Redefinition of the human kappa opioid receptor gene (OPRK1) structure and association of haplotypes with opiate addiction

Abstract

The kappa opioid receptor (KOR) plays a role in stress responsivity, opiate withdrawal and responses to cocaine. KOR activation by its endogenous ligand dynorphin A(1-17) decreases basal and drug-induced striatal levels of dopamine. The complete structure of the human KOR gene (hOPRK1) has not been previously determined. This study: (i) characterized the genomic structure of the hOPRK1 gene; (ii) identified single nucleotide polymorphisms (SNPs) in the hOPRK1 gene; and (iii) investigated possible associations of these variants with vulnerability to develop heroin addiction. Analysis of 5'-RACE cDNA clones revealed the presence of a novel exon 1 ranging in length from 167 to 251 nucleotides in the 5' 5'-untranslated region of the hOPRK1 mRNA. We found that the hOPRK1 gene has four major exons and three introns, similar to rodent OPRK1 genes. Direct sequencing of amplified DNA containing all four exons and intron 1 of the hOPRK1 gene were evaluated for polymorphisms in 291 subjects (145 former heroin addicts and 146 controls). Twelve SNPs were identified, nine novel variants and three previously reported SNPs. Using logistic regression with opioid dependence as the dependent variable, the 36G>T SNP exhibited a point-wise significant association (P = 0.016) with disease status. The number of haplotypes seen in the three ethnic groups were nine, six and five for African-Americans, Caucasians, and Hispanics, respectively, with corresponding significance levels for differences in haplotype frequencies between cases and controls of P = 0.0742, 0.1015 and 0.0041. Combining ethnicities by Fisher's method yields an empirical significance level of P = 0.0020.

Fig. 1

Exon/intron organization of the 5’-UTR of the human OPRK1. The novel exon 1 identified is shown as striped. The human OPRK1-specific primers P2 and P4 were used in 5’-RACE PCR, and P5 and P7 were used in 3’-RACE PCR. Primers P3/P4 were used for PCR amplification and sequencing of exon 2 and its flanking regions. Exons and introns have been numbered to reflect this novel structure of the hOPRK1 gene.

Fig. 2

(a) Nucleotide sequence of 5’-UTR and relative positions of 5’-ends of the human OPRK1 cDNA-RACE clones. Positions of the human OPRK1 exon 1, intron 1, and exon 2 (partial) are shown in comparison with the exon 1/intron 1/exon 2 structure of the mouse OPRK1 gene [32,41]. The exonic regions are in uppercase. The ATG translation initiation codons in the human and mouse OPRK1 mRNA are underlined. The positions of three major transcription start sites are designated by ▼ The location of the SNPs are designated in bold and marked with an asterisk. The functional transcription binding sites for murine Ikaros-1 and E-box are shown as boxed nucleotides [28]. Alignment of human and mouse OPRK1 gene sequences was created using the Multiz program at the UCSC website [52] (www.genome.ucsc). (b) The 3’-end and partial sequence of 3’-UTR (capital letters) of the human OPRK1 with downstream genomic sequence (lower case letters) are shown in comparison with the 3’-end of the mouse OPRK1 mRNA [38] (GenBank Accession number AF490606). Potential polyadenylation sites are shown in bold. Numeration in the sequences designates position of nucleotides by starting from the first nucleotide followed the TGA codon. The sequence of the novel 5’-UTR of the OPRK1 mRNA has been deposited in GenBank (accession number AY466378).