Allelic Expression Imbalance of Human mu Opioid Receptor (OPRM1) Caused by Variant A118G

Abstract

As a primary target for opioid drugs and peptides, the mu opioid receptor (OPRM1) plays a key role in pain perception and addiction. Genetic variants of OPRM1 have been implicated in predisposition to drug addiction, in particular the single nucleotide polymorphism A118G, leading to an N40D substitution, with an allele frequency of 10 -32%, and uncertain functions. We have measured allele-specific mRNA expression of OPRM1 in human autopsy brain tissues, using A118G as a marker. In 8 heterozygous samples measured, the A118 mRNA allele was 1.5-2.5-fold more abundant than the G118 allele. Transfection into Chinese hamster ovary cells of a cDNA representing only the coding region of OPRM1, carrying adenosine, guanosine, cytidine, and thymidine in position 118, resulted in 1.5-fold lower mRNA levels only for OPRM1-G118, and more than 10-fold lower OPRM1 protein levels, measured by Western blotting and receptor binding assay. After transfection and inhibition of transcription with actinomycin D, analysis of mRNA turnover failed to reveal differences in mRNA stability between A118 and G118 alleles, indicating a defect in transcription or mRNA maturation. These results indicate that OPRM1-G118 is a functional variant with deleterious effects on both mRNA and protein yield. Clarifying the functional relevance of polymorphisms associated with susceptibility to a complex disorder such as drug addiction provides a foundation for clinical association studies.Drug addiction is a complex disorder with a strong genetic component (1, 2). Serving as a primary target for opioid drugs and peptides, the mu opioid receptor (OPRM1) mediates the effects of morphine and heroin (3, 4). By impinging on dopaminergic pathways, OPRM1 also plays a role in addiction to other drugs of abuse, such as cocaine, nicotine, and alcohol (5, 6).Because of its central role in drug addiction, numerous studies have addressed potential contributions of polymorphisms in the gene encoding OPRM1 to addiction susceptibility (7,8). Among multiple single nucleotide polymorphisms (SNPs) 2 in OPRM1, C17T (A6V), and A118G (N40D) are well studied nonsynonymous SNPs located at the N terminus of the receptor. In particular, A118G, with an allele frequency of 10 -32% in different ethnic groups (9), has been associated with susceptibility to heroin, nicotine, and alcohol addiction (10 -12). However, other studies have failed to corroborate these associations (13, 14), possibly as a result of population admixture.The A118G polymorphism has also been linked to differences in pharmacological properties of OPRM1. In a Swedish population, the G118 variant was shown to correlate with poor response to naltrexone in the treatment of alcoholism (15). Moreover, OPRM1-antagonist naloxone elicited an increased cortisol response in individuals with a G118 allele (16,17). In transfected cells, OPRM1-Asp 40 was reported to have 3-fold higher affinity for ␤-endorphin than OPRM1-Asn 40 (18), suggesting a gain of function, but subsequent studies have failed to corroborate these results...

Endogenous Opioid Blockade and Impulsive Responding in Alcoholics and Healthy Controls

“…Our model raises the possibility that NTX reduces impulsive choice in proportion to the degree of k-receptor mediated effect relative to m-receptor mediated effect. This notion is supported by data showing that people with the A118G m-receptor polymorphism show reduced m-receptor expression (Zhang et al, 2005), and more effective NTX-mediated abstinence from alcohol (Oslin et al, 2003). However, our small sample sizes make it difficult to conclusively attribute the impulsive choice differences we find between the AA and CS groups to propensity to alcoholism per se, or other related factors, such as depression.…”

“…Given that NTX acts at both m-and k-opioid receptors, which exert opposing effects on forebrain DA release (Spanagel et al, 1992;Herz and Spangel, 1995;Margolis et al, 2006), differential effects of NTX on DA levels could be due to differences in relative m-and k-mediated effects of NTX, as depicted in Figure 6. Relative m-receptor to k-receptor blockade effects would be expected to differ in subjects with low circulating levels of endogenous m-receptor ligands, as is the case with alcoholics and their offspring (Govoni et al, 1983;Vescovi et al, 1992;del Arbol et al, 1995;Dai et al, 2005), or in subjects with low levels of m-receptor expression, as is found in subjects with low frontal DA levels (Berthele et al, 2005) owing to their catechol-O-methyltransferase genotype (Meyer-Lindenberg et al, 2005), and in subjects with the A118G polymorphism of the m-receptor (Zhang et al, 2005). This latter polymorphism has also shown a functional differentiation in the therapeutic response to NTX (Oslin et al, 2003), and has distinguished alcoholics from non-alcoholics in some populations (Bart et al, 2005).…”

Altered Levels of Basal Cortisol in Healthy Subjects with a 118G Allele in Exon 1 of the Mu Opioid Receptor Gene

“…These cells were chosen because, in the mu opioid receptor, amino-acid position 40 is a putative site of glycosylation. Further, a recent report on human receptors harvested from post-mortem brain tissue has found that some of the functional effects seen by Bond et al may be related to a decrease in mu opioid receptor mRNA expression in subjects with a variant 118G allele (Zhang et al, 2005). Other studies have also identified reduced receptor expression in transfected cell lines of receptors encoded by the 118G allele, a finding also noted in our ongoing studies (Beyer et al, 2004;Kroslak et al, 2003).…”

Allelic expression of serotonin transporter (SERT) mRNA in human pons: lack of correlation with the polymorphism SERTLPR

“…We have developed a robust procedure for allele-specific measurement of mRNA expression using a fluorescence-based primer extension assay to quantify real-time PCR amplification. 51,52 We have successfully applied this approach to identifying functional cis-acting polymorphisms in MDR1 53 and OPRM. 52 In the present study, we used this method to measure AEI for SERT mRNA in 48 postmortem pons tissues sections from individuals heterozygous for a marker SNP (rs1042173) in the 3 0 UTR of SERT mRNA.…”

“…51,52 We have successfully applied this approach to identifying functional cis-acting polymorphisms in MDR1 53 and OPRM. 52 In the present study, we used this method to measure AEI for SERT mRNA in 48 postmortem pons tissues sections from individuals heterozygous for a marker SNP (rs1042173) in the 3 0 UTR of SERT mRNA. Although we have detected a limited degree of allelic variations in mRNA expression, this did not correlate with the SERTLPR promoter polymorphism.…”