The association of DAT gene methylation with striatal DAT availability in healthy subjects

The dopamine transporter (DAT) is a sodium chloride-dependent transmembrane protein on the presynaptic dopaminergic nerve terminal [1], regulating levels of dopamine by reuptake [2, 3]. As DAT binding is correlated with the density of dopaminergic neurons [4], decreased DAT uptake represents dopaminergic neurodegeneration such as PD [4], a neurodegenerative disease with death of dopaminergic neurons in the substantia nigra pars compacta [5, 6]. The most sensitive imaging techniques for the diagnosis of neurodegeneration are positron emission tomography (PET) and single photon emission computed tomography (SPECT) by using ligands that report nigrostriatal dopaminergic function [3], which allow quantifying dopaminergic system in human brain [7].

Epigenetic change is a heritable phenotype change without alterations in the DNA sequence, such as DNA methylation. DNA methylation is an epigenetic process that adds methyl groups to cytosines of the genome, mainly in CpG sites [8]. As DNA methylation inhibits gene expression by preventing transcription factors from binding to DNA [9], DNA methylation is inversely associated with protein expression [10]. Functioning of nigrostriatal dopaminergic neurons is influenced by DAT, and genetic variations in the gene encoding DAT contribute to differences in reward processing [11, 12]. However, in a previous study by Wiers et al., striatal DAT availability measured by ¹¹C-cocaine PET did not show any significant association with DAT methylation of peripheral blood in healthy subjects [13], which is the only report that investigated the association between them. Therefore, we aimed to investigate the action of DAT methylation on DAT protein expression measured by PET and reward scales measured with the Generalized Reward and Punishment Expectancy Scales (GRAPES) [14].

Thirty-five healthy males, with an age range between 20 and 30 years, and a mean age of 24.4 ± 2.7 years, were included in this study. Coordinates and the percentage of methylated alleles (%) for all SLC6A3 CpG sites are presented in Table 1. The mean percentages of methylation (%) were 9.9 (cluster A), 76.1 (cluster B), 91.6 (cluster C), 75.1% (cluster D), respectively. The mean percentage of methylation (%) from each cluster did not show any association each other (Fig. 1). The mean percentage of methylation (%) from each cluster was not associated with DAT availability of the ventral striatum, showing a trend of positive correlation between that from cluster C and DAT availability of the ventral striatum without correction (rho = 0.3712, p = 0.0281), which was not significant after correction for multiple comparison. In addition, the mean percentage of methylation (%) from cluster C was positively correlated with GRAPES A: reward expectancy scale (rho = 0.7178, p < 0.0001) (Figs. 2, 3).

In this study, the degrees of methylation from cluster C (Exon 7) of DAT in peripheral blood showed a trend of positive correlation with DAT availability of the ventral striatum and were significantly correlated with reward scales of GRAPES A: reward expectancy scale.

This is the first study that investigated the association between the degree of methylation of DAT and DAT availability of PET in healthy subjects. Previously, the association of methylation of DAT gene with DAT availability was investigated in patients with attention-deficit hyperactivity disorder (ADHD) [13], which is the only study of DAT methylation and PET. Although methylation of DAT gene was not significantly different between patients with ADHD and healthy subjects, DAT availability measured by ¹¹C-cocaine PET was negatively correlated with the degree of DAT methylation in patients with ADHD, not in healthy subjects [13]. This correlation was significant in both caudate nucleus, putamen, and ventral striatum [13]. However, the underlying mechanisms of insignificancy between DAT availability and DAT methylation in healthy subjects remained unclear. In subjects with alcohol dependency, the effect of DAT methylation was investigated with reward processing [10] and depressive symptoms [17] using functional magnetic resonance imaging. DAT methylation in promoter region predicted activation of nucleus accumbens during the anticipation of high and low loss in healthy subjects, not in subjects with alcohol dependency [10]. In addition, subjects with alcohol dependency showed that activity of amygdala to alcohol cue was correlated with DAT methylation only those with low depression scores [17]. Also, higher DAT methylation predicted the relapse of alcohol dependency [17].

Epigenetic change is a heritable phenotype change without alterations in the DNA sequence, such as DNA methylation. DNA methylation is a process that adds methyl groups to cytosines of the genome, mostly in CpG sites [8]. DNA methylation inhibits gene expression by preventing transcription factors from binding to DNA [9]. Therefore, DNA methylation has shown to be inversely associated with protein expression [10]. In rodent, DAT methylation was negatively associated with striatal DAT expression [18]. In humans, only one study reported the association between DAT methylation and DAT availability measured by ¹¹C-cocaine PET as explained above [13]. Inconsistent with the study by Wiers et al., we demonstrated a trend of positive association between DAT methylation and DAT availability measured by ¹⁸F-FP-CIT PET. In addition, the mean percentage of methylation (%) from cluster C was strongly correlated with GRAPES A: reward expectancy scale, similar to the study by Muench et al., which predicted striatal responses during reward processing by DAT methylation in healthy subjects, not in alcohol-dependent individuals [10]. DAT methylation in alcohol-dependent individuals [10], or ADHD [13], showed the difference with that in healthy subjects. Therefore, DAT methylation might have an implication on treatment of alcohol dependency or ADHD.

There are several points that should be considered in these results. First, it is unclear that whether DAT methylation obtained from peripheral blood has an association with DAT methylation of brain. In this regard, Wiers et al. proved the significant correlation between DAT methylation of substantia nigra and DAT methylation from peripheral blood both in healthy subjects and patients with ADHD, however, showing moderate correlation in both promotor region (r = 0.44), and CpG site 230 (r = 0.53) of healthy subjects [13]. Second, the mean percentage of methylation was calculated from each cluster and considered as the degree of DAT methylation level. However, the degree of DAT methylation may be different within each cluster, and the region of each cluster may be different. Third, the radiopharmaceuticals used in measurement of DAT availability were different in each study. Theoretically, as DNA methylation inhibits or regresses the binding of transcription factors [9], DNA methylation might be negatively associated with protein expression [10]. However, at least with regard to DAT, this association is unclear until now as we showed a trend of positive association between DAT methylation and DAT availability, and Wiers et al. showed no significant association [13]. The mechanisms underlying these findings might be complex and likely vary across genes. Also, as a small number of subjects were included in this study, further studies of a larger number of subjects with a broader age range should be carried out in the future.

DAT methylation from peripheral blood showed a trend of positive correlation with DAT availability of ventral striatum in healthy subjects; however, it was not significant after correction for multiple comparison. The degrees of methylation from cluster C of DAT in peripheral blood were significantly correlated with reward scales of GRAPES A: reward expectancy scale. The association between DAT methylation and DAT expression needs to be investigated further.