MDR1 Genotypes and Haplotypes Are Closely Associated with Postoperative Fentanyl Consumption in Patients Undergoing Radical Gastrectomy

Fentanyl is a powerful opioid analgesic, and its analgesic effect is greatly different among individuals. This study was aimed at exploring the effects of multidrug resistance gene-1 (MDR1) genetic variation on postoperative fentanyl consumption. A total of 135 patients, who planned to undergo radical gastrectomy with general anesthesia, were studied. The subjects received patientcontrolled analgesia (PCA) by intravenous fentanyl within 48 hours after operation and maintained a numerical rating scale (NRS) score ≤ 3. The consumption and side effects of fentanyl were recorded within 24 hours and 48 hours after the operation. Single nucleotide polymorphisms (SNPs) of all patients with MDR1 1236C>T, 2677G>T/A, and 3435C>T were screened by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) or DNA sequence analysis after PCR. There was no difference in postoperative fentanyl consumption among patients having 2677G>T/A and 3435C>T polymorphisms (all P > 0:05). MDR1 1236C>T polymorphisms and haplotypes combined by three SNPs, however, significantly affected postoperative fentanyl consumption (all P < 0:05). Moreover, 1236TT genotype carriers consumed more fentanyl during 24 hours (P = 0:038) and 48 hours (P = 0:003) postoperatively. The MDR1 TTT haplotype carriers needed more fentanyl compared with the CGC haplotype carriers during the first 48 hours after surgery (P = 0:017). Nausea, vomiting, and dizziness were not found to have significant differences among the above three SNPs and their haplotypes (P > 0:05). MDR1 1236C>T polymorphism and haplotypes were factors contributing to the individual variability in postoperative fentanyl consumption.


Introduction
Fentanyl is a widely used narcotic analgesic, but its analgesic efficacy has a large interindividual variation among patients [1,2]. The analgesic effect of fentanyl is affected by many factors, and the gene polymorphism is one of the main factor [3][4][5]. Gene polymorphism plays an important role in the pharmacokinetics and pharmacodynamics of fentanyl [5]. Drug metabolizing enzymes and targets of fentanyl are both likely affected by gene polymorphisms, which in turn affect the analgesic effect of fentanyl [6,7]. The genes contributing to drug transporters of fentanyl, however, remain largely unexplored. P-Glycoprotein (P-gp) which is the product of the MDR1 gene, acts as a transmembrane efflux pump in the bloodbrain barrier, liver, kidney, and intestine [8]. The expression and function of P-gp are regulated by MDR1 gene SNPs, including exon 1236C>T, 2677G>T/A, and 3435C>T SNPs [9][10][11]. 1236C>T and 3435C>T are synonymous mutations. They may affect the P-gp function by altering protein folding [9]. In contrast, 2677G>T/A is a nonsynonymous mutation due to the substitution of amino acid alanine with serine or threonine [9]. MDR1 mRNA expression in heart tissue is significantly elevated in 2677AT or TT genotype carriers [10], and the homozygous T allele of 3435C>T reduces P-gp expression in the upper gastrointestinal tract [11]. On the other hand, these three SNPs are in strong linkage disequilibrium and form haplotypes. Haplotypes containing mutant alleles have been shown to exhibit large structural modifications that result in conformational changes in the binding site and subsequent reductions in P-gp activity [12,13].
Fentanyl is one of the substrates of P-gp [14][15][16]. Human and animal studies have shown that P-gp affects the absorption, distribution, excretion, and clinical effects of fentanyl [14][15][16]. Several authors have identified that MDR1 1236C>T and 3435C>T SNPs have significant effects on postoperative fentanyl consumption [7,17]. However, other studies cannot identify such associations [18] or obtain the opposite results [19]. Overall, the effects of three MDR1 SNPs on fentanyl efficacy are not definite. It may be the result of considering only one of the three SNPs. Since SNPs are in incomplete linkage disequilibrium, it may be necessary to consider the SNP combination to accurately assess the role of common polymorphisms in the efficacy of fentanyl. No studies, so far, have analyzed whether haplotypes of MDR1 1236C>T, 2677G>T/A, and 3435C>T led to variability in the dosage of fentanyl among patients. Hence, in this study, we evaluated whether genetic polymorphisms of 1236C>T, 2677G>T/A, and 3435C>T and their haplotypes affect postoperative fentanyl requirements for analgesia.

Materials and Methods
2.1. Ethical Statements. The research plan was reviewed and approved by the institutional ethics committee of the Third Xiangya Hospital. Each patient received information about the aim of the study and provided written informed consent. Moreover, participants were assured that the participation is completely voluntary and personal information is not disclosed to third parties.

Patient Selection.
Since the liver and intestines affect the metabolism and absorption of fentanyl, we chose patients who were scheduled for radical gastrectomy by general anesthesia in the present study. Exclusion criteria were as follows: patient refusal; ASA classification more than III; patients with severe liver injury, renal impairment, diabetes mellitus, cardiovascular disease, psychiatric disorders, chronic pain, and pregnancy or lactation; patients taking drugs that are substrates of P-gp; long-term use of analgesic; allergy to fentanyl; or drug abuse.

Anesthesia and Postoperative
Analgesia. General anesthesia was induced with 0.05 mg/kg midazolam, 0.2 mg/kg etomidate, 0.12 mg/kg vecuronium, and 4-6 μg/kg fentanyl. Anesthesia was maintained by 1%-2% sevoflurane inhalation, continuous infusion of propofol (4-12 mg·kg -1 ·h -1 ), and intermittent intravenous bolus of 0.05 mg/kg vecuronium. During surgery, intravenous infusion of fentanyl was started before skin incision, appended 2 μg/kg each time intraoperatively, and administration stopped once the surgeons opened the abdominal cavity. The total dose of fentanyl was less than 12 μg/kg, and the residual effect on postoperative fentanyl dose was avoided. After surgery, all patients were administered 8 mg ondansetron to prevent postoperative nausea and vomiting. Then, patients were extubated and observed in the postanesthesia care unit (PACU) for 2 hours. The antagonist of muscle relaxation was not used. The NRS score was recorded after extubation. Patients were considered to have inadequate pain control if NRS > 3, and fentanyl (20 μg per time) was intravenously given until NRS ≤ 3. Patients then received patient-controlled intravenous analgesia(PCIA) with fentanyl during the first 48 hours after surgery. In the analgesic pump, 30 μg/kg fentanyl was filled in normal saline, with a total volume of 240 mL. The pump was programmed to administer a 1.5 mL/h background dose, 20 μg bolus of fentanyl solution, and 5-minute lockout time. Postoperative pain was controlled with NRS ≤ 3 at rest. All enrolled patients used no opioid receptor antagonists and other analgesic drugs except for fentanyl after operation.
2.4. Genotyping. Genomic DNA from peripheral blood leukocytes was extracted using E.Z.N.A.™ SQ Blood DNA Kit (D5032-02). The primer design software Oligo 6 designed primers (Table 1). 1236C>T and 3435C>T were genotyped by the PCR-RFLP method (Figures 1(a) and 1(b)), and MDR1 2677G>T/A was screened by DNA sequencing (Figure 1(c)). The restriction enzyme BsuRI (Lot: 00143447, Thermo Scientific) was used to distinguish the T allele from the C allele of 1236C>T, and the restriction enzyme MboI (Lot: 00098836, Fermentas) was employed to distinguish the T allele from the C allele of 3435C>T.

Data Collection.
During the first 24 hours and 48 hours, patients' age, gender, BMI, length of incision, duration of surgery, and postoperative fentanyl consumption were collected. The NRS was recorded at 4 hours, 8 hours, 12 hours, 24 hours, and 48 hours after the operation. The incidence of side effects, including nausea, vomiting, respiratory depression, and lethargy, was recorded in the first 48 hours.
2.6. Statistical Analysis. The sample size of this research was calculated based on the frequency of MDR1 genotypes. According to the allele frequency of MDR1 gene SNPs (1236C>T, 2677G>T/A, and 3435C>T) and their haplotypes previously reported in Chinese [7,20], it was required that the sample size of 108 patients showed an absolute difference of 20% in the difference of fentanyl dose (power = 80%, α = 0:05). As a result of clinical experience and preliminary experiment, absolute difference of 20% was chosen as a minimum detectable difference. PASS 11 software (NCSS, Kaysville, UT) was used to calculate statistical power samples.

Journal of Nanomaterials
Clinical research and genotyping were blinded to the clinical outcome of patients. Data were analyzed using SPSS 19.0. P < 0:05 was statistically significant. All continuous variables were checked for normal distribution, and data were presented as means ± standard deviation or medians and range. Comparison of postoperative fentanyl consumption was performed using Kruskal-Wallis H test. Allele and genotype frequencies were estimated by gene counting. The devi-ation from the Hardy-Weinberg equilibrium was evaluated by chi-square tests.

Results
3.1. General Patient Information. In this study, 147 patients were recruited, and 12 of them were excluded because of patient quitting (n = 5), unavailability of blood samples   Tables 2 and 3 show the demographics and clinical data of the patients. There was no significant difference in demographic and clinical data among different MDR1 genotypes and haplotypes (all P > 0:05).

Genotypes and Allele
Frequency of MDR1 SNPs. Of 135 patients, 27 cases were 1236CC genotype, 66 cases were 1236CT, and 42 cases were 1236TT. The mutation frequency of allele T was 55.6%. 2677G>T/A polymorphism developed six genotypes: GG, GT, GA, TA, TT, and AA. Among the 135 patients, 33 cases were carriers of the GG genotype, 37 cases were carriers of the GT genotype, 20 cases were carriers of the GA genotype, 13 cases were carriers of the TA genotype, 25 cases were carriers of the TT genotype, and seven cases were carriers of the AA genotype, and the mutation frequency of the allele T and allele A was 37.0% and 17.4%, respectively. There were 53 carriers of the 3435CC genotype, 62 carriers of the CT genotype, and 20 carriers of the TT genotype, and the mutation frequency of allele T was 37.8%. The allelic frequencies of 1236C>T, 2677G>T/A, and 3435C>T conformed to the Hardy-Weinberg equilibrium (P > 0:05).

Linkage Disequilibrium and Haplotype Analysis.
Haploview software used the existing genotype data to calculate linkage disequilibrium statistics and infer the population haplotype model. Haploview software showed that there was strong linkage disequilibrium of MDR1 1236C>T, 2677G>T/A, and 3435C>T. Four haplotypes (TTT, CGC, CAC, and TGC) which were constructed by three MDR1 SNPs represented 90.7% of all haplotypes observed in this study.

Correlation between the Three SNPs of MDR1 Gene and Their Haplotypes and Postoperative Fentanyl Consumption.
In this study, there was no statistically significant difference in postoperative fentanyl consumption in 2677G>T/A and 3435C>T at 24 hours and 48 hours after surgery (P > 0:05). However, there was a significant difference in postoperative fentanyl consumption among the polymorphisms of 1236C>T. Specifically, the 1236TT genotype carriers needed more fentanyl for postoperative analgesia within the first 24 hours and 48 hours after surgery (P = 0:038 and P = 0:003, respectively) ( Table 4). Among the haplotypes of the MDR1    Journal of Nanomaterials gene, the TTT haplotype consumed more fentanyl than the CGC haplotype within the first 48 hours after surgery (P = 0:017) ( Table 5).

Correlation between Side Effects and the Three MDR1
SNPs and Their Haplotypes. The main side effects of fentanyl were nausea, vomiting, and dizziness at postoperative 48 hours. There was no significant difference in nausea, vomiting, and dizziness within 48 hours postoperatively among 1236C>T, 2677G>T/A, 3435C>T, and their haplotypes (P > 0:05) ( Table 6). None of the patients experienced respiratory depression or other serious side effects.

Discussion
In this study, we found that MDR1 1236C>T SNP and the MDR1 gene haplotypes constructed from 1236C>T, 2677G>T/A, and 3435C>T were predictive of individual variations in postoperative fentanyl consumption. Moreover, 1236TT carriers and MDR1 TTT haplotype carriers needed more postoperative fentanyl in patients undergoing radical gastrectomy.
The MDR1 1236C>T mutation is more commonly detected in Chinese Han. In our study, the frequency of the 1236T variant was 55.6%, and Xiao et al. [20] report that the frequency of the 1236T variant is 67.8% in Chinese Han. The MDR1 1236C>T mutation may induce a change in the substrate binding site and reduce the P-gp activity [9,21]. Zhang et al. [7] has found that patients with the TT genotype of 1236C>T consume significantly more fentanyl than those with the CC and CT genotypes within 24 hours and 48 hours after lower segment caesarean section surgery, and we got the same results within 24 hours and 48 hours after radical gastrectomy. Elkiweri et al. [22] have indicated that fentanyl concentration in the rat's brain is decreased in the presence of the P-gp inhibitor verapamil. P-gp is a transmembrane efflux pump from inside the cell [8]. These results could not be explained by P-gp distributed in the blood-brain barrier but by P-gp in the liver. The decrease of P-gp activity in hepatocytes of the TT genotype groups means that fentanyl is accumulated in the liver and metabolized by hepatic microsomal enzymes, resulting in increased consumption of fentanyl [7].
In addition, we also found that there was no significant difference in postoperative fentanyl consumption among the 2677G>T/A and 3435C>T genotypes, which was consistent with Kim et al.'s study [18]. In another study [21], 2677G>T/A and 3435C>T are not associated with fentanyl's pharmacokinetics and pharmacodynamics. The above The haplotype TTT required more fentanyl than the CGC haplotype at 48 hours postoperatively. MDR1 1236C>T, 2677G>T/A, and 3435C>T constructed four main haplotypes. In this study, the proportions of TTT, CGC, CAC, and TGC haplotypes were 32.2%, 23.3%, 17.8%, and 17.4%, respectively, which were consistent with previous studies [23]. These data showed that the TTT haplotype was the predominant genotype of Chinese Han populations. No studies have analyzed whether MDR1 haplotypes contribute to the individual variability in postoperative fentanyl consumption. We found that the MDR1 gene haplotypes constructed from the SNPs, 1236C>T, 2677G>T/A, and 3435C>T, could predict the individual variability in postoperative fentanyl consumption. TTT haplotype carriers needed more fentanyl within the first 48 hours after surgery, while CGC haplotype carriers needed less fentanyl. It has been reported that the efflux ability of P-gp in cells transfected with MDR1 CGC is higher than that in cells overexpressing the MDR1 TTT variant haplotype [12]. Thus, our results could also be explained by the transport activity of P-gp in hepatocytes, which were decreased in the TTT haplotype and increased in the CGC haplotype, leading to differences in fentanyl metabolism. Overall, using the commonly detected haplotypes (1236C>T, 2677G>T/A, and 3435C>T) could be more efficient compared to just studying only one polymorphism. Our study provided preliminary evidence that the MDR1 haplotype could influence fentanyl response.
Postoperative nausea, vomiting, and dizziness are common side effects of fentanyl. Our data showed that the fre-quency of nausea, vomiting, and dizziness was 34.8%, 17.8%, and 27.4%, respectively. No significant differences in the aforementioned side effects were found among 1236C>T, 2677G>T/A, 3435C>T, and their haplotypes within 24 hours and 48 hours postoperatively. Severe side effects, such as respiratory depression, were not detected in this study.
There were some limitations in our study. First, the 2677G>T/A allele G could mutate to allele A or T, and the frequency of the allele A variant was low. We only observed seven AA carriers in this study, while limited sample sizes might affect the objectivity of this research to a certain extent. Second, whether the MDR1 1236C>T SNP and haplotypes affect the efflux ability of P-gp-mediated fentanyl in hepatocytes is only speculation and needs further verification. However, our findings provided preliminary evidence that the MDR1 1236C>T polymorphism and haplotypes composed of 1236C>T, 2677G>T/A, and 3435C>T greatly contributed to the individual variability in postoperative analgesia with fentanyl. As far as we know, this is the first study to explore the effect of MDR1 gene haplotypes on fentanyl analgesia. It is possible to improve the perioperative pain management by preoperative MDR1 genotyping.

Conclusion
The MDR1 gene 1236C>T polymorphism and haplotypes composed of 1236C>T, 2677G>T/A, and 3435C>T greatly contributed to the individual variability of postoperative fentanyl consumption in patients undergoing radical gastrectomy.

Data Availability
The data used to support the findings of this study may be released upon application to the Department of Anesthesiology, Third Xiangya Hospital, Central South University, which can be contacted at xy3irb@163.com.