Differential expression and transcription factor binding associated with genotype at a pharmacogenetic variant in .

Background: The functional mechanism is unknown for many genetic variants associated with substance use disorder phenotypes. Rs678849, an intronic variant in the delta-opioid receptor gene (), has been found to predict regional brain volume, addiction risk, and the efficacy of buprenorphine/naloxone in treating opioid use disorder. The variant has also been implicated as an expression quantitative trait locus (eQTL) for several genes.

Objectives: The objective of this study was to identify functional differences between the two alleles of rs678849 . We hypothesized that the two alleles of rs678849 would have different effects on transcriptional activity due to differential interactions with transcription factors.

Methods: 15bp regions containing the C or T alleles of rs678849 were cloned into luciferase constructs and transfected into BE(2)C neuroblastoma cells to test the effect on transcription. Electrophoretic mobility shift assays (EMSA) using nuclear lysates from BE(2)C cell or human postmortem medial prefrontal cortex were used to identify proteins that differentially bound the two alleles.

Results: At 24 hours post-transfection, the C allele construct had significantly lower luciferase expression than the T allele construct and empty vector control (ANOVA < .001). Proteomic analysis and supershift assays identified XRCC6 as a transcription factor specifically binding the C allele, whereas hnRNP D0 was found to specifically bind the T allele.

Conclusion: These functional differences between the C and T alleles may help explain the psychiatric and neurological phenotype differences predicted by rs678849 genotype and the potential role of the variant as an eQTL.