Background
Serotonin deficiency was one of the earliest hypothesis of depression causality [
1,
2]. This came from the discovery of a class of antidepressants, the selective serotonin re-uptake inhibitors (SSRIs), which block the reuptake of the neurotransmitter and therefore increasing its availability. SSRIs are thus commonly prescribed to treat depression, however the response rate remains relatively modest (around 50%) [
3]. The serotonin transporter (5-HTT), encoded by the serotonin transporter gene (
SLC6A4), is a principle regulator of serotonergic neurotransmission and the active target of SSRIs [
4]. Thus, the role of
SLC6A4 and its (dys)regulation has been a major focus of depression research.
One feature of
SLC6A4 which has been the subject of much research, is the upstream gene-linked polymorphic region,
5-HTTLPR. A 44 bp insertion/deletion variable number tandem repeat (VNTR), results in either short (S) or long (L) alleles. The S allele results in lower
SLC6A4 transcriptional levels and therefore reduced reuptake of serotonin [
5,
6]. There is some evidence that
5-HTTLPR genotype affects vulnerability to a broad range of behavioural disorders including depression [
1]. However, these associations have not been consistently found [
7], suggesting that other regulatory mechanisms and factors are likely to contribute to depression risk [
8‐
10].
Approximately 1 kb downstream from
5-HTTLPR lies another widely studied regulatory element, a CpG island that spans the upstream promoter of exon 1 and the transcriptional start site [
11]. DNA methylation of this compact region of CpG sites has been associated with reduced expression [
11]. Previous, generally small studies have investigated the association between depression and
SLC6A4 DNA methylation in peripheral tissues, but with conflicting findings [
12‐
20]. It is now clear that genetic variation is an important regulator of DNA methylation across the genome [
21], yet only a few studies have examined whether
5-HTTLPR influences methylation and/or the association between methylation and depression [
15,
16]. To date the effects of other
SLC6A4 genetic variants on DNA methylation and on the potential link between depression and methylation, have yet to be investigated.
Using data gathered as part of a large general population cohort study of older persons, this study firstly investigated whether SLC6A4 genetic variation across the gene influences promoter DNA methylation and secondly, whether depression is associated with DNA methylation independently and/or in combination with genetic variation.
Discussion
Our study investigated the relationship between SLC6A4 promoter methylation and six polymorphisms (5-HTTLPR, rs140700, rs42151417, rs6354, rs25528, rs25531), plus a combined variant (5-HTTLPR/rs25531) independently and in the context of late-life depression. We found marginal and nominally significant evidence that six genetic variants influenced site-specific methylation at four out of 11 CpG units. Whilst methylation was not independently associated with depression, specific genotypes were found to modify the association between depression and methylation at several CpG units. In particular, for individuals with the SS or S’S genotypes of 5-HTTLPR and 5-HTTLPR/rs25531 respectively, depression was significantly associated with lowermethylation at CpG 21 and CpG 25.26. On the other hand, for individuals carrying the LL or L’L’ genotypes, depression was nominally associated with higher methylation at CpG 21. These findings were not confounded by sex, age, antidepressant use, or other factors examined including living situation and comorbidities.
Differential
SLC6A4 methylation at the promoter CpG island has been correlated with risk factors and adversities related to depression. Associations has been observed between maternal depression and decreased infant
SLC6A4 promoter methylation [
34]. In adults, increased methylation has been associated with childhood trauma [
35] and stress (early life and recent) [
30,
36]. An inverse correlation between
SLC6A4 mRNA levels and promoter methylation has been demonstrated [
11]. Several studies support our findings, reporting no association between
SLC6A4 methylation in blood and depression in adults, in either Caucasian [
12] or three Asian (
n = 108;
n = 286;
n = 100) [
14,
15,
17] populations, as well as in buccal mucosa from Caucasian adolescents (
n = 150) [
16] and EBV-transformed lymphoblasts (
n = 192) [
18]. In contrast, three studies found positive associations between peripheral
SLC6A4 methylation in blood and depression in Caucasian (
n = 57) [
13] and Asian adults (
n = 151;
n = 84) [
19,
20], two of which had overlapping assay regions with our study [
19,
20]. Interestingly, Shi et al. (2016) found significantly highermethylation at two CpG sites (∆ = 2.52 and 0.15) corresponding to CpG 25.26 in our study, which we failed to find, despite this unit having the highest level and variability of methylation.
It is increasingly clear that genetic variation plays a critical role in the regulation of DNA methylation [
21]. Such loci, referred to as methylation quantitative trait loci (mQTLs), may also modify the association between DNA methylation risk of disease [
37,
38]. Previous studies that have investigated the extent to which
SLC6A4 genotype can influence DNA methylation in the context of depression, have focused primarily on the
5-HTTLPR polymorphism. Several reported no significant association with mean methylation [
12,
16,
17], which is supported by our findings. However, we did observe a trend for an association between
5-HTTLPR SS genotype and higherCpG 21 methylation (
p = 0.077), which is in concordance with a study in post-stroke depression [
15]. In addition,
rs25531, which has been shown to modify the functionality of
5-HTTLPR [
27,
29] was also associated with site-specific methylation at CpG unit 16–20. Here we have shown for the first time that four other polymorphisms (
rs4251417, rs140700, rs6354 and
rs25528) in
SLC6A4 may potentially regulate DNA methylation in the region.
Modifying effects of
5-HTTLPR genotype on the association between
SLC6A4 methylation and several depression-related adversities, including childhood abuse [
12] and stress [
36], have been demonstrated previously. The few studies which have investigated this in the context of depression, have found that the S allele in combination with higher
SLC6A4 methylation increased the risk of depressive symptoms in adolescents [
16] and adults following stroke [
15]. This contrasts with our current study which observed lower methylation at CpG units 21 and 25.26 in depressed older individuals with the SS genotype. However, a recent study by Iga et al. (2016) (average age: 42.2–45.0 years) found that the L allele was associated with higherperipheral blood methylation in depressed patients [
13]. More broadly, as an inverse correlation exists between
SLC6A4 methylation and mRNA levels [
11], our finding of decreased methylation in depressed individuals is consistent with the observation of increased peripheral
SLC6A4 mRNA in un-medicated MDD patients [
39‐
41].
The vast majority of epigenetic studies of
5-HTTLPR did not consider
SL genotype individually, which makes it hard to determine the real (in)consistency of data [
8]. Similar to Iga et al., we found highermethylation at CpG 21 in depressed older individuals with the LL genotype, although this was only nominally significant in our study. Interestingly, previous findings also indicate that age can influence the association between
5-HTTLPR and mental health [
8].
Given the larger sample size, our findings further strengthen the evidence that
5-HTTLPR plays a role in modifying the association of methylation with depression. We also found significant modifying effects from the triallelic
5-HTTLPR/rs25531 polymorphism. Whilst
5-HTTLPR/rs25531 modification effects have not been studied in relation to the CpG island, a recent study found that carriers of the S′ allele had lowermethylation at a neighboring Alu retrotransposon (AluJb) in association with stress [
42], which is in a similar direction of effect found in our study.
This is also the first study to examine the potential modifying effects of other polymorphisms throughout the SLC6A4 gene on the depression-methylation association. Here, we report modifying effects from rs140700, rs4251417, rs6354, but these associations did not remain significant following multivariate adjustment. This may be because of the relatively small sample size with these genotypes, in particular because there were too few minor homozygotes to include in the analysis, with the comparison instead been only between major homozygotes and heterozygotes. Therefore, a larger sample size able to capture the minor homozygote is needed to further investigate these associations. Overall, the effects of genotype and methylation may combine to exert another layer of regulation in modifying risk of depression. Further investigation is needed to examine the underlying mechanisms and its function in influencing depression risk.
Our study of 302 participants is larger than all previous studies investigating
SLC6A4 methylation and genetic variation in depression. However only subtle differences in DNA methylation levels have been observed in peripheral tissues in association with psychiatric disorders, suggesting that an even larger sample size may be required to fully reveal true associations. We were able to consider several polymorphisms throughout the gene in combination with promoter methylation in our study, and adjust for a wide variety of confounding factors including sex, age and antidepressant use. Our participants were from the general population, thus making our findings more generalisable, as opposed to studies focused only on depressed hospital patients. Late-life depression covers a range of mild to severe depressive symptoms [
43], so the CES-D scale to assess depressive symptoms, together with the DSM-IV classification of MDD (American Psychiatric Association, 1994), has helped ensure non-depressed individuals in this study did not have significant levels of sub-clinical symptomatology. Whilst our results may be generalisable to the older population, it may not be the case for earlier onset depression and non-Caucasian populations. Contributors to the etiology of depression may vary by age of onset, with late-life depression more frequently comorbid with physical and psychiatric conditions such as cardiovascular disease and stroke [
44]. Depressive symptoms are also more frequent amongst the oldest old, which may be explained by factors associated with aging, such as higher proportion of women and increased physical impairment [
44]. This is consistent with our finding that depressed individuals are significantly more likely to be female, older and functionally impaired (Table
1). DNA methylation patterns have been reported to vary with age, sex and ethnicity [
45,
46]. Specifically, global hypomethylation has been reported in females [
46] and with increasing age [
45]. Hence, decreases in
SLC6A4 methylation, as observed in this study, may be specifically associated with late-life depression.
Other limitations to our study are the cellular heterogeneity of blood, the cross-sectional design of this study and potential residual confounding from factors for which information was not available or collected. Our study focused on white blood cells, which contain a heterogenous assortment of cell types. Given the cell-type-specific nature of methylation, variation in cellular composition may lead to distinct methylation profiles between cell types, potentially confounding methylation-related analyses [
47]. This may account for the lack of associations observed between depression and
SLC6A4 methylation in our study. Cellular heterogeneity cannot be controlled for in a candidate gene study such as ours. Epigenome-wide association studies, on the other hand, have the ability to computationally adjust for cellular heterogeneity [
48], with the added advantage of being able to interrogate large proportions of the (epi)genome without an a priori hypothesis.
Finally, as an association study, we are unable to draw any conclusions about the functionality or causality of our findings. Using peripheral methylation to examine a brain-based disorder has its limitations, as methylation profiles can also be distinct across different tissues. A few studies have reported correlations between DNA methylation levels in blood and post-mortem brain tissue, although these are likely to be gene-specific [
49,
50]. Changes in peripheral tissues, such as in inflammatory markers, have been observed in depression, therefore becoming increasingly recognised as a systemic disease [
51,
52]. Further, both
SLC6A4 methylation and genetic variants have been correlated with brain structural changes, including the hippocampus and corpus callosum, in depressed individuals [
12,
53‐
55]. As such, our study may provide support for
SLC6A4 methylation as a biomarker of depression, indicating that such a biomarker would need to consider
SLC6A4 methylation in combination with genetic variation.