Abstract
SLC6A4, encoding serotonin transporter (5-HTT), is a key molecule to elucidate the pathophysiology of psychiatric disorders because it regulates the concentration of serotonin, which is related to emotional behavior, in the brain. We have previously shown the promoter hypermethylation of SLC6A4 in the affected monozygotic twin discordant for bipolar disorder (BD). We also confirmed hypermethylation of specific two CpG sites (named CpG3 and CpG4) in lymphoblastoid cell lines (LCLs) and postmortem brains of patients with BD. However, the sample size in the previous study was relatively small, and LCLs have the possibility to be subjected to the artificial DNA methylation changes. In the present study, we tested DNA methylation levels of CpG3 and CpG4 in the SLC6A4 promoter using genomic DNA of peripheral blood cells (PBCs) of healthy controls (n = 454) and patients with BD (n = 447) from the Japanese population. We also examined DNA methylation levels of SLC6A4 promoter in schizophrenia (SZ, n = 411). In addition, we analyzed the relationship between DNA methylation level and genotype of 5-HTTLPR (5-HTT linked polymorphic region), which is a known functional DNA polymorphism of SLC6A4 promoter. As a result, we found a significant higher DNA methylation at CpG3 in patients with male BD and male SZ harboring particular L alleles compared to male controls. To examine the functional significance of DNA methylation at CpG3, we performed the luciferase reporter assay using in vitro methylated constructs and found that DNA methylation of CpG3 suppressed the transcriptional activity. Finally, using MRI images, we found a significant positive relationship between methylation level at CpG3 and the relative volume of right amygdala superficial region. These results suggest that hypermethylation at the CpG3 site has pathophysiological consequence such as down-regulation of transcription and volume change of the amygdala.