Functional impact of a single nucleotide polymorphism in the OPRD1 promoter region

The δ-opioid receptor mediates rewarding effects of many substances of abuse. We reported an increased frequency of the minor G-allele of single nucleotide polymorphism (SNP) rs569356 [the only variant identified so far in the promoter region of the δ-opioid receptor gene (OPRD1)] in subjects with opioid dependence. In this study, we examined the functional significance of this variant. OPRD1 promoter region harboring SNP rs569356 was amplified by polymerase chain reaction (PCR) and inserted into a firefly luciferase reporter vector. HEK293 cells were co-transfected with these constructs and a renilla luciferase vector to control for transfection efficiency. Expression of firefly luciferase (driven by the OPRD1 promoter) was measured by a dual luciferase reporter assay and normalized by renilla luciferase expression. Moreover, alleles altering expression were further assessed for binding of human brain nuclear proteins by electrophoretic mobility shift assay (EMSA). The minor G-allele was associated with significantly greater expression levels of firefly luciferase than the major A-allele of SNP rs569356 (P=0.003). EMSA also showed specific gel shift bands, suggesting that SNP rs569356 is situated in the binding site of potential transcription factors. These results suggest that the minor G-allele of SN