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Opioid drugs are among the most commonly used and effective human analgesics. To date, the clinical benefits of opioid analgesics have not been fully realized due to substantial individual variations in the responses to opioids, insufficient drug dosing, and a high rate (up to 66%) of adverse events. As such, there is a substantial need to identify the genetic and molecular biological mechanisms that mediate individual responses to opioid therapy. Recent discoveries show that genetic variations in the μ-opioid receptor (OPRM1) gene locus play an essential role in inter-individual responses. The majority of genetic association studies have focused on the A118G polymorphism, which codes for a non-synonymous change in OPRM1 exon 1. In addition to the A118G polymorphism, another functional SNP (rs563649), which is located within an alternatively-spliced OPRM1 isoform (MOR-1K), has been identified. The MOR-1k isoform codes for 6TM OPRM1 isoforms that display excitatory rather than the inhibitory cellular effects, which are characteristic of the canonical 7TM isoforms. Thus, stimulation of the 6TM isoforms may engage the molecular mechanisms mediating opioid-dependent hyperalgesia, tolerance and dependence. Future clinical and basic studies that seek to identify the functional genetic variants within OPRM1 locus, and associated molecular mechanisms, will result in a better understanding of individual responses to opioid therapy and ultimately to the development new pharmacotherapeutics and diagnostic tools.
Opioid analgesics are the most widely used drugs to treat moderate to severe pain. Unfortunately, these agents produce significant individual side effects consisting of miosis, pruritus, sedation, nausea and vomiting, cognitive impairment, constipation, hypotension and life-threatening respiratory depression (Rowlingson and Murphy, 2000; Ready,2000; Inturrisi,2000; Goldstein,2002). Even when the correct clinical protocol is followed for the use of opioids, an effective and safe dose of an opioid for an individual patient can be difficult to predict, which reflects the considerable inter-individual variability in the clinical responses to opioid analgesics. In fact, the minimal effective analgesic concentration (MEAC) for opioids, such as morphine, pethidine, alfentanil and sufentanil, vary among patients by factors of 5 to 10(Glass,2000; Camu and Vanlersberghe,2002). As such, there is a substantial need to develop new biological markers that will provide valid and reliable predictions of individual responses to opioid therapy.
Results from animal and human studies suggest that variations in opioid analgesia and side-effect profiles are influenced by multiple factors, including genetic variability. Among genetic factors, allelic variants in the gene locus coding for μ-opioid receptor (MOR) have been suggested to play a critical role in the variation in the responses to MOR agonists(Thompson et al,2000; Duguay et al,2004; Klepstad et al,2004). MOR1 is the primary target of both endogenous and exogenous opioid analgesics, which mediates basal nociception as well as μ-opioid receptor agonists responses(Matthes et al,1996; Sora et al,1997;Uhl et al,1999;Edwards et al,2006). Both animal and human studies have indicated that reduced basal nociceptive sensitivity is associated with greater opioid analgesia(Mogil et al,1999),(Edwards et al,2006). Identified genetic polymorphisms in the human OPRM1 gene, which codes for MOR1, are the primary candidates for sources of clinically relevant variability in opiate sensitivity and baseline nociception(Mogil,1999;Uhl et al,1999;Han et al,2004). Several polymorphisms have been found in the promoter, coding and intron regions of the gene that are associated with pharmacological and physiological effects mediated by MOR1 stimulation (for review see(Lotsch and Geisslinger, 2005)). However, one of the most commonly investigated MOR genetic variants involves a single nucleotide polymorphism (rs1799971) in exon 1 of OPRM1, in which an adenine to guanine substitution exists at position 118 (A118G or 118A→G). This missense mutation causes a substitution of asparagine for aspartic acid at amino acid position 40 in the N-terminal extracellular domain, which corresponds to the loss of a putative glycosylation site (Bond et al, 1998). The functional molecular consequences of this non-synonymouse change has been widely studied, but has not been fully elucidated. Early studies reported that the 118G receptor displayed increased binding affinity and potency for endogenous ligands(Bond et al, 1998), though other studies failed to replicate this finding or found impaired signaling through the receptor (Befort et al, 2001; Beyer et al, 2004). Consistent with the latter findings, the majority of the human genetic association studies that have examined the responses to opioids show that carriers of the 118G variant allele demonstrate poor responses’ for opioids (Table 1), although the opposite or no effect have been reported as well(Lotsch and Geisslinger, 2005). Furthermore, in twin studies few functional non-synonymous OPRM1 mutations have also been identified (Table 2). It is unclear how common these rare familial mutations are in the human population and it is not known if they aggregate within specifc haplotypes. Since OPRM1 gene locus is situated within two large haploblocks, with one long functional halotype spanding the entire gene locus, the joint effect of several functional SNPs and interactions between them have been described(Shabalina et al,2009). Collectively, the current literature provides evidence for the existence of other functional SNPs within the OPRM1 gene locus in addition to A118G.
The MOR1 receptor is a member of G-protein-coupled receptors (GPCRs) family. It has an extracellular N-terminus and intracellular C-terminus, with seven membrane-spanning domains that comprise the binding pocket for exogenous drugs. MOR1 induces analgesia via pertussis toxin (PTX)-sensitive inhibitory G protein (Gαi/o), which inhibits cAMP formation and Ca2+ conductance and activates K+ conductance, leading to hyper-polarization of cells thereby, exerting an inhibitory effect(North,1986;Crain and Shen, 2000). The major form of OPRM1, also called MOR-1, is coded by exons 1, 2, 3 and 4, whereas exon 1 codes for first transmembrane domain and exon 2 and 3 code for the second through seventh transmembrane domains(Pasternak,2004). There is growing evidence from rodent studies for an important role of alternatively-spliced forms of OPRM1 in mediating opiate analgesia(Pasternak,2004). The synergistic activities of these splice variants has been proposed to explain the complex pharmacology of μ-opioids(Pasternak,2004). Though there is substantial discrepancy between the genomic organization of mouse OPRM1 and the genomic organization of human OPRM1, (see Unigene databases,(Pasternak,2004;Kvam et al, 2004;Pan;Doyle et al,2007)), there are human homologues for each, or at least majority of the mouse OPRM1 exons(Shabalina et al,2009). There are two common splicing patterns of OPRM1 that involve the C-terminus and N-terminus. C-terminus variants contain exons 1, 2 and 3 and code for all seven transmembrane domains, but differ structurally and functionally at the intracellular domain, a region important in signal transduction following receptor activation. There are also a number of variants that differ in their N-terminus, some of which encode for truncated receptors. All reported mouse N-terminus variants are initiated from exon 11. Exon 11 is located approximately 30 kb upstream of exon 1 and is under the control of a different upstream promoter, suggesting alternative regulation of transcription. Three of these variants are predicted to code for truncated receptors with only six transmembrane domains (6TM). Thus, the plausible biological role and functional significance of these truncated receptors represents an intriguing experimental and clinical question.
During the last few years, several human alternatively-spliced forms coding for 6TM receptor variants have been reported. First, a human MOR isoform MOR-3 has been cloned that is selectively activated by opiate alkaloids, but is insensitive to opioid peptides(Cadet et al, 2003). Unlike the mouse 6TM isoform, MOR-3 mRNA is lacking exon 11 and seems to use a promoter upstream to exon 2. At the functional level, COS-1 cells transfected with the MOR-3 receptor exhibited a dose-dependent release of nitric oxide (NO) following treatment with morphine, but not with DAMGO. Importantly, naloxone blocks the effect of morphine on COS-1 cells transfected with MOR-3(Cadet et al, 2003). Similar to the mouse 6TM receptor isoform, this isoform lacks an amino acid sequence of ~90 amino acids that constitute the extracellular N-terminal and the first transmembrane domain TMH1 and part of the first intracellular loop, but retains the ligand binding pocket that is distributed across conserved TMH2, TMH3, and TMH7 domains. Additionally, the MOR-3 isoform possesses unique intracellular C-terminal amino acid sequences that have been hypothesized to serve as coupling or docking domains required for constitutively expressed NO synthase (NOS) activation(Kream et al,2007). Northern blot and RT-PCR results reveal the expression of this MOR-3 variant in human vascular tissue, mononuclear cells, polymorphonuclear cells, and human neuroblastoma cells(Cadet et al, 2003). Following the first reports of the human 6TM MOR isoform MOR-3, there has been a increase in the number of identified and characterized OPRM1 isoforms that code for a 6TM receptor.
In our recent work, we applied new comparative genomic approaches that generated evidence that the human OPRM1 gene is orthologous to the mouse OPRM1 gene and is thus much more complex than previously appreciated(Shabalina et al,2009). We hypothesized that all 20 of the reported mouse exons, or a substantial number of these exons, have analogous exons within the human OPRM1 gene locus. Appling a unique set of bioinformatic comparative analytical approaches to mouse and human genomic OPRM1 gene, we identified 10 new potential exons, and two new putative promoters(Shabalina et al,2009). Our results suggest that at least one new alternatively spliced form of the OPRM1 gene, MOR1K, codes for a receptor variant that results in greater sensitivity to noxious stimuli and poorer responses to morphine(Shabalina et al,2009). Importantly, the MOR1K isoform codes for a 6TM rather than 7TM G protein coupled receptor (GPCR). Although similar to the MOR-3 variant reported by Cadet(Cadet et al, 2003), MOR-1K codes for a truncated 6TM MOR receptor with a different intracellular domain and tissue distribution. In contrast to MOR-3, MOR-1K is expressed in brain tissues, but not in vascular tissues or leukocytes. Unlike the mouse MOR-1K isoform, where transcription is initiated from an alternative promoter upstream to exon 11, The 5’ start site of MOR-1K isoform has been mapped to a novel OPMR1 promoter region that is located upstream of exon 13(Shabalina et al,2009). We also reported that agonists of the 6TM receptor produce dose-dependent excitatory rather than the classic inhibitory cellular effects observed for the major 7TM MOR1 isoform. Specifically, stimulation of the MOR-1K 6TM isoform with morphine produces a dose-dependent increase in intracellular cAMP and Ca++, and increases the cellular release of nitric oxide(Gris et al, 2010). These observations provided the basis for the intriguing hypothesis that the “excitatory shift” in MOR signaling depends, at least in part, on the stimulation of the 6TM isoform, and thus contributes to opioid-induced hyperalgesia, tolerance and dependence (Gris et al, 2010).
We are also conducting a comprehensive bioinformatic study of the molecular structure of human exon 11. We demonstrated that the exon 11 region shows the strongest conservation between man and mouse genomes (Shabalina et al,2009). Shortly after this bioinformatc prediction, the human exon 11 was cloned (Xu et al,2009). Four new human isoforms MOR-1G1, MOR-1G2, MOR-1I and MOR-1H were identified and were shown to contain exon 11 spliced either to exon 2 or to exon 1 through a variable 3’splice site. These isoforms code for either the extended 7TM receptor isoforms or the 6TM receptor isoform that are identical to the MOR-1K isoform we cloned(Shabalina et al,2009). Importantly, the cloned exon 11 corresponds to our predicted exon 11 and the predicted 3’ splice site reported by Pan and coworkers is situated between two alternative 3’ spliced sites of the cloned exon we identified. The 5’ spliced site of the cloned exon 11 was not mapped by Pan and coworkers(Xu et al,2009), but instead was predicted by using bioinformatic approaches (Fig.6). The successful cloning of human exons 11 and 13 by Pan and coworkers(Xu et al,2009) and by our(Shabalina et al,2009) groups validates our approach to predicting the extended OPRM1 structure(Shabalina et al,2009).
We also recently proposed that SNPs located within the new functional elements of OPRM1 gene affect the activity or regulate the relative amount of corresponding alternatively-spliced OPRM1 forms that are contribute to human variability to opioid responses. Our association analysis between a set of SNPs densely situated within the newly identified functional elements and measures of human pain perception and analgesic responses to morphine identified four new potentially functional SNPs (unpublished data, Shabalina et al,2009). Importantly, all significantly associated SNPs are situated within the new functional elements of OPRM1. This suggests that alternative exons represent an important target for genetic variability and may be a significant source of variation in common clinical phenotypes, including responses to opioid drugs. Since modifications of the function of the main receptor form may have dramatic consequences on genetic fitness and may not reach significant frequency in the general population, genetic variations in alternative exons that are expressed at low levels or only under specific conditions may lead to more subtle phenotypic differences that underlie the observed variations in MOR1-dependent phenotypes. In addition to the substantial biological significance of these findings, these results are likely to be of substantial clinical importance, as conceptually new tactics can now be considered for the identification of new functional SNPs that define the analgesic efficacy and side effects of opioids commonly used in clinical practice.
Thus, we demonstrated that SNP rs563649, which is located within a structurally conserved internal ribosomal binding site (IRES) in the 5'UTR of the novel isoform MOR-1K, affects both mRNA levels and translation efficiency of these variants(Shabalina et al,2009). The higher receptor expression is driven by the minor T allele and is associated with greater sensitivity to noxious stimuli and poorer responses to morphine(Shabalina et al,2009), confirming the potential excitatory cellular effects of 6TM MOR isoform. We propose to use similar approaches to identify the molecular genomic mechanisms that underlie other functional polymorphisms within the OPRM1 gene locus and MOR alternatively spliced isoforms.
Marked individual variability in the pharmacokinetics of opioids as well as genetic variations in opioid-metabolizing enzymes, transporter and signal transduction elements have been reported(Somogyi et al,2007). Several genes contribute to the pharmacodynamics and pharmacokinetics of opioids. For example, morphine, is particularly vulnerable to glucuronidation in the liver and brain by UDP-glucurosyltransferase activity (mainly by UGT2B7) resulting in morphine-3-glucuronide (M3G) and M6G, which are the main metabolites of morphine(Lotsch et al, 2002). Following morphine administration, M3G levels are increased but it is biologically inactive. In concert, levels of M6G increase to a lesser extent but this substatnace is an agonist of MOR1, with an anlagesic potency greater than morphine morphine(Dahan et al, 2008). The transporter P-glycoprotein (PGP), wich is encoded by the ABCB1 gene, also plays an important role in the pharmacokinetics of morphine. It facilitates the uptake of morphine, but not M6G, into the brain(Thompson et al;Bostrom et al, 2005; Hamabe et al,2006). The most consistent genetic contributors to morphine response reported in the literature so far include ABCB1, catechol-O-methyl transferase (COMT) and melanocortin-1 receptor (MC1R)(Somogyi et al,2007;Diatchenko et al, 2007). Thus combined effects of genes affecting pharmacokinetics and pharmacodynamics of opioids should be considered in clinical association studies that examine opioid responses.
A focused study of the OPRM1 gene locus is a critical first step in advancing our understanding of genetic differences in the responses to opioids. MOR1 is the major binding target for most clinically used opioid therapeutics and is a potent source of the currently recognized genetic variability of responses to opioids; yet, there is a striking deficiency in our knowledge of the genomic and genotypic structure of OPRM1. Because of the inherent difficulties in studying the molecular genetics of OPRM1, such as the very low abundance of the mRNA, the unusual sequence structure that is not optimal for current molecular biological tools, and some degree of divergence between human and mouse transcriptomic structure, this very important drug target lags behind the vast majority of other gene loci in terms of our understanding of its molecular and genetic structure.
Furthermore, it is crucially important to study the molecular mechanisms that underlie the observed associations mediated by specific SNPs in order to identify the functional allelic variants rather than variants that are merely associated genetic markers. This will permit a major advancement in our understanding of opioid receptor biology, pharmacodynamic responses to opioid administration, and will enable the development of reliable pharmacogenomic tests that predict the efficacy and side-effects of opioids.
MOR agonists are the most widely used analgesics, prescribed for both acute postoperative pain and chronic pain conditions; yet, there is substantial individual variation in drug responses of which we have very limited understanding and ability to control. Thus, the identification of new functional SNPs within alternative exons of OPRM1 is of considerable importance to the field of medical genetics. Existing evidence suggests that recently studied OPRM1 SNPs are not just another group of genetic variations that affect OPRM1 function, but instead are very important contributors to the OPRM1 genetic variability and receptor function. Further analysis of OPRM1 gene locus for the presence of functional SNPs within new potential alternative exons and other functional elements is required. The intriguing hypothesis that the analgesic efficacy and/or side effect profile of morphine are associated with and predicted by SNPs identified in new alternative exons and other functional elements rather than constitutive exons (Shabalina et al,2009) is an area of future investigation. Further genotypic analysis of the OPRM1 gene locus and receptor function hold great promise in elucidating a set of genetic markers that can be used to r predict the safety and efficacy of opioids on an individual basis.
Authors did not have any writing assistance.
Role of the funding source
This work was supported in part by NIDCR and NINDS grants RO1-DE16558, UO1-DE017018, NS41670 and PO1 NS045685. NIDCR and NINDS were not involved in the study design, in the collection, analysis and interpretation of data; in the writing of the manuscript; or in the decision to submit the manuscript for publication.
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Conflict of interest statement
Authors declare no conflict of interests.
Luda Diatchenko, Centre for Neurosensory Disorders, University of North Carolina at Chapel Hill, Chapel Hill, USA.
J. Elliott Robinson, Department of Neurology, University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, USA.
William Maixner, Centre for Neurosensory Disorders, University of North Carolina at Chapel Hill, Chapel Hill, USA.