Neuropsychopharmacology (Link to full-length article)

Author(s): Robert A Moyer, Danxin Wang, Audrey C Papp, Ryan M Smith, Linda Duque, Deborah C Mash and Wolfgang Sadee

Abstract

The dopamine receptor D2 (encoded by DRD2) is implicated in susceptibility to mental disorders and cocaine abuse, but mechanisms responsible for this relationship remain uncertain. DRD2 mRNA exists in two main splice isoforms with distinct functions: D2 long (D2L) and D2 short (D2S, lacking exon 6), expressed mainly postsynaptically and presynaptically, respectively. Two intronic single-nucleotide polymorphisms (SNPs rs2283265 (intron 5) and rs1076560 (intron 6)) in high linkage disequilibrium (LD) with each other have been reported to alter D2S/D2L splicing and several behavioral traits in human subjects, such as memory processing. To assess the role of DRD2 variants in cocaine abuse, we measured levels of D2S and D2L mRNA in human brain autopsy tissues (prefrontal cortex and putamen) obtained from cocaine abusers and controls, and genotyped a panel of DRD2 SNPs (119 abusers and 95 controls). Robust effects of rs2283265 and rs1076560 on reducing formation of D2S relative to D2L were confirmed. The minor alleles of rs2283265/rs1076560 were considerably more frequent in Caucasians (18%) compared with African Americans (7%). Also, in Caucasians, rs2283265/rs1076560 minor alleles were significantly overrepresented in cocaine abusers compared with controls (rs2283265: 25 to 9%, respectively; p=0.001; OR=3.4 (1.7-7.1)). Several SNPs previously implicated in diverse clinical association studies are in high LD with rs2283265/rs1076560 and could have served as surrogate markers. Our results confirm the role of rs2283265/rs1076560 in D2 alternative splicing and support a strong role in susceptibility to cocaine abuse.

Commentary

by Wolfgang Sadee
Numerous clinical studies have implicated several genetic variants in the dopamine D₂ receptor (DRD2) as risk factors in CNS disorders or as potential biomarkers predictive of the response to antipsychotic drugs.  We previously found that two intronic DRD2 single nucleotide polymorphisms (SNPs), both in high linkage disequilibrium (LD) with each other, alter the splicing of D₂ mRNA, resulting in higher formation of the long D_2L isoform at the expense of the short D_2S isoform, the latter predominantly located presynaptically. In the present work, we first replicated the finding that the two intronic SNPs indeed change the D₂ mRNA splicing pattern in human brain tissues and then investigated if the SNPs are associated with cocaine abuse.  Previous work has shown that the D_2S physically interacts with the dopamine transporter (DAT), a primary target of cocaine, thus facilitating the functional expression of DAT in the presynaptic membrane. Given the presynaptic receptor function of D_2S, an effect on cocaine abuse appeared likely, and indeed was suggested by previous studies using other DRD2 SNPs.  Our clinical sample was derived from the Miami Dowd County Brain Endowment Bank, with controls and cocaine overdose subjects proven to have been heavy cocaine abusers.  The result was quite remarkable: the intronic SNPs both scored with odds ratios exceeding 3 (threefold greater risk to die of a cocaine overdose) in an allele test.  Moreover, homozygous carriers appeared to have even higher risk but the sample size was too small to yield sufficient statistical power.  This odds ratio represents a very substantial risk that needs to be validated in follow-up studies.  As the two intronic SNPs are in strong LD with a series of additional DRD2 SNPs, we searched the literature for clinical associations published previously with other DRD2 SNPs - presumably surrogate markers for the intronic SNPs.  A majority of these studies indeed found significant clinical associations with a variety of phenotypes, involving SNPs in high LD with our intronic SNPs, supporting the notion that they have substantial clinical relevance.  Our study leaves a number of questions open, for example, do the DRD2 splicing variants facilitate the initiation of cocaine abuse or play a main role in promoting severe abuse, with death by overdose a strong risk. An obvious follow-up study should address whether the intronic DRD2 SNPs interact with functional variants in DAT, a study now ongoing in our laboratory.