2022 Volume 71 Issue 3 Pages 424-430
Alcohol dependence is an addiction that causes psychological and physical dependence and makes it difficult for those affected to control their intake of alcohol. The μ (mu), δ (delta) and κ (kappa) opioid receptors are thought to be associated with the development of alcohol dependence. Research has shown that μ opioid receptor knockout mice exhibit reduced alcohol consumption and reduced preference for alcohol. In this study, we confirmed whether mu opioid receptor (OPRM1) polymorphisms (IVS2+691C/G, −172G/T and −1748G/A) indeed have an effect on alcohol dependence formation in 64 patients, with 73 healthy people used as a control group. We compared the genotype, allele, carrier (minor allele carriers versus major allele homozygous carriers) and haplotype frequencies between these groups. In addition, the 1510A allele of acetaldehyde dehydrogenase 2 (ALDH2) polymorphism (1510G/A) causes poor metabolism of acetaldehyde, a major metabolite of alcohol. We also focused on ALDH2 1510G/G (ALDH2 *1/*1) carriers in the subjects. Three OPRM1 and one ALDH2 genotypes were determined by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. No significant differences were found in the frequency of OPRM1 polymorphisms between those suffering from alcohol dependence and the control group. We concluded that the three OPRM1 polymorphisms (IVS2+691C/G, −172G/T and −1748G/A) were not likely to be risk factors for alcohol dependence in a Japanese population. This report is the first in a Japanese population. Nevertheless, further analysis of the opioid receptor gene in a large sample size is required.
アルコール依存症とは，飲酒の自制が困難となる薬物中毒であり，オピオイド受容体との関連が考えられている。μオピオイド受容体欠損マウスは，アルコール自己投与の減少とアルコールに対する嫌悪を示すことが確認されている。本研究では，μオピオイド受容体（OPRM1）遺伝子多型IVS2+691C/G，−172G/Tおよび −1748G/Aがアルコール依存形成に与える影響を調べるため，遺伝子型，アレルおよびマイナーアレル保持者別頻度，ハプロタイプ解析，連鎖不平衡解析を含めた検討を行った。なお，OPRM1遺伝子多型の影響についてより詳細に検討するため，アルコール感受性に関わる2型アルデヒド脱水素酵素遺伝子多型ALDH2 *1/*1を有する対象者に着目した解析も行った。アルコール依存症患者64人，コントロール75人を対象とした。遺伝子多型の解析には，制限酵素断片長多型法（PCR-RFLP）を用いた。解析の結果，患者群および健常者群間における遺伝子型，アレルおよびマイナーアレル保持者別，全てのハプロタイプ頻度において，有意な差異は認められなかった。今回の結果より，着目した3つのOPRM1遺伝子多型がアルコール依存症発症の危険因子となる可能性は低いことが示唆された。今後は，同遺伝子上の他の多型や，他のオピオイド受容体にも着目し，アルコール依存症との関与についてより詳細に検討する必要があると考えられる。
Alcohol dependence (AD) is an addiction that causes both psychological and physical dependence and makes it difficult for those affected to control their intake of alcohol.1) This disease tends to aggregate within families and it has been shown that the risk of AD in relatives of probands is increased about twofold.2) Regarding the prevalence of AD in Japan, it has been shown that the proportion of people with lifetime experience of AD is 1.9% for males and 0.2% for females, and the total number of patients is estimated to be 1.07 million.3)
Mu (μ), delta (δ) and kappa (κ) opioid receptors represent the initially classified main receptor subtypes. It has been shown that μ opioid receptor knockout mice exhibit reduced alcohol consumption and reduced preference of alcohol.4) Additionally, since 2019, nalmefene hydrochloride hydrate has been used in Japan as a medication to reduce alcohol consumption in patients with AD. Nalmefene is an opioid receptor modulator, which is a μ and δ opioid receptor antagonist and a κ opioid receptor partial agonist,5)–7) and helps reduce the urge to drink alcohol. Drinking releases β-endorphin which activates μ opioid receptors, resulting in the release of dopamine.6),7) As a result, the reward system activates,6) initiating a sense of happiness and the like which can ultimately lead to addiction. Therefore, opioid receptors are a known risk factor for AD.
Finding disease related genes has the potential to improve outcomes for patients in various ways, such as developing new tests and medications. Accordingly, we focused on the several known OPRM1 (opioid receptor mu 1) gene polymorphisms which have not yet been the target of AD related studies in a Japanese population and we can survey by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. The human OPRM1 gene is located at 6q24-25, spanning approximately 50 kb over and consisting of 4 exons.8) We investigated whether the IVS2+691C/G (rs2075572), −172G/T (rs6912029) and −1748G/A (rs12205732) polymorphisms of the OPRM1 gene are susceptibility factors for AD for the first time in a Japanese population. Figure 1 shows the polymorphism positions in the OPRM1 gene.9)
In addition, the acetaldehyde dehydrogenase 2 (ALDH2) gene polymorphism (rs671: 1510G/A) 1510A allele (ALDH2 *2) causes poor metabolism of acetaldehyde, a major metabolite of alcohol.1) 1510G allele (ALDH2 *1) were significantly associated with AD in a Japanese population.1) Furthermore, as most AD patients have 1510G/G (ALDH2 *1/*1), we also focused on the 1510G/G carriers among the subjects.
This study was approved by the ethics committee of Azabu University, Japan (2359). Written informed consent was obtained from 64 AD subjects (57 males; 7 females) diagnosed with DSM-IV diagnostic criteria, and 75 healthy people (23 males; 52 females) in the control group.
2. DNA AnalysisThe three OPRM1 gene polymorphisms were genotyped by means of PCR-RFLP according to the methods of Bergen et al.,10) Dinh et al.11) and Nikolai.12) The ALDH2 gene polymorphism was genotyped according to the method described by Wu et al.13) The PCR conditions were arranged in this study with T100 Thermal Cycler (Bio-Rad Laboratories, Inc. Hercules, CA). The PCR cycling conditions were as follows: IVS2+691C/G (An initial denaturation for 5 min at 95°C, followed by 30 cycles of denaturation for 1 min at 95°C, annealing for 1 min at 61°C, and extension for 1 min at 72°C, followed by the final extension for 7 min at 72°C); −172G/T (An initial denaturation for 2 min at 94°C, followed by 35 cycles of denaturation for 40 sec at 94°C, annealing and extension for 1 min at 72°C, followed by the final extension for 3 min at 72°C, in a shuttle cycle.); −1748G/A (As for −172G/T except for annealing for 40 sec at 61°C); and ALDH2 1510G/A (An initial denaturation for 10 min at 95°C, followed by 35 cycles of denaturation for 30 sec at 95°C, annealing for 30 sec at 60°C, and extension for 30 sec at 72°C, followed by the final extension for 7 min at 72°C). The PCR products were digested with HinfI (New England Biolabs, Tokyo, Japan), Bpu1102I (Takara, Shiga, Japan) or MboII (New England Biolabs, Tokyo, Japan). The digested products were subjected to electrophoresis on agarose gels or polyacrylamide gels with the ethidium bromide staining method in Figures 2–4.
Lane M, 20 bp DNA Ladder; Lane 1, G/G genotype; Lane 2, C/G genotype; Lane 3, C/C genotype. (20 bp was often not visible.)
Lane M, 100 bp DNA Ladder; Lane 1, G/G genotype; Lane 2, G/T genotype. (T/T genotype was not shown.)
Lane M, 100 bp DNA Ladder; Lane 1, G/A genotype; Lane 2 and 3, G/G genotype. (A/A genotype was not shown.)
The Hardy-Weinberg disequilibrium was assessed using a chi-square test. We compared the OPRM1 genotypes and alleles between AD subjects and controls by performing statistical analysis using a chi-square test with Yate’s correction. In addition, the subjects were divided into two groups for each of the polymorphisms. We compared minor allele carriers with major allele homozygotes carriers of the OPRM1 gene, i.e., C/G+G/G versus C/C of IVS2+691C/G, G/T+T/T versus G/G of −172G/T and G/A+A/A versus G/G of −1748G/A. This was performed to investigate the effect of the minor allele. Haplotype frequencies and linkage disequilibrium (LD) coefficients were calculated by gPLINK v. 2.050 (http://zzz.bwh.harvard.edu/plink/index.shtml)14) and Haploview v.4.2 (http://www.broad.mit.edu/mpg/haploview/index.php).15) In this study, p < 0.05 was considered to be statistically significant.
The results of OPRM1 gene genotype and allele frequencies in AD subjects and controls are shown in Table 1. The OPRM1 gene genotype frequencies were as follows: IVS2+691C/G (Alcohol+Control; C/C: 40+52, G/C: 22+21, G/G: 2+2); −172G/T (G/G: 51+60, G/A: 13+15, A/A: 0+0) and −1748G/A (G/G: 51+60, G/A: 13+15, A/A: 0+0). The OPRM1 genotype distribution was in Hardy-Weinberg equilibrium (IVS2+691C/G; AD subjects: χ2(1) = 0.245, p = 0.620; Control: χ2(1) = 0.00480, p = 0.945 and −172G/T or −1748G/A; Alcoholic: χ2(1) = 0.818, p = 0.366; Control: χ2(1) = 0.926, p = 0.336). As a result of the analysis, it was established that there were no significant differences in OPRM1 genotype (IVS2+691C/G: χ2(2) = 0.540, p = 0.763; −172G/T and −1748G/A: χ2(2) = 0.028, p = 0.986) and allele frequencies (IVS2+691C/G: χ2(1) = 0.613, p = 0.434; −172G/T and −1748G/A: χ2(1) = 0.002, p = 0.966) between AD subjects and controls as shown in Table 1.
| SNP | Subject | n | Genotype (%) | Allele (%) | Carrier (%) | ||||
|---|---|---|---|---|---|---|---|---|---|
| IVS2+691C/G (rs6912029) |
C/C | C/G | G/G | C | G | C/C | C/G+G/G | ||
| AD | 64 | 40 (62.5) | 22 (34.4) | 2 (3.1) | 102 (79.7) | 26 (20.3) | 40 (62.5) | 24 (37.5) | |
| Control | 75 | 52 (69.3) | 21 (28.0) | 2 (2.7) | 125 (83.3) | 25 (16.7) | 52 (69.3) | 23 (30.7) | |
| χ2(2) = 0.540 p = 0.763 | χ2(1) = 0.613 p = 0.434 | χ2(1) = 0.721 p = 0.396 | |||||||
| −172 G/T (rs2075572) |
G/G | G/T | T/T | G | T | G/G | G/T+T/T | ||
| AD | 64 | 51 (79.7) | 13 (20.3) | 0 (0) | 115 (89.8) | 13 (10.2) | 51 (79.7) | 13 (20.3) | |
| Control | 75 | 60 (80.0) | 15 (20.0) | 0 (0) | 135 (90.0) | 15 (10.0) | 60 (80) | 15 (20) | |
| χ2(2) = 0.028 p = 0.986 | χ2(1) = 0.002 p = 0.966 | χ2(1) = 0.002 p = 0.963 | |||||||
| −1748G/A (rs12205732) |
G/G | G/A | A/A | G | A | G/G | G/A+A/A | ||
| AD | 64 | 51 (79.7) | 13 (20.3) | 0 (0) | 115 (89.8) | 13 (10.2) | 51 (79.7) | 13 (20.3) | |
| Control | 75 | 60 (80.0) | 15 (20.0) | 0 (0) | 135 (90.0) | 15 (10.0) | 60 (80) | 15 (20) | |
| χ2(2) = 0.028 p = 0.986 | χ2(1) = 0.002 p = 0.966 | χ2(1) = 0.002 p = 0.963 | |||||||
Carrier (%): major allele homozygous carriers vs minor allele carriers (%)
We also compared minor allele carriers with major allele homozygotes carriers in the OPRM1 gene as shown in Table 1, but there were also no significant differences (C/G+G/G versus C/C of IVS2+691C/G: χ2(1) = 0.721, p = 0.396; G/T+T/T versus G/G of −172G/T and G/A+A/A versus G/G of −1748G/A: χ2(1) = 0.002, p = 0.963). Moreover, we performed the same as above analysis in the group of ALDH2 1510G/G (ALDH2 *1/*1) carriers in AD subjects and controls as shown in Table 2. However, in this case too, no significant differences were observed (IVS+691C/G: genotypes: χ2(2) = 0.146, p = 0.930; alleles: χ2(1) = 0.07, p = 0.793; −172G/T and −1748G/A; genotypes: χ2(2) = 0.187, p = 0.911; alleles: χ2(1) = 0.403, p = 0.526). There were also no significant differences between minor allele carriers and major allele homozygotes carriers as shown in Table 2 (C/G+G/G versus C/C of IVS2+691C/G: χ2(1) = 0.167, p = 0.682; G/T+T/T versus G/G of −172G/T and G/A+A/A versus G/G of −1748G/A: χ2(1) = 0.453, p = 0.501).
| SNP | Subject | n | Genotype (%) | Allele (%) | Carrier (%) | ||||
|---|---|---|---|---|---|---|---|---|---|
| IVS2+691C/G (rs6912029) |
C/C | C/G | G/G | C | G | C/C | C/G+G/G | ||
| AD | 62 | 39 (62.9) | 21 (33.9) | 2 (3.2) | 99 (79.8) | 25 (20.2) | 39 (62.9) | 23 (37.1) | |
| Control | 48 | 32 (67) | 14 (29) | 2 (4) | 78 (81.25) | 18 (18.75) | 32 (67) | 16 (33) | |
| χ2(2) = 0.146 p = 0.93 | χ2(1) = 0.069 p = 0.793 | χ2(1) = 0.167 p = 0.682 | |||||||
| −172 G/T (rs2075572) |
G/G | G/T | T/T | G | T | G/G | G/T+T/T | ||
| AD | 62 | 51 (82.3) | 11 (17.7) | 0 (0) | 113 (91.1) | 11 (8.9) | 51 (82.3) | 11 (17.7) | |
| Control | 48 | 37 (77.1) | 11 (22.9) | 0 (0) | 85 (88.5) | 11 (11.5) | 37 (77.1) | 11 (22.9) | |
| χ2(2) = 0.187 p = 0.911 | χ2(1) = 0.403 p = 0.526 | χ2(1) = 0.453 p = 0.501 | |||||||
| −1748G/A (rs12205732) |
G/G | G/A | A/A | G | A | G/G | G/A+A/A | ||
| AD | 62 | 51 (82.3) | 11 (17.7) | 0 (0) | 113 (91.1) | 11 (8.9) | 51 (82.3) | 11 (17.7) | |
| Control | 48 | 37 (77.1) | 11 (22.9) | 0 (0) | 85 (88.5) | 11 (11.5) | 37 (77.1) | 11 (22.9) | |
| χ2(2) = 0.187 p = 0.911 | χ2(1) = 0.403 p = 0.526 | χ2(1) = 0.453 p = 0.501 | |||||||
Carrier (%): major allele homozygous carriers vs minor allele carriers (%)
As a result of haplotype analysis, no significant difference was found between AD subjects and controls in all haplotype frequencies as shown in Table 3 (global p = 0.5717).
| Haplotype | IVS2+691C/G (rs2075572) |
−172 G/T (rs6912029) |
−1748G/A (rs12205732) |
Frequency | χ2 | Haplotype p value |
|
|---|---|---|---|---|---|---|---|
| AD | Control | ||||||
| 1 | G | T | A | 0.09449 | 0.09396 | 0.0002244 | 0.988 |
| 2 | G | G | G | 0.1102 | 0.07383 | 1.103 | 0.2935 |
| 3 | C | G | G | 0.7953 | 0.8322 | 0.621000 | 0.4307 |
Haplotype distributions were not significantly different between AD subjects and controls (global p = 0.5717).
Figure 5 shows the LD analysis results. It was found that between −172G/T and −1748G/A was in complete LD (D' = 1, r2 = 1). They were suggested between IVS2+691C/G and −172G/T or IVS2+691C/G and −1748G/A are not complete LD by their scores (D' = 0.903, r2 = 0.406).
(a): D' value = 0.903 or 1, (b): r2 value = 0.406 or 1
rs6912029: −172G/T polymorphism, rs2075572: IVS2+691G/C polymorphism, rs12205732: −1748G/A polymorphism.
In the present study, the results suggest that the IVS2+691C/G, −172G/T and −1748G/A of the OPRM1 gene polymorphisms are unlikely to be risk factors for AD. However, as a part of the interpretation of the results, it should be noted that there were no −172T/T and −1748A/A genotype carriers in the population of 139 Japanese participants in this study. In a previous study with an Asian population, there were also no −172T/T carriers in a population of 238 Malay participants.16) Nevertheless, 8 carriers of −172T/T were found in a population of 610 Chinese and 135 Indian participants.16) In a further study, −172T/T and −1748A/A carriers were found at 2.0% each in a population of 52 Japanese participants.17) It is considered that −172T/T and −1748A/A carriers are rare but not nonexistent in Asian populations. Genotype frequencies in this study connect haplotypes and LD analyses results. If a complete LD relationship is found, one polymorphism can be used instead of the other as a tag SNP. Such an approach may lead to a reduction in the time and effort required for genetic research. In this study, 2 haplotypes and a complete LD relationship between −172G/T and −1748G/A were found. Conversely, the previous study’s scores were D' = 0.864, r2 = 0.746 and 4 haplotypes in a Japanese population of 52 participants were found.17) Further research in other ethnic groups is needed.
Association studies of the OPRM1 gene polymorphisms and diseases including AD have been carried out. Many previous studies from around the world regarding opioid receptor genes have focused on the OPRM1 gene polymorphism 118A/G. In fact, an association between 118A/G and AD in a Japanese population has already been demonstrated.1) In another previous study, it was suggested that representative polymorphisms should be surveyed in LD blocks.18) IVS2+691C/G polymorphism is located in intron 2, −172G/T and −1748G/A polymorphism is located in the 5' flanking promoter region in the OPRM1 gene. It was supposed that at least four or five LD blocks exist in the OPRM1 gene.9),18) In the current research, we wanted to investigate the polymorphisms in 5 areas. However, the PCR-RFLP methods of many OPRM1 polymorphisms have still not been reported efficiently or restriction enzymes suitable for the variants do not exist. Therefore, methods other than PCR-RFLP should be taken into consideration. Moreover, genotype frequencies vary depending on ethnicity. For example, it has been shown that there are associations between both the OPRM1 gene −2044C/A (rs17180961) and AD in a European American population (p = 0.0036).19) However, there are no 2044A carriers in an east Asian population by NCBI website (https://www.ncbi.nlm.nih.gov/snp/rs17180961?vertical_tab=true#frequency_tab).20) Consequently, we should also consider further investigations into polymorphism frequencies among different ethnic groups.
In addition, it is necessary to increase sample sizes and focus on the δ and κ opioid receptor genes polymorphisms, and other the OPRM1 gene polymorphisms in order to investigate AD in more detail.
There is no potential conflict of interest to disclose.
This research was supported by a research project grant awarded by Azabu University.
The authors would like to express their gratitude to Mr. Jonathan Lynch (Azabu University) for proofreading the English version of the manuscript.
Special thanks also to Ms. Teruko Honda (Azabu University) for helpful advice.
