Methylation of the SLC6a2 gene promoter in major depression and panic disorder

Abstract

Reduced function of the noradrenaline transporter (NET) has been demonstrated in patients with major depressive disorder (MDD) and panic disorder. Attempts to explain NET dysfunction in MDD and panic disorder by genetic variation in the NET gene SLC6a2 have been inconclusive. Transcriptional silencing of the SLC6a2 gene may be an alternative mechanism which can lead to NET dysfunction independent of DNA sequence. The objective of this study was to characterise the DNA methylation state of the SLC6a2 gene promoter in patients with MDD and panic disorder. SLC6a2 promoter methylation was also analysed before and after antidepressant treatment. This study was performed with DNA from blood, using bisulphite sequencing and EpiTYPER methylation analyses. Patients with MDD or panic disorder were not found to differ significantly from healthy controls in the pattern of methylation of the SLC6a2 gene promotor. While significant correlations between methylation levels at some CpG sites and physiological measures were identified, overall the variation in DNA methylation between patients was small, and the significance of this variation remains equivocal. No significant changes in SLC6a2 promoter methylation were observed in response to antidepressant treatment. Further in-depth analysis of alternative mechanisms of transcriptional regulation of the SLC6a2 gene in human health and disease would be of value.

Figure 1. CpG methylation in Region 1 of the SLC6a2 promoter in humans.

The methylation status of SLC6a2 promoter Region 1 (-515 bp to -225 bp) in leukocytes was analysed by bisulphite sequencing in 4 healthy subjects compared to 4 subjects with MDD or panic disorder (#1-4). A) A schematic representation of Region 1. The individual CpG sites analysed in Region 1 are indicated as enlarged boxes with their positioning relative to the transcription start site (+1) indicated with an arrow. B) Bisulphite sequencing data for MDD, panic disorder and healthy controls. Each row of boxes represents a single cloned allele, and each box represents a single CpG dinucleotide. Black boxes indicate methylated cytosine residues, white boxes indicate unmethylated cytosine residues. For healthy subjects #1-4, 10 individual clones were sequenced. For MDD subjects #1-4, between 7 and 12 individual clones were sequenced. For panic disorder subjects #1-4, between 5 and 9 individual clones were sequenced for each subject.

Figure 2. CpG methylation in Region 2 of the SLC6a2 promoter in humans.

The methylation status of the SLC6a2 promoter Region 2 (-180 bp to +167 bp) was analysed by bisulphite sequencing in 4 healthy subjects compared to 4 subjects with MDD and panic disorder. A) A schematic representation of Region 2. The individual CpG sites analysed in Region 2 are indicated as enlarged boxes with their positioning relative to the transcription start site (+1) indicated with an arrow. B) Bisulphite sequencing data are presented as in Figure 1. For healthy subjects #1-4, 10 individual clones were sequenced. For MDD subjects #1-4, between 8 and 9 individual clones were sequenced. A different MDD patient (#5) replaced sample #3 due to insufficient sample. For panic disorder subjects #1-4, between 8 and 10 individual clones were sequenced.

Figure 3. CpG methylation of Region A of the human SLC6a2 promoter.

Representation of the methylation percentages for CpG sites in Region A of the SLC6a2 gene determined by EpiTYPER analysis. CpG site. CpG sites are numbered 5’- 3’ along the X-axis, with percentage methylation on the Y-axis. Usable data could not be obtained for all CpG sites for all samples. All groups exhibited a similar methylation profile across the region, and no difference between groups for the region as a whole was determined to be significant by two-way ANOVA.

Figure 4. CpG methylation of Region B of the human SLC6a2 promoter.

Representation of the methylation percentages for CpG sites in Region B of the SLC6a2 gene determined by EpiTYPER analysis. CpG sites are numbered 5’- 3’ along the X-axis, with percentage methylation on the Y-axis. Usable data could not be obtained for all CpG sites for all samples. The overall methylation of Region B in subjects with MDD was determined to be significantly greater than all other groups (p<0.05).

Figure 5. Analysis of methylation in Region A pre- and post-SSRI treatment.

Representation of the methylation percentages for CpG sites in Region A of the NET gene determined by EpiTYPER analysis in human leukocytes pre- and post-SSRI treatment. CpG sites are numbered 5’- 3’ along the X-axis, with percentage methylation on the Y-axis. Usable data could not be obtained for all CpG sites for all samples. Samples pre- and post-SSRI treatment exhibited a similar methylation profile across the region, and no differences in average methylation of CpG sites in response to SSRI treatment were determined to be significant by paired t-test. *Statistically significant site-specific difference in methylation with SSRI treatment.

Figure 6. Analysis of methylation in Region B pre- and post-SSRI treatment.

Representation of the methylation percentages for CpG sites in Region B of the NET gene determined by EpiTYPER analysis in human leukocytes pre- and post-SSRI treatment. CpG sites are numbered 5’- 3’ along the X-axis, with percentage methylation on the Y-axis. Usable data could not be obtained for all CpG sites for all samples. Samples pre- and post-SSRI treatment exhibited no significant difference in methylation profile across the region.