Differential expression and transcription factor binding associated with genotype at a pharmacogenetic variant in OPRD1

ABSTRACT

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 (OPRD1), 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 in vitro. 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 p < .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.

KEYWORDS:

• OPRD1; delta-opioid receptor; pharmacogenetics

Disclosure Statement

The authors report no conflict of interest.

Additional information

Funding

R.C.C. was supported by NIDA K01 DA036751. R.C.C., W.H.B, and G.A.D. were supported by NIDA R01 DA044015. R.C.C. and W.H.B. were supported by a Pennsylvania CURE grant. B.C.R. was supported, in part, by a 2017 NARSAD Young Investigator Grant (#26634) from the Brain and Behavior Research Foundation as the Patrick A. Coffer Investigator, funding for which was generously provided by Ronald and Kathy Chandonais; National Institute on Drug Abuse [K01 DA036751, R01 DA044015]; Pennsylvania Department of Health [CURE].