Genetic and epigenetic analysis of the serotonin 2A receptor in the context of cocaine abuse

Abstract

Despite more than 2 million American cocaine users monthly, there is no approved drug for treating cocaine use disorder. Cocaine use disorder has a multifactorial aetiology, including both genetic and environmental factors. Both cocaine use and genetic variations demonstrably alter DNA methylation and gene expression in the brain in a complex manner. How these factors interact in the context of cocaine abuse in humans is unknown. We propose that we can identify potential drug targets for treating cocaine use disorders by examining genetic, epigenetic, and expression changes in the brains of individuals that abused cocaine. In this study, we identified the interaction between the epigenetics changes (DNA CpG methylation) and genetic variants (SNPs) in the HTR2A gene in the context of cocaine addiction by using brain tissue collected from individuals that overdosed on cocaine (N = 14) and healthy matched controls (N = 16). We generated DNA CpG methylation profiles in eight regions of HTR2A harbouring frequent SNPs, measuring both allelic and total methylation, and compared these methylation profiles with HTR2A mRNA expression. Furthermore, we examined the influence of common variants rs6311 and rs6313 on cocaine abuse, methylation, and gene expression. We found evidence that rs6311 regulates HTR2A methylation, consistent with earlier studies. Furthermore, the minor alleles for rs6311 and rs6313 are associated with significantly increased expression of a splice isoform in which exon 2 is truncated in both cocaine and control samples. These results reveal specific roles for HTR2A in the context of cocaine abuse, highlighting opportunities to modulate this target for treating cocaine use disorder.

Keywords: DNA methylation; HTR2A; cocaine addiction; genetics; rs6311; rs6313; serotonin; single nucleotide polymorphisms.

Figure 1.

Boxplot of the methylation score of rs6311 genotypes. The methylation score for A/A homozygotes was significantly higher than methylation scores for G/G and A/G genotypes (* p-value < .05).

Figure 2.

Relative expression of truncated exon 2 splice isoform to total HTR2A expression in cocaine group vs. control group (left panel) and the total expression of HTR2A in the cocaine group vs. control group, normalized to β-actin. Note: higher Ct values indicate lower expression.

Figure 3.

Relative expression of truncated exon 2 splice isoform to total HTR2A expression in cocaine and control groups with respect to rs6311 genotypes (left panel) the total expression of HTR2A in cocaine group vs. control group with respect to rs6311 genotypes, normalized to β-actin. Note: higher Ct values indicate lower expression.

Figure 4.

Relative percentage of truncated exon 2 splice isoform (as estimated by AUC), versus E2⁺ and E2⁻ isoforms with respect to rs6311 genotypes. A/A homozygotes express significantly more of the E2^tr isoform relative to G/G homozygotes and heterozygotes (*p < 0.05).