychopathology, including MDD [1,2], and on brain structure [3]. The hippocampus may be a highly relevant brain structure, given the widely reported associations among childhood trauma, dysregulated physiological and emotional stress responses, and structural changes in the hippocampus in stress-related disorders [3–6].

Current neurobiological theories of MDD postulate that altered serotonin (5-HT) neurotransmission may represent a biological risk factor, which is more likely to be expressed in the presence of adversity [7]. Consistent with this model, researchers have identified a number of 5-HT polymorphic genes that may increase the risk of developing a mental disorder. The most widely studied is the 5-HTTLPR polymorphism of the 5-HT transporter (SLC6A4) gene. The s allele of the 5-HTTLPR polymorphism has been associated with increased risk for MDD in the presence of early adversity (e.g., [8]). Although research is still inconsistent (e.g., [9]), some of the inconsistencies can likely be attributed to methodological differences [10,11]. Imaging studies showed that carriers of the s allele of the 5-HTTLPR polymorphism show amygdala hyper-reactivity upon exposure to emotional stimuli [12]. Results of these latter studies are consistent with SLC6A4 deletion studies in rodents [13,14] and further support the role of 5-HT (including SLC6A4) in brain development [15]. Moreover, we found that, in patients with MDD, those with a history of childhood maltreatment and at least one s allele of the 5-HTTLPR had significantly smaller hippocampal volumes [16]. The underlying mechanism accounting for such gene by environment (GxE) interactions are, however, unknown.

One of the potential mechanisms by which gene and environment interact and affect brain development is via environmentally induced stable changes in genetic expression [7,17,18]. These changes in stable expression are most probably caused by epigenetic mechanisms [7,17,18]. Following the observation of tissue-specific DNA methylation changes in the hippocampal glucocorticoid receptor gene in post-mortem brains of victims of childhood abuse [19], a number of researchers have studied DNA methylation processes in peripheral tissues. With regard to the 5-HT system, an increasing number of studies have found associations between peripheral methylation in the SLC6A4 gene and both early life adversity (see [7]) and depression [18]. Specifically, studies demonstrated that early stress, including a history of childhood abuse, was associated with altered levels of peripheral methylation in SLC6A4 gene promoter regions later in life [20–24]. In addition, we found that peripheral DNA methylation at the SLC6A4 gene promoter in white blood cells of healthy adults was associated with lower in vivo measures of brain 5-HT synthesis in the orbitofrontal cortex, irrespective of 5-HTTLPR genotype [25]. The functional relevance of DNA methylation in SLC6A4 promoter regulation was further demonstrated by an in vitro experiment that showed that SLC6A4 methylation resulted in loss of promoter activity in transient transfection promoter-luciferase reporter assays [25]. Taking these findings together suggests that DNA methylation at the SLC6A4 promoter is not limited to peripheral tissues and is paralleled in the brain, where it may be one of the physiological mechanisms underlying how early stress could translate into altered brain development [26]. Hippocampal changes may be particularly relevant, since this brain region is densely innervated with 5-HT and is highly involved in stress regulation [3,5].

The aim of the present study was to test the hypothesis that DNA methylation in specific CpG sites at the SLC6A4 promoter in peripheral cells is associated with childhood trauma, depressive symptomatology, and smaller hippocampal volume. To this end, we assessed DNA methylation at the SLC6A4 promoter in whole blood DNA in depressed patients and in age- and sex-matched healthy controls, all of whom previously underwent high-resolution structural Magnetic Resonance Imaging (MRI) to measure hippocampal volume [27]. Secondary aims of the study were to examine the association between demographic and (sub)clinical characteristics of depression and between DNA methylation at the SLC6A4 promoter. We focused on DNA methylation in a region of the SLC6A4 promoter and associated specific CpG sites that were previously shown to be most strongly associated with in vivo measures of 5-HT synthesis [25].

Materials and Methods

Participants

Sixty-nine adult participants were included. The sample included 33 adult patients with MDD, recruited from the mental health services of either the Tallaght Hospital or St. James’s Hospital, Dublin, Ireland. MDD diagnosis was based on DSM-IV criteria and was confirmed by an independent psychiatrist using the SCID interview. Thirty-six healthy controls from the local community were also recruited and the groups were balanced for age and sex (Table 1). Participants were excluded (1) if they were taking antipsychotics or mood stabilizers, (2) if they were not between the ages of 18 and 65, or (3) if they had a history of neurological or comorbid psychiatric disorders (Axis I or Axis II), other severe medical illness, head injury, or substance abuse. Demographic variables as well as inclusion and exclusion criteria were documented by using a standardized questionnaire and a structured interview conducted by a psychiatrist.

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Table 1. Characteristics of the sample.

https://doi.org/10.1371/journal.pone.0119061.t001

Written informed consent was obtained from all participants following a verbal and written detailed description of the study, which was designed and performed in accordance with the ethical standards laid out by the Declaration of Helsinki and which was approved by the ethics committees of (1) St. James and Tallaght Hospitals, Dublin, Ireland, (2) McGill University, Montreal, Canada, and (3) Queen’s University, Kingston, Canada. Specialists in psychiatry assessed all participants with regard to their ability to consent, and only patients who had full capacity to understand the study procedures and to provide consent took part in the study.

Definition of Hippocampus

In the present study, we assessed whole hippocampal volume, as well as hippocampal subregions including the dentate gyrus and the cornu ammonis, as these have different functions and seem to have different stress sensitivities [33, 34].

Hippocampal subfields volumes were assessed automatically with the program FreeSurfer. We identified the main hippocampal subfields belonging to the cornu ammonis and dentate gyrus: CA1, CA2/3, and CA4/DG (Fig. 1). It was previously shown that automatically calculated volumes of CA2/3 and CA4/DG are strongly correlated with those volumes derived from manual delineation, with a correlation coefficient of. 91 (p ≤. 0002) and. 83 (p ≤. 0028), respectively [35].

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Fig 1. Example for hippocampal subfield delineation.

Shown are subfields CA1, CA2/3, CA4/DG, subiculum and presubiculum. The program FreeSurfer automatically assessed volumes of subfields which were then manually viewed and checked for quality.

https://doi.org/10.1371/journal.pone.0119061.g001

Results

Patients and controls were well matched (Table 1). Smoking behaviour (defined either categorically [present/absent] and/or by the number of cigarettes smoked per day did not differ between patients and controls, nor did it correlate with any of the methylation measures, and it was thus left out of further analyses. A regression model including age, sex, abuse, diagnosis, and whole hippocampal volume as predictors (N = 69) explained 29% variance of SLC6A4 methylation (F(5,63) = 5.12, p = .001). Being male, increased age, greater childhood abuse, and smaller hippocampal volume were each associated with greater percentage of SLC6A4 methylation across the investigated promoter region (Table 2, Fig. 2–3, and Fig. A in S1 File). MDD diagnosis was not significantly associated with DNA methylation (p = .28). The model had adequate stability (range r²:. 27–.42) and a large effect size (f2 = 0.41). We also tested for associations between the same independent variables (sex, age, abuse, hippocampal volume, MDD) and SLC6A4 site-specific methylation. Hippocampal volume was found to be very similar to the association observed with methylation across the whole region (CpG 5&6: F(5,63) = 4.29, p = .002; CpG 11&12: F(5,63) = 3.6, p = .006). Sex, however, was not significantly associated with site-specific SLC6A4 methylation. There was also no significant association between abuse and methylation in CpG 5&6 (p = .08), nor between age and methylation in CpG 11&12 (see Table A and Table B in S1 File) while controlling for the other main variables in the equations. The interactions between (1) hippocampus and MDD, (2) abuse and MDD, and (3) hippocampus, MDD, and abuse did not provide a significant additional contribution to the regression models.

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Table 2. Parameters of the investigated regression model that includes sex, age, MDD diagnosis, childhood abuse, and hippocampal volume as independent variables, and SLC6A4 methylation as the outcome variable.

https://doi.org/10.1371/journal.pone.0119061.t002

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Fig 2. Scatterplot showing the association between hippocampal volumes and methylation of SLC6A4.

There was a negative correlation between both variables indicating that smaller hippocampal volumes were associated with higher levels of methylation.

https://doi.org/10.1371/journal.pone.0119061.g002

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Fig 3. Scatterplot showing the association between total childhood abuse and methylation of SLC6A4.

There was a positive correlation between both variables indicating that more abuse is associated with higher levels of methylation.

https://doi.org/10.1371/journal.pone.0119061.g003

Results were overall very similar when the different hippocampal subfields CA1, CA2/3 and CA4/DG, rather than the whole hippocampus, were used as variables in the models (see Table C, Table D, and Table E in S1 File).

Further examination of the regression diagnostics of the model with the best fit showed very low multicollinearity (indicating low correlations among predictors) (VIF = 1.4, criteria for multicollinearity usually > 10), thereby further supporting the stability of the models (see Table A in S1 File).

Discussion

The present study showed for the first time that methylation in a regulatory region of the SLC6A4 gene in whole blood DNA is independently associated with hippocampal volume, childhood adversity, being male, and an older age. Almost one third of the variance in SLC6A4 methylation was explained by these variables.

Childhood abuse was previously found to be associated with altered levels of peripheral methylation states in SLC6A4 promoter regions later in life [20–22,24]. The present study confirmed these findings and showed that the results were most pronounced in those CpG sites specifically associated with brain 5-HT synthesis [25].

Among the selected variables of investigation, lower hippocampal volume was most strongly associated with SLC6A4 regulatory region methylation state across CpG sites, as well as in the specific, a priori-selected CpG sites [25]. Interestingly, this was the case for the whole hippocampus and also for CA1 as well as the hippocampal subfields gyrus dentate and CA2/3. The relatively strong association between peripheral methylation of SLC6A4 regulatory region and hippocampal volumes expands on our previous study showing that childhood adversity interacts with the polymorphism in the promoter region of the SLC6A4 gene on hippocampal volumes [16]. It suggests that SLC6A4 methylation may be an underlying physiological mechanism of how gene and environment interact to affect hippocampal development.

Although the present study was cross-sectional and correlational, and causality as well as formal mediation models could thus not be tested, it could be speculated that peripheral methylation of SLC6A4 regulatory region might affect hippocampal development in different ways, one of which may be through disruption of 5-HT homeostasis, thereby affecting 5-HT as neurotrophic factor [26]. Specifically, the 5-HT system is known to play a prominent role in neurogenesis, which takes part in the gyrus dentate [43,44]. Thus, increased methylation of SLC6A4 owing to environmental factors like childhood abuse [7]—which appears to be functionally relevant [25]—might alter the neurotrophic properties of 5-HT, which in turn has consequences for brain development.

The 5-HT system is highly interactive with other biological systems. Being one of the brain areas implicated in MDD, the hippocampus may have a particular role in the interactions between the 5-HT system and the hypothalamic-pituitary-adrenal (HPA) axis [27,45]. Lower mRNA expression of cortisol-inducible genes, which can be seen as a marker for a blunted cortisol response, was previously found to be associated with smaller hippocampal volumes in MDD patients [27]. Thus, it could also be hypothesized that stress-induced alterations in peripheral SLC6A4 methylation states may affect hippocampal volume through modulation of HPA axis functioning. Further research is necessary to understand the interplay of HPA-axis–associated (epi)genetic factors, the role of mediators and moderators, and their effects on different brain regions.

In our present study we did not find that MDD diagnosis was significantly associated with methylation. Such a finding is in line with the hypothesis that SLC6A4 methylation represents risk for developing depression and that other biological or social risk factors, as well as protective factors, need to be taken into account too [7].

The present observation that higher DNA methylation is associated with age is of interest given the results of a previous study comparing genome-wide methylation rates between centenarians and newborns, which showed that age-associated increase of DNA methylation of the regulatory regions is gene specific [40]. The present study is the first to show age-related methylation increases in the SLC6A4 gene specifically in a sample with a much smaller age range. With regard to sex, in a recent review [38], it was explored whether methylation of DNA might play a role in contributing to the observed sex differences in the prevalence of stress-related mental disorders like posttraumatic stress disorder (PTSD) and depression. It was suggested that sex differences in methylation should be investigated in future studies in order to find an explanation for sex differences in mental disorders [38]. Yet, research since that study has been inconclusive; a previous study showed that females had lower SLC6A4 methylation than men [46]. About one third of the participants of that study had a history of MDD, and the large majority of the participants had no current MDD symptomatology. In another study in healthy young adults, no sex differences were found. In the present study, males had significantly higher methylation in SLC6A4 compared to females, independent of childhood abuse or MDD diagnosis. These differing results highlight that there is no convincing evidence that methylation in the SLC6A4 gene contributes to sex differences in prevalence of depression.

Interestingly, MDD patients currently receiving SSRIs showed increased methylation of SLC6A4 compared to patients who were currently medication-free, while controlling for the other predictors. In an experimental study, mice treated with fluoxetine after an experimental brain injury showed increased neurogenesis, increased methylation, and increased histone H3 acetylation in the dentate gyrus [47]. This finding might point toward an acute effect of SSRIs on DNA methylation, but needs to be extended to human studies using longitudinal designs.

We did not find SLC6A4 genotype to be associated with DNA methylation, which is consistent with our previous study in healthy volunteers [25], with a study that used participants with various levels (sub)clinical depressive symptoms [48], and with one that used a large sample of healthy adults [49].

A previous study found that higher SLC6A4 methylation was associated with increased responses to loss or other trauma in carriers of the ll genotype [23]. The observation in the present study of higher methylation levels in carriers of the ll genotype who were also exposed to childhood trauma, relative to s carriers with or without childhood trauma, is consistent with this previous finding [23].

As in our previous study, mRNA expression was not related to DNA methylation [25], nor was it associated with any of our outcome measures. The relationship between SLC6A4 methylation and mRNA expression appears to be complex; some studies report an association or report that the association is genotype-specific [50], while in others there is no association between mRNA expression and SLC6A4 methylation (e.g., [49]). The non-significant associations may be caused by degradation owing to sensitivity to fast processing time [51]. In contrast, DNA is more robust and methylation remains more constant over time. This supports the conclusion that DNA methylation in peripheral cells might be a more reliable biomarker than mRNA [7,25,52].

Moreover, in the present study we confirmed that the hippocampus, and the cornu ammonis and dentate gyrus volumes in particular, are significantly smaller in patients with MDD compared to healthy controls, which is in agreement with another study on hippocampal subfields and the results of some meta-analyses [34,53].

The results of our study are consistent with those of our PET study [25] and with the majority of studies from other research groups, showing that higher SLC6A4 methylation is associated with increased behavioural depressive symptomatology or early life adversity [20,21,24,48]. Interestingly, however, a very recent study showed that SLC6A4 methylation was positively correlated with hippocampal volume [54]. Notably, methylation rates in the Dannlowski et al. [54] study were on average 48%, while in most of the SLC6A4 methylation studies, including ours, methylation rates lie between 4 and 15% [20,21,24,25,48]. The observed discrepancy is likely due to differences in methodology. For instance, the location of the methylation region in Dannlowski et al. [54] differed from the one used in our study. Moreover, while our study sample consisted of MDD patients and controls, the sample in Dannlowski et al. [54] consisted of healthy individuals with very low depression scores and who were free of lifetime psychiatric disorder and therefore possibly low on early adversity (although trauma was not assessed in that study). Finally, differences in statistical imaging methodology may have also contributed to a results discrepancy.

Nevertheless, while there is now emerging evidence for the relevance of peripheral SLC6A4 methylation for brain processes as shown by research from different groups, these findings taken together suggest that the association is not a simple, static one-to-one correlation. Rather, the strength and direction of effect seems to be a complex, dynamic interplay between other biological factors or experiences and variable across specific methylation regions / CpG sites within a gene.

One particular strength of the study was that the investigated SLC6A4 region and primary CpG sites were chosen a priori, based on previous work and validation in vivo and in vitro in an independent sample [25]. The finding that the associations between states of methylation of SLC6A4 and hippocampal volume and abuse were primarily observed in the same CpG sites that were previously associated with brain 5-HT synthesis [25] points to the possibility that site-specific SLC6A4 methylation may be a biomarker for 5-HT–associated stress-related psychopathology.

The findings of the present study rest upon the following limitations. First, the CTQ was filled out retrospectively, which could have led to inaccurate recollection of events, thereby affecting CTQ scores. Thus the interpretation of the trauma effect should be interpreted with caution. Nevertheless, the scores obtained in the current sample are overall similar to those obtained in many other studies using the same questionnaire. Second, it is important to mention that the observed associations in the present study were correlational and cross-sectional. Hence, we cannot draw a conclusion about cause and effect, nor test formal mediation models. Intervention studies are currently under way to further determine the direction of effect. Third, although the observation of the association between early life trauma and methylation is consistent with other studies, we cannot rule out that the association is directly due to or mediated by abuse or environmental stress in adulthood. Fourth, the control group had very low levels of childhood abuse. Hence, this did not allow us to investigate the association between abuse and SLC6A4 methylation in the control group. On the other hand, our regression analyses showed an association between childhood abuse and SLC6A4 methylation while controlling for depressive symptoms. Fifth, the present study was not primarily designed to investigate the effect of different medication types on peripheral methylation of SLC6A4. Thus, we did not randomize treatment prior to study participation, and the link between medication and methylation could therefore be confounded by other clinical characteristics. Future studies could be designed specifically to study the effects of (previous) antidepressant treatment on methylation and also to investigate, with longitudinal designs, whether those subjects who have higher SLC6A4 methylation respond best to SSRIs, compared to those with lower SLC6A4 methylation.

Notwithstanding these limitations, this is one of the first studies using MDD patients and controls that shows an association between methylation states at the SLC6A4 gene promoter with hippocampal volumes, specifically in the subfields dentate gyrus and CA2/3. The findings expand on our previous reports of interactions between childhood abuse and the s-allele of the serotonin transporter polymorphism and on the association between peripheral SLC6A4 methylation and 5-HT synthesis measured with Positron Emission Tomography. The present study suggests that epigenetic mechanisms may biologically underlie environment by gene interactions that influence hippocampal development. This might in turn, directly or through brain development, make individuals more vulnerable to developing depression or other stress-related disorders.

Supporting Information