The Opposing Roles of IVS2+691 CC Genotype and AC/AG Diplotype of 118A>G and IVS2+691G>C of OPRM1 Polymorphisms in Cold Pain Tolerance Among Opioid-Dependent Malay Males on Methadone Therapy

The opioidergic neurotransmission system is an important processor of painful stimuli [1‐4]. The μ-opioid receptor (OPRM1) is a primary target for clinically important opioid analgesics, including methadone. Studies among healthy subjects showed a possible role of the opioid receptor mu 1 gene (OPRM1) in altered pain sensitivity [5, 6]. Genetic factors that affect the density, function, and, consequently, the signaling efficacy of μ-opioid receptors may contribute to inter-individual variations in the pain response to opioids [7‐9].

Southeast Asia is a highly populated and cultural-diverse region with ethnic Malays constituting the largest population group, mainly populating countries including Malaysia, Indonesia, and the southern part of the Philippines. Males who are ethnic Malays make up the majority of opioid-dependent patients on methadone treatment in Malaysia [10]. We have previously reported that a majority of opioid-dependent Malay males on methadone therapy are cold pain sensitive and that the pain is frequently associated with poor sleep quality [11]. One postulated mechanism for altered pain sensitivity in this susceptible population is polymorphisms in the OPRM1 gene.

Some of the most frequently studied polymorphisms of OPRM1 include 118A>G (dbSNP rs1799971), IVS2+691G>C (dbSNP rs2075572), and IVS2+31G>A (dbSNP rs9479757). The association between 118A>G polymorphism and pressure pain was first reported by Fillingim et al. [6] and Lotsch et al. [5], but subsequent studies failed to replicate these preliminary reports in different experimental pain models and in different populations [12‐14]. Similarly, the roles of other polymorphisms on pain sensitivity, including IVS2+691G>C and IVS2+31G>A, have been mixed. For example, the IVS2+691G>C polymorphism did not increase morphine requirements in patients with pain caused by malignant disease [7] and did not have significant association with 24-h post-operative opioid requirement [15]. To summarize, available studies have raised a great deal of speculation over the role of OPRM1 polymorphisms in pain sensitivity and one postulation is that this phenomenon may be gender and ethnic dependent [16].

Recent studies indicate OPRM1 polymorphisms are associated with side effects including changes in libido and insomnia [17], and also methadone-related deaths [18, 19]. On the other hand, the role of OPRM1 polymorphisms on pain sensitivity among opioid-dependent patients on methadone is unclear because of limited evidence. Thus far, only one study has explored this [20] but unfortunately the low frequencies of variants in the study samples were a limitation. A better understanding of OPRM1 polymorphisms in pain and opioid response of a specific population has implications for the treatment of pain and addictive disease, and clinical management of each in the presence of the other. In the present study, we aimed to investigate the influence of common OPRM1 polymorphisms on pain responses among opioid-dependent Malay males on methadone maintenance therapy (MMT).

The 118G allele frequency of 40.2% in our study is in good agreement with previous smaller scale studies of this polymorphism among Malay drug addicts in Malaysia [24] and heroin-dependent Malays in Singapore [25], although ours was much higher compared to intravenous drug users on MMT [26]. Likewise, frequency of variant IVS2+691C allele of 83.1% in our subjects is similar to those reports from heroin-dependent Malays in Singapore [25]. The reported frequency in our study seems to have contrasted with those of the Chinese (73.1%) and Indian (75%) Singaporeans [25], as well as the Chinese (69.3%) in Hong Kong [27]. The above reports indicate that variability in frequency of 118G and IVS2+691C allele in populations on methadone treatment may be an ethnic-dependent phenomena.

In the current study, there was no association seen between 118A>G variations with cold pain threshold and pain tolerance. Instead, IVS2+691G>C polymorphisms using different models were shown to affect cold pain tolerance, but not pain threshold and pain intensity. These include homozygous CC genotype (a significant shorter pain tolerance time in CC vs. GG/GC) and combined homozygous 118 AA genotype and heterozygous IVS2+691 GC genotype (AC/AG diplotype; a significant longer pain tolerance time with this diplotype vs. without this diplotype). We have previously reported that a majority of opioid-dependent Malay males on methadone therapy are cold pain sensitive [11] and OPRM1 polymorphisms might be responsible for this altered sensitivity based upon our study results. What are the implications of this? Any inter-individual variations in opioid-induced hyperalgesia among methadone users may weaken or strengthen their determination to abstain [28], and the knowledge of OPRM1 polymorphisms in such a circumstance may help to provide a prediction. In addition, this knowledge on OPRM1 polymorphisms may help to guide the methadone dosages and, therefore, avoid adverse drug reactions.

Studies by Wang et al. [17] indicated that IVS2+691G>C polymorphisms might affect the side effects of methadone including change in libido and insomnia. Our study suggests that variations in IVS2+691G>C might also affect pain tolerance. The actual functions of IVS2+691G>C are still under investigations but available data support its role in affinity of transcriptional regulatory factors for the intronic DNA sequence [29‐31]. The DNA intronic sequence could involve in alternative DNA splicing and this results in creations of different isoforms of human OPRM1 gene [30, 32]. It may also directly alter mRNA levels and, therefore, affect expression of OPRM1 gene [29‐31]. Based on our results, we hypothesized the increased expression of IVS2+691 CC variant resulted in a higher OPRM1 availability and because of chronic sensitization by prolonged opioid use, it may then facilitate opioid tolerance, hyperalgesia and subsequently lower the pain tolerance. However, tolerance normally indicates a rightward shift in a dose–response curve. Since participants have been treated with methadone anywhere from 0.3 to 9 years, sensitization may, therefore, also be quite variable or absent. It is possible that those participants in the program with a shorter stay were different than those with longer treatment times.

On the other hand, the AC/AG diplotype of 118 and IVS2+691 was associated with a longer cold pain tolerance time. Opioid-related adverse effects such as hyperalgesia were probably less likely to occur in patients with AC/AG diplotype at 118 and IVS2+691 given that the mechanisms of opioid-induced adverse events involve OPRM1. Thus, although 118A>G polymorphism alone does not influence cold pain tolerance, the 118 variant may play an indirect role in pain modulation. A strong linkage disequilibrium (LD) between studied polymorphisms and unstudied polymorphisms has been shown to exist [15, 33], and resulted in altered binding affinity between endogenous (and/or exogenous) opioids and OPRM1. As a consequence, there was less hyperalgesia and, therefore, an increase in cold pain tolerance.

There were associations, although not significant, where variants of 118G allele and GC haplotype of 118 and IVS2+691 caused a higher cold pain intensity, but 118 AA genotype resulted in a lower cold pain intensity. At this juncture, it is difficult to explain the differential effects on pain intensity with these polymorphisms and further studies with larger samples are needed. Besides individual polymorphisms, haplotype and diplotype analyses are also necessary to clarify the influence of OPRM1 on inter-individual variations in pain sensitivity among opioid-dependent patients on methadone therapy. There is also the possibility that heightened pain sensitivity is a result of the prolonged opioid use itself or opioid-induced hyperalgesia. The heightened pain sensitivity is a form of latent hyperalgesia to chronic opioid misuse, and subsequent methadone therapy does not exacerbate hyperalgesia but does not make it worse either [34, 35].

Some limitations to this study need to be highlighted. There are a number of OPRM1 polymorphisms (such as rs1074287, rs6912029, rs12209447, rs510769, rs3798676, rs7748401, rs495491, rs10457090, rs589046, rs3778152, and rs563649) but we only studied two common variants. Only Malay males were included but we aimed to reduce the confounding effects of gender and ethnicity on cold pain response [36‐44]. Furthermore, Malay males are the majority of opioid-dependent patients on methadone treatment in Malaysia. There were other confounding factors such as induction of tolerance to methadone effects, methadone clearance and genetic variations of other pain-related genes and genes related to pharmacokinetics and pharmacodynamics of methadone, which may be important. Only a tonic pain model using cold pain was studied here and phasic pain may differ qualitatively, neurologically, and functionally from tonic pain stimuli [45]. However, CPT has been shown to be the better pain-induction technique for methadone studies [20, 46, 47]. Lastly, our analyses did not correct for multiple testing because of single gene being studied. There was a significant LD between loci and it was 47.3% of the theoretical maximum but there was a weak correlation between single nucleotide polymorphisms [D′ = 0.473, r ² = 0.031, x ² (df) = 9.176 (1), P = 0.0026]. In the view that correction was not performed for multiple tests, these two findings would be considered nominally significant. It is possible that our result might be a false-positive association partly because of limitation in sample size. Therefore, our study may be considered explorative in nature and it would require replication by other investigators. Nevertheless, our results can be considered as a basis for further larger scale study.

In summary, our study indicates that the IVS2+691 CC genotype was associated with a shorter cold pain tolerance time but AC/AG diplotype of 118A>G and IVS2+691G>C was associated with a longer cold pain tolerance time in opioid-dependent Malay males on methadone therapy. It can be postulated that haplotypes of OPRM1 may be associated with altered binding affinity. The OPRM1 polymorphisms may serve as an important variable for developing directed interventions and to guide management of pain among opioid-dependent patients on MMT with genetic risk for suffering pain. Personalized medicine based on pharmacogenetics may be able to maximize the benefits of methadone pharmacotherapy and minimize their harms.