Methylation Patterns of the HTR2A Associate With Relapse-Related Behaviors in Cocaine-Dependent Participants

Abstract

Relapse during abstinence in cocaine use disorder (CUD) is often hastened by high impulsivity (predisposition toward rapid unplanned reactions to stimuli without regard to negative consequences) and high cue reactivity (e.g., attentional bias towards drug reward stimuli). A deeper understanding of the degree to which individual biological differences predict or promote problematic behaviors may afford opportunities for clinical refinement and optimization of CUD diagnostics and/or therapies. Preclinical evidence implicates serotonin (5-HT) neurotransmission through the 5-HT_2A receptor (5-HT_2AR) as a driver of individual differences in these relapse-related behaviors. Regulation of 5-HT_2AR function occurs through many mechanisms, including DNA methylation of the HTR2A gene, an epigenetic modification linked with the memory of gene-environment interactions. In the present study, we tested the hypothesis that methylation of the HTR2A may associate with relapse-related behavioral vulnerability in cocaine-dependent participants versus healthy controls. Impulsivity was assessed by self-report (Barratt Impulsiveness Scale; BIS-11) and the delay discounting task, while levels of cue reactivity were determined by performance in the cocaine-word Stroop task. Genomic DNA was extracted from lymphocytes and the bisulfite-treated DNA was subjected to pyrosequencing to determine degree of methylation at four cytosine residues of the HTR2A promoter (-1439, -1420, -1224, -253). We found that the percent methylation at site -1224 after correction for age trended towards a positive correlation with total BIS-11 scores in cocaine users, but not healthy controls. Percent methylation at site -1420 negatively correlated with rates of delay discounting in healthy controls, but not cocaine users. Lastly, the percent methylation at site -253 positively correlated with attentional bias toward cocaine-associated cues. DNA methylation at these cytosine residues of the HTR2A promoter may be differentially associated with impulsivity or cocaine-associated environmental cues. Taken together, these data suggest that methylation of the HTR2A may contribute to individual differences in relapse-related behaviors in CUD.

Keywords: A-1438G; HTR2A; attentional bias; cocaine use disorder; impulsivity; methylation; rs6311.

Figure 1

Cocaine users demonstrate hypomethylation of the HTR2A promoter versus healthy controls. (A) HTR2A gene is represented as the gray line and contains the promoter region, exon 1, exon 2, and the initial segment of the coding sequence of the gene. Transcription factor binding sites are represented in blue and green. CpG islands and SNPs are represented as red lines and transcription initiation sites are in pink. Translation start site is represented as a black line. The silencer sequence is annotated in tan and the coding sequence is in maroon. Comparison of percent methylation between healthy controls and cocaine users for (B) CpG site -1439, (C) -1420, (D) -1224 and (E) -253. *p < 0.05 versus healthy controls.

Figure 2

Levels of impulsivity correlate with percent methylation at site -1224 of the HTR2A promoter in cocaine users. (A) Impulsivity was determined using the Barratt Impulsiveness Scale (BIS-11). *p < 0.05 versus healthy controls. (B) Correlations and p values of percent methylation of the HTR2A promotor with total BIS scores at specific sites within the HTR2A promoter are represented in the table. Graphical representation of the correlations between percent methylation of the HTR2A promotor with total BIS-11 scores at specific sites within the HTR2A promoter for healthy controls (C–F) and cocaine users (G–J). The number of individuals used in each correlation are indicated on the bottom left of the graph.

Figure 3

Delay discounting rates correlate with percent methylation at site -1420 of the HTR2A promotor in healthy controls. (A) Impulsive choice was determined using the delay discounting task in healthy controls and cocaine users. *p < 0.05 versus healthy controls. (B) Correlations and p values of percent methylation of the HTR2A promoter with the delay discounting rates at specific sites within the HTR2A promoter are represented in the table. Graphical representation of the correlations between percent methylation of the HTR2A promotor and delay discounting rates at specific sites within the HTR2A promoter for healthy controls (C–F) and cocaine users (G–J). The number of individuals used in each correlation are indicated on the bottom left of the graph.

Figure 4

Attentional bias correlates with percent methylation at site -253 of the HTR2A promotor in cocaine users. Attentional bias was determined using the cocaine-word Stroop task in cocaine users. (A) Correlations and p values of percent methylation of the HTR2A promotor with mean reaction times (msec) at specific sites within the HTR2A promoter are represented in the table. (B–E) Graphical representation of the correlations between percent methylation of the HTR2A promotor and attentional bias at specific sites within the HTR2A promoter for cocaine users. The number of individuals used in each correlation are indicated on the bottom left of the graph.