No association between peripheral serotonin-gene-related DNA methylation and brain serotonin neurotransmission in the healthy and depressed state

Abstract

Background: Methylation of serotonin-related genes has been proposed as a plausible gene-by-environment link which may mediate environmental stress, depressive and anxiety symptoms. DNA methylation is often measured in blood cells, but little is known about the association between this peripheral epigenetic modification and brain serotonergic architecture. Here, we evaluated the association between whole-blood-derived methylation of four CpG sites in the serotonin transporter (SLC6A4) and six CpG sites of the tryptophan hydroxylase 2 (TPH2) gene and in-vivo brain levels of serotonin transporter (5-HTT) and serotonin 4 receptor (5-HT₄) in a cohort of healthy individuals (N = 254) and, for 5-HT_4, in a cohort of unmedicated patients with depression (N = 90). To do so, we quantified SLC6A4/TPH2 methylation using bisulfite pyrosequencing and estimated brain 5-HT₄ and 5-HTT levels using positron emission tomography. In addition, we explored the association between SLC6A4 and TPH2 methylation and measures of early life and recent stress, depressive and anxiety symptoms on 297 healthy individuals.

Results: We found no statistically significant association between peripheral DNA methylation and brain markers of serotonergic neurotransmission in patients with depression or in healthy individuals. In addition, although SLC6A4 CpG2 (chr17:30,236,083) methylation was marginally associated with the parental bonding inventory overprotection score in the healthy cohort, statistical significance did not remain after accounting for blood cell heterogeneity.

Conclusions: We suggest that findings on peripheral DNA methylation in the context of brain serotonin-related features should be interpreted with caution. More studies are needed to rule out a role of SLC6A4 and TPH2 methylation as biomarkers for environmental stress, depressive or anxiety symptoms.

Keywords: 5-HT; Depression; Early life stress; Epigenetics; Human brain imaging; Mood disorders; PET; Serotonin 4 receptor; Serotonin transporter; TPH2; Tryptophan hydroxylase 2.

Fig. 1

Overview of the data and methods used in this study. a, b, c, d depict the primary analyses, in which a latent variable model was used to determine the association between peripheral TPH2 and SLC6A4 methylation and brain levels of 5-HTT and 5-HT₄. e, f, g describe the sensitivity analyses evaluating the association between SLC6A4/TPH2 methylation and measures of environmental stress, depressive and anxiety symptoms. h and i show sensitivity analyses used to evaluate potential influence of blood cell proportions in the A-G analyses. Abbreviations: 5-HT₄: serotonin 4 receptor; 5-HTT: serotonin transporter; CpG: CpG site; TSS: transcription start site; SLE: stressful life events; PBI: parental bonding inventory; BDI: Beck’s depressive index; GAD10: generalized anxiety disorder 10-item; CATS: childhood abuse trauma scale; HAMD6: Hamilton depressive rating scale 6; PSS: perceived stress scale; CpG_LV: latent variable including all CpG methylation values

Fig. 2

Associations between peripheral SLC6A4 methylation and brain 5-HTT binding (a) or 5-HT₄ binding (b). Blue dashed boxes depict the SLC6A4 CpG sites and the covariates included in the model. The light blue boxes indicate the CpG and covariate effects on the latent variable (5-HTT_LV or 5-HT_4LV). Dark grey boxes to the right represent the observed 5-HTT or 5-HT₄ binding in the brain regions of interest. β values refer to the parameter estimates; they are reported either with their respective p-values or with their 95% confidence intervals. Dashed arrows connecting brain regions show interregional correlations, while dashed circles on the brain regions show error estimates. For representation purposes, PET and MR scanner covariates are not reported in the a and b models. Similarly, although included in the 5-HT₄ latent variable model, 5-HTTLPR/rs25531 and BDNF rs6265 genotypes are not reported in (b). Scatter plots in c and d depict the relation between SLC6A4 methylation and 5-HTT_LV or 5-HT_4LV in healthy controls (c, d), while the relation between SLC6A4 methylation and 5-HT₄ binding in patients with MDD is shown in (e)

Fig. 3

Associations between peripheral TPH2 DNA methylation and brain 5-HTT binding a or 5-HT₄ binding b in the healthy cohort. Orange dashed boxes to the left depict the TPH2 CpG sites and the covariates included in the model. Rs45706210 stands for TPH2 rs45706210 G/T SNP. For representation purposes, PET and MR scanner covariates are not reported in the a and b models. Similarly, although included in the 5-HT₄ latent variable model, 5-HTTLPR/rs25531 and BDNF rs6265 genotypes are not reported in (b). Scatter plots in c and d depict the relation between TPH2 methylation and 5-HTT_LV or 5-HT_4LV in healthy controls (c, d), while the relation between TPH2 methylation and 5-HT₄ binding in patients with MDD is showed in (e)