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 invivo
brain levels of serotonin transporter (5-HTT) and serotonin 4 receptor (5-HT4) in a cohort of healthy individuals
(N = 254) and, for 5-HT4, 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.