Abstract
Objectives: To evaluate the effect of COMT and OPRM1 gene polymorphisms on the preoperative pain sensitivity in tumor patients. Methods: 300 cases of cancer patients undergoing elective surgery were included, and the Val158 Met loci of COMT gene and OPRM1 loci of A118 G gene were genotyped by PCR-RFLP. Pain threshold and pain tolerance threshold were measured using electrical stimulation to investigate the preoperative pain sensitivity in patients with different genotypes. Results: For the COMT gene, the pain threshold and pain tolerance threshold of patients with M allele both decreased (both P < 0.001); for PPRM1 gene, pain threshold and pain tolerance threshold of patients with G allele decreased (both P < 0.001). We also found that there was an interaction between the two genes. Conclusion: Gene polymorphisms of COMT and OPRM1 were correlated with the preoperative pain sensitivity of cancer patients. The patients with M allele of COMT and G allele of OPRM1 had higher preoperative pain sensitivity.
Keywords: Pain measurement, COMT, Opioid receptors; SNP, pre-operative care
Instruction
Surgery is an important therapeutic measure for cancer patients, but the sensitivity of preoperative pain has a significant effect on the cooperation in operation and quality of life after surgery [1]. Preoperative pain sensitivity has significant individual differences, which related to the genetic factors of patients [2]. Previous studies have shown that various genetic polymorphisms were correlated with pain sensitivity, such as KCNJ6 gene [3], opioid receptor μ1 gene [4], COMT gene [5], OPRM1 [6] and 5-serotonin receptor gene [7]. But the findings are inconsistent, and most studies only focused on the effect of a single gene, paying little attention to the combined roles of genes. Research confirmed that the impact of gene polymorphisms on the disease trait was minor, so the gene-gene interaction could achieve a more comprehensive understanding of the relationships between genetics and complex traits. Previous studies showed that, gene polymorphisms of OPRM1 and COMT were associated with pain sensitivity [5,6]. Therefore, this study focused on the effects of OPRM1 and COMT polymorphisms on the preoperative pain sensitivity in cancer patients and the interactions between the two genes.
Materials and methods
The study was approved by the Medical Ethics Committee of Shengjing Hospital of China Medical University, and all patients had signed informed consent.
Case selection
300 cases of cancer patients undergoing surgery in the departments of oncology, gynecology or general surgery of Shengjing Hospital of China Medical University from February 2010 to December 2013 were selected, without previous surgery, aged from 22 to 57 years, with a mean age of 41.3±12.4 years old, including 218 cases of male and 82 cases of female. The mean body mass index (BMI) was 25 kg/m2; ASA grade: 178 cases of grade I, 122 cases of grade II. There were no history of smoking and alcohol abuse, no abnormity in liver and kidney function, no history of diabetic, spiritual and neurological diseases, no complications of acute and chronic pain, no recent application of sedative and analgesic drugs.
Genetic testing
5 ml peripheral blood was collected before surgery and anticoagulated by EDTA; phenol-chloroform extraction method was used to extract DNA. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was used for the genotyping of Val158Met loci in COMT gene and A118G loci in OPRM1gene. PCR primer sequences and PCR-RFLP conditions were shown in Table 1.
Table 1.
Primer sequences and PCR products
| SNPs | Primer sequence | PCR product | Annealing temperature | Endonuclease |
|---|---|---|---|---|
| Val158Met | upstream:5’-GGATGGTGGATTTCGCTCGC-3’ | 117 | 55 | Bsh1236I |
| downstream:5’CTGGTGGGGAGGACAAAGTGC-3’ | ||||
| A118G | upstream:5’-GGTCAACqTGTCCCACTI’AGATCGC-3’ | 193 | 58 | Bsh1236I |
| downstream:5’-AATCACATACATGACCAGGAAGTTT-3’ |
Determination of the pain threshold and pain tolerance
Patients calmed for 10 min after burglary; using MEB-5100 type electric stimulator (Nihon Kohden Corporation, Japan), pain threshold and pain tolerance threshold were measured. Patients were in supine position, and the electrodes were fixed to the deltoid muscle of right upper arm; two electrodes were both 5 mm × 5 mm, with an electrode spacing of 1.5 cm, stimulation frequency of 50 Hz and pulse width of 0.5 ms [8]. Current intensity started from 0, increasing 0.2 mA each time; when gradually turning up the output current, we observed and inquired the reaction of the objects, and respectively recorded the current intensity when a slight tingling began and the pain cannot be tolerated (pain threshold and pain tolerance threshold). The test was repeated three times, with an interval of 3 min; the mean value was calculated.
Statistical analysis
SPSS 16.0 statistical software was used for analysis; Measurement data were presented as mean ± standard deviation (Mean ± SD); factorial analysis of two factors and three levels was performed; a weighted analysis of variance was performed between groups. Count data were compared using the chi-square test. Hardy-Weinberg equilibrium was tested by chi-square test. P < 0.05 was considered statistically significant.
Results
Hardy-Weinberg equilibrium test
Genotyping results of A118G and Val158Met were shown in Table 2; the distribution of genotypes was consistent with Hardy-Weinberg equilibrium (all P > 0.05).
Table 2.
Genotype and allele frequency of these two SNPs
| SNPs | Genotype | Allele | |||
|---|---|---|---|---|---|
| A118G | AA | AG | GG | A | G |
| 144 (48.0%) | 114 (38.0%) | 42 (14.0%) | 0.67 | 0.33 | |
| V158M | VV | VM | MM | V | M |
| 138 (46.0%) | 115 (38.3%) | 47 (15.7%) | 0.65 | 0.35 |
Comparison of patients with different genotypes in general
According to different genotypes, the age, gender, BMI and other indicators were compared, and there were no significant differences between genotypes (Table 3).
Table 3.
General comparison of different genotypes
| Genotype | N | Sex (M/F) | Age (Year) | BMI (Kg/m2) |
|---|---|---|---|---|
| OPRM1 gene | ||||
| AA | 144 | 104/40 | 41.1±12.1 | 18.4±2.8 |
| AG | 114 | 85/29 | 40.8±12.0 | 18.2±2.4 |
| GG | 42 | 29/13 | 41.6±12.4 | 18.3±2.4 |
| P value | 0.122 | 0.887 | 0.433 | |
| COMT gene | ||||
| VV | 138 | 106/32 | 40.9±11.9 | 18.1±2.7 |
| VM | 115 | 85/30 | 41.7±12.1 | 18.4±2.2 |
| MM | 47 | 32/15 | 41.4±12.2 | 18.2±2.1 |
| P value | 0.683 | 0.342 | 0.338 |
Comparison of pain threshold and pain tolerance among different genotypes
For both OPRM1 and COMT genes, compared to wild homozygote, the pain threshold and pain tolerance threshold of mutant homozygote carriers were significantly lower (Table 4).
Table 4.
Comparison of pain threshold and pain tolerance among different genotypes
| Genotype | N | Pain threshold (Mean ± SD, mA) | Pain tolerance (Mean ± SD, mA) |
|---|---|---|---|
| OPRM1 gene | |||
| AA | 144 | 2.2±0.8 | 5.4±1.2 |
| AG | 114 | 1.6±0.7 | 4.3±1.1 |
| GG | 42 | 1.5±0.6 | 4.2±1.4 |
| P value | < 0.001 | < 0.001 | |
| COMT gene | |||
| VV | 138 | 2.1±0.7 | 5.4±1.2 |
| VM | 115 | 1.5±0.8 | 4.4±1.0 |
| MM | 47 | 1.4±0.6 | 4.3±0.9 |
| P value | < 0.001 | < 0.001 |
Gene-gene interaction analysis
Factorial analysis showed that there was an interaction between COMT gene and OPRM1 gene (P < 0.05). Patients with the two kinds of mutant alleles had a more significant decrease in the pain threshold and pain tolerance threshold (Table 5).
Table 5.
Interaction analysis between OPRM1 and COMT genes
| Genotype combinations | N | Pain threshold | Pain tolerance |
|---|---|---|---|
| AA-VV | 74 | 2.3±0.9 | 5.5±1.3 |
| AA-VM | 44 | 2.2±1.0 | 5.4±1.2 |
| AA-MM | 26 | 2.0±1.1 | 5.0±1.1 |
| AG-VV | 51 | 2.2±0.9 | 5.3±1.2 |
| AG-VM | 49 | 1.7±0.9 | 4.5±1.3* |
| AG-MM | 14 | 1.6±0.9* | 4.4±1.2* |
| GG-VV | 13 | 2.0±0.9 | 4.8±1.6 |
| GG-VM | 22 | 1.6±0.8* | 4.4±1.2* |
| GG-MM | 7 | 1.5±0.8* | 4.3±1.2* |
P < 0.05, compared to AA-VV group.
Discussion
The study found that, polymorphisms of OPRM1 gene and COMT gene were correlated with pain sensitivity in cancer patients, and there was an interaction between the two genes.
Although pain threshold and pain tolerance threshold have different forming mechanisms and asynchronous changes, they are both the specific and sensitive indicators of pain sensitivity in clinical, which can accurately reflect the changes in pain sensitivity [9]. Continuous electrical stimulation at the same point in the deltoid muscle can objectively assess the body’s pain threshold and pain tolerance threshold [9]; therefore this study chose to stimulate the central part of the deltoid muscle of right forearm and used electrical stimulator to assess the preoperative pain threshold and pain tolerance threshold, thus ensuring the reliability of the observed indicators.
Factorial analysis showed that there was a interaction between polymorphisms of COMT gene and OPRM1 in pain sensitivity; one factor was fixed in the further weighted analysis of variance, and the difference of another factor between different levels was analyzed, indicating that pain sensitivity was increased in mutant homozygote group and the pain sensitivity of patients carrying mutations in both alleles increased more apparent.
Some studies showed that GOMT-V158M gene affected the pain by changing COMT activity and regulating psychology, stress and other factors [10-11]. Studies have also indicated that with respect to COMT single nucleotide polymorphism, low activity state of COMT was an important regulator of pain sensitivity [12,13]. But the factors that affect the state of COMT activity are various; on the COMT gene, the polymorphisms of multiple loci may affect COMT activity. In this study, only the common V158M polymorphisms were studied, and the pain sensitivity was not fully explained; so further research remains to be conducted.
In summary, gene polymorphisms of COMT-V158 M and OPRM1-A118 G were correlated with the preoperative pain sensitivity in cancer patients. Patients carrying double mutant homozygote of COMT and OPRM genes had higher pain sensitivity before surgery.
Acknowledgements
This study was supported by the Liaoning province science and technology plan project (No. 2012408002) and by the National Natural Science Foundation (No. 81271538) and by Outstanding Scientific Fund of Shengjing Hospital (No. 201208).
Disclosure of conflict of interest
None.
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