In the absence of prophylactic antiemetic medication, the overall risk of developing nausea and/or vomiting (PONV) in the postoperative period is approximately 30%. In high-risk groups, the likelihood of PONV events may even increase individually up to 80%.1–3 Many efforts have been made to prevent this unpleasant side effect of anesthesia, but a considerable percentage of patients do not respond satisfactorily despite prophylactic or therapeutic measures.4–7
On the other hand, PONV may be successfully prevented or treated by administering 5-hydroxytryptamine Type 3 (5-HT3) receptor antagonists.8 This finding, together with the fact that the activation of 5-HT3 receptors on vagal gastrointestinal afferents or in the central chemoreceptor trigger zone may provoke acute emesis, may indicate an involvement of the serotonin system in the pathogenesis of PONV.9,10
Besides the multifactorial genesis of PONV, which is currently regarded as a major cause of possible treatment failure, a genetic component may play an important role. In many patients affected by extreme or repeated PONV events, a family history of common nausea or vomiting has been documented.11–13 It has therefore been hypothesized that genetic variations in 5-HT3 genes may modulate susceptibility to nausea or vomiting. There are several 5-HT3 receptor genes (5-HT3A–E) with high sequence homology.14,15 In particular, the genes for the subunits 5-HT3A (HTR3A) and 5-HT3B (HTR3B) are located close together on chromosome 11q23.1. The 5-HT3B subunit is considered only to be effective in conjugation with 5-HT3A and may specifically modify its function. Both subunits are coexpressed in diverse cerebral and intestinal regions and possibly form the 5-HT3 receptor as a heteromeric complex.16–20
The possible contribution of genetic HTR3A and HTR3B polymorphisms to nausea and vomiting has already been examined in a cohort of patients undergoing cancer chemotherapy, and it has been demonstrated that patients who were homozygous for the −100_ −102delAAG deletion in the promoter region of the HTR3B gene experienced vomiting more frequently.21,22
In this study, we investigated both HTR3A and HTR3B genes in patients with postoperative vomiting (POV) after general anesthesia. The frequencies of the identified genetic variants were compared with those in a control group of patients without POV.
The study was approved by the Leipzig University ethics committee.
A total of 205 adult patients who met the following criteria were considered for the subsequent study.
- ASA physical status I–III
- Elective (nonemergency) surgery with a duration of at least 60 min
- General (balanced) anesthesia with volatile anesthetics
- No use of nitrous oxide
- Orotracheal intubation
Patients were ineligible if they had suffered preoperatively from gastroesophageal reflux, increased intraabdominal pressure, relevant gastrointestinal obstruction, or vestibular dysfunction, or if they had been treated with emetogenic drugs (e.g., chemotherapy). This preliminary inclusion was performed in the preanesthetic interview before surgery where the patients had to give their first consent on the standardized anesthetic interview protocol.
General anesthesia was induced IV with propofol (2 mg/kg), fentanyl (2–3 μg/kg) or sufentanil (0.5–0.6 μg/kg), and rocuronium (0.6 mg/kg) for orotracheal intubation and was maintained with isoflurane or desflurane (minimum alveolar anesthetic concentration 0.9–1.1) in an air/oxygen mixture under standardized monitoring conditions (including neuromuscular monitoring). If necessary, the patients received repeated boli of the opioid and the neuromuscular blocker used. Postoperatively, piritramide was allowed in all patients for pain management; cholinesterase inhibitor-based reversal drugs were not administered.
After general anesthesia, the patients were stratified into two study arms. Patients in the subsequent POV group developed vomiting in the recovery room, where they were required to have vomited at least once during an observation period of 6 h (i.e., “early onset”). The severity of vomiting was not considered. Intraoperative PONV prophylaxis (dolasetron 12.5 mg IV and 8 mg dexamethasone IV according to the department's standard operating procedure) did not lead to exclusion from this group.
Patients were allocated to the control group if they did not complain of any nausea or vomiting within a 24-h postoperative follow-up survey. They were excluded from the control group if they received any antiemetic prophylaxis or therapy intraoperatively or postoperatively. The patient data of both groups were collected by the anesthesiologist responsible for the recovery room.
The inclusion criteria were reexamined by the authors 24 h after surgery. Patients with “later onset” nausea and/or vomiting (>6 h after surgery) were not included either in the POV group or in the control group.
The definitive written informed consent was obtained when the POV and control patients were no longer taking all sedatives and opiates. After this, 10 mL EDTA anticoagulated venous blood samples were taken for further DNA testing. The individual data collected comprised gender, height, weight, daily use of tobacco and alcohol, medical history, history of PONV, accompanying medication, and type of surgery. Based on these data, the putative risk of developing PONV was evaluated according to Apfel simplified risk score.23–25
Genomic DNA was prepared using the Invisorb blood giga kit (Invitek GmbH, Berlin, Germany). Specific DNA regions were amplified by polymerase chain reaction in 25 μL containing 100 ng DNA, 200 μM dinucleotide triphosphate, 1 μM of each primer, 10X buffer, 1 U Ampli Taq Gold™ polymerase (Roche, Branchburg, NJ), or Bio-X-ACT™ polymerase (Bioline GmbH, Luckenwalde, Germany) for long template amplification.
Primer pairs were designed for amplification of the nine exons, the exon/intron boundaries, and the 5′ untranslated region of the HTR3A and HTR3B genes (seven and four reactions, respectively). In the POV group, subsequent sequence analysis was performed using a big-dye primer cycle sequencing kit on an ABI 310 capillary DNA sequencer (Applied Biosystems, Forster City, CA). Eleven regions in the HTR3A gene and nine in the HTR3B gene were sequenced in a forward and reverse direction (primer sequences and reaction conditions are available on request).
If HTR3A and HTR3B variants were identified in the POV group, their occurrence in the control group was examined by using denaturing high performance liquid chromatography (DHPLC) on automated instrumentation equipped with a DNASep column (Transgenomic, San Jose, CA). DHPLC was performed with three additionally designed polymerase chain reaction amplifications in the HTR3A region and four in the HTR3B region. For DHPLC running of the specific DNA sections, individual analytical gradient conditions were compiled. The runs of direct sequencing and heterozygous DHPLC profiles were checked by visual inspection. All primers and reaction conditions are available on request.
All statistical analyses were performed using SPSS for Windows (V. 14, SPSS, Chicago, IL). The level of significance was set to 0.05 for all tests.
In the absence of normality (Kolmogorov-Smirnov test), the variables of groups were compared using the Mann–Whitney U-test. Frequencies were compared using the χ2 test (two-tailed). If the expected cell count was <5, Fisher's exact test was applied.
To quantify the influence of specific genotypes on POV, a multivariate logistic regression was performed for global analysis of the POV and control groups. Because there were no data relevant to our hypothesis in the literature, we decided to use a backward stepwise approach for this pilot study. In an initial univariate analysis, the genetic variants (calculated on chromosomes) and classic risk factors for POV were tested for significance between the groups by cross tabs. In the following analysis, only significant or almost significant variables (P < 0.1) were included in the logistic regression model. Based on “maximum likelihood estimators” (Hosmer-Lemeshov test), all those variables were subsequently excluded, which did not significantly advance the multivariate logistic regression model (i.e., if the −2 log likelihood was significantly minimized). Interactions between the variables were not analyzed. Predictors of POV are reported as odds ratios (enclosed by a 95% confidence interval).
A total of 189 patients (95 POV patients and 94 control patients) were ultimately included in this noninterventional cohort study. The groups investigated were comparable with respect to their ASA status, duration of general anesthesia, and postoperative opioid administration (Table 1).
The patients underwent surgery in the following anatomical regions: abdominal cavity (POV: n = 31, controls: n = 35), extra-/retroperitoneal cavity (POV: n = 12, controls: n = 30), bones and joints ([orthopedic surgery] POV: n = 27, controls: n = 15), face and neck (POV: n = 15, controls: n = 9), brain (POV: n = 3, controls: n = 2), thoracic cavity (POV: n = 3), and others ([neuroplasty, mastectomy] POV: n = 4, controls n = 3).
Demographic Data and Risk Factors
As expected, the percentage of known emetogenic factors was significantly higher in the POV group than in the controls (Table 1). This was reflected in median risk scores of 3 in the POV group and 2 in the control group. No patient in either group had a risk score of zero, because almost all received opioids in the postoperative period. The POV group included approximately 2.5 times more female patients. The protective effect of smoking was confirmed; the body mass index had no influence on the occurrence of POV.26,27 The consumption of alcohol reduced the risk significantly. The higher consumption of alcohol in the control group was probably due to the higher proportion of male patients.
Genetic Polymorphisms in the HTR3A Gene (5-HT3A Subunit)
By analyzing the sequence of the HTR3A gene, we identified 16 different single nucleotide polymorphisms in the POV group and 10 in the control group (Fig. 1, Table 2), but there was no significant difference in the total number of polymorphisms. Five of the 16 variants were located in the coding region of the gene: c30C>T, c576G>A, c684C>T, c831G>A, and c1377A>G.
Genetic Polymorphisms in the HTR3B Gene (5-HT3B Subunit)
Of the overall 19 polymorphisms detected, 18 were identified in the POV group and 16 in the control group (Fig. 2, Table 3). Seventeen single nucleotide polymorphisms, a 3-base pair deletion variant (−100_ −102delAAG in the promoter region), and a 2-base pair deletion in intron 5 (c5+201_+202delCA) were found. Four of five variants of the coding regions led to an amino acid substitution: c386A>C (pTyr129Ser), c466A>C (pSer156Arg), c547G>A (pVal183Ile), and c784T>A (pTyr262Asn).
Genetic “Risk Variations” for POV
The following variables were included in the multivariate logistic regression model (P < 0.1): gender, history of PONV, smoker status, and the subsequent genetic variants: HTR3A: c5-56G>T, c1377A>G; HTR3B: c3-45T>C, c5+201_+202delCA, and c6-137C>T. As a result, the HTR3A variant c1377A>G as well as female gender, nonsmoker status, and history of PONV were independently associated with a higher risk of POV in this model. In contrast, two genetic HTR3B variants significantly reduced the incidence of POV: c5+201_+202delCA and c6-137C>T (Table 4).
This pilot study aimed at investigating the genes for the 5-HT3A and 5-HT3B subunits (HTR3A and HTR3B) of the serotonin receptor for genetic variants in a cohort of POV patients (more precisely, those who developed vomiting in the early postoperative period of 6 h) and controls. We expected an initial insight into the possible genetic background of POV susceptibility. As previously demonstrated in cancer patients treated with chemotherapeutics, HTR3A and HTR3B polymorphisms may contribute to a higher incidence and intensity of nausea and vomiting.21,22,28 For example, patients who were homozygous for the HTR3B −100_−102AAG deletion of the noncoding promoter region experienced vomiting more frequently under chemotherapy.22 We did not confirm this specific finding, probably because the investigated cohorts differed between the studies. We supposed that the external triggers of nausea and vomiting would be milder in patients exposed to the conditions of general anesthesia. We therefore expected a different and comparatively greater genetic impact of HTR3A and HTR3B variants in POV patients.
About 5000 base pairs per POV patient were screened and various genetic variants were identified in the promoter, coding, and exon/intron boundaries of both genes.
We found in the logistic regression analysis that three genetic variants (HTR3A: c1377A>G; HTR3B: c5+201_+202delCA; c6-137C>T) were independently able to influence the incidence of POV significantly. Our pilot study was also not, however, powered to demonstrate a significant change in the allelic frequencies of these variants in the χ2 test with Bonferroni correction for multiple comparisons. According to a power analysis,29 a prospective study that aims to investigate all three genetic variants would require a sample size of about 300 patients in each group (600 patients, 1200 chromosomes) to demonstrate significant differences with a statistical power of 0.8.
The results should not, therefore, be overvalued. The amount of variants detected (16 in the HTR3A gene, 19 in the HTR3B gene) reflected a remarkable genetic heterogeneity. How strong their functional effect on POV or PONV is, or whether there is a functional impact at all, cannot be answered in this study. Most of the variants were located beyond the protein coding region or did not lead to an amino acid exchange, but they may have regulatory effects on mRNA synthesis. Furthermore, the genetic variants identified were primarily related to the phenotype “POV patient,” but it has to be remembered that this phenotype is not uniform and that several factors may contribute to the putative genetic association. All these aspects should be addressed in subsequent adequately powered gene association studies. POV and control patients should then consistently meet classic risk factors and influencing surgical factors as cross-matched pairs.
In conclusion, this analysis should be regarded as a preliminary study and a starting point for future targeted genetic investigations, all the more because the complete sequencing of two genes (HTR3A and HTR3B), as performed, involves considerable effort and cost. The results confirm the multifactorial genesis of POV and suggest a possible association of POV with genetic HTR3A and HTR3B variants.
The authors thank Kerstin Krist and Dr. Thilo Busch for their expert technical assistance.
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