|Year : 2018 | Volume
| Issue : 3 | Page : 109-114
Polymorphism of serotonin transporter gene (SLC6A4) and its relation to tramadol dependence
Heba Baz1, Dina Mahmoud1, Sara A Naser2, Walaa Rabee1, Nahla Fawzy1, Ola El-Sissy1, Samir A Magd2, Dalia Enaba2
1 Department of Clinical and Chemical Pathology, Cairo University, Giza, Egypt
2 Department of Psychiatry, Cairo University, Giza, Egypt
|Date of Submission||07-Mar-2018|
|Date of Acceptance||22-Mar-2018|
|Date of Web Publication||11-Oct-2018|
Department of Psychiatry, Kasr Al Ainy Cairo University, Building 7540 Street 23 From 9 Street Mokattam, Cairo 11439
Source of Support: None, Conflict of Interest: None
Background Many genes have been proven to be linked to substance use disorder, on top were serotonin receptor and transporter genes.
Aim The aim of this study was to determine the frequency of 5-HTTVNTR allele variants of the serotonin transporter gene and the presence of polymorphic serotonin receptor gene (5-HTR1A) in tramadol-dependent patients in comparison with controls and to explore the association between the groups of tramadol dependent with and without psychiatric comorbidity and 5-HTTVNTR allele variants.
Patients and methods This was a cross-sectional case–control outpatient study. The study sample consisted of 90 patients, with 60 tramadol dependents (55 males and five females) and 30 healthy controls. All patients fulfilled the Diagnostic and Statistical Manual of Mental Disorders, 4th ed., Criteria for substance dependence (on tramadol). Genotyping of the 5-HTTVNTR gene and 5-HT1A receptor gene promoter was done employing real-time PCR and PCR/RFLP, respectively.
Results A significant association was found between the 5-HTT gene 10 allele polymorphism and tramadol dependence. No significant association was observed with the 5-HT receptor gene polymorphism.
Conclusion This study detected an association between the presence of 5-HTTVNTR less transcriptional-efficient genotypes and tramadol dependence, and these genotypes increase psychiatric comorbidity with tramadol dependence, which suggests that the modulation of the serotoninergic system might be implied in psychiatric problems in tramadol dependence.
Keywords: 5-HTR1A gene, 5-HTTVNTR allele gene, serotonin, serotonin receptor, tramadol, transporter gene
|How to cite this article:|
Baz H, Mahmoud D, Naser SA, Rabee W, Fawzy N, El-Sissy O, Magd SA, Enaba D. Polymorphism of serotonin transporter gene (SLC6A4) and its relation to tramadol dependence. Egypt J Psychiatr 2018;39:109-14
|How to cite this URL:|
Baz H, Mahmoud D, Naser SA, Rabee W, Fawzy N, El-Sissy O, Magd SA, Enaba D. Polymorphism of serotonin transporter gene (SLC6A4) and its relation to tramadol dependence. Egypt J Psychiatr [serial online] 2018 [cited 2018 Oct 21];39:109-14. Available from: http://new.ejpsy.eg.net/text.asp?2018/39/3/109/243026
| Introduction|| |
Tramadol is the third most abused substance in Egypt after cannabinoids and alcohol (Hamdi et al., 2013). This warrants a focused research on its basis.
Tramadol has inhibitory actions on the 5-hydroxytryptamine type 2C (5-HT2C) receptor. This could be responsible for reducing depressive and obsessive-compulsive symptoms in psychiatric patients with comorbid neurological illnesses (Ogata et al., 2004). The antidepressant effect may be also owing to its reuptake inhibition effect on monoamine norepinephrine and serotonin (5-HT), making it similar to action of dual-action drugs like mirtazapine and venlafaxine drugs. This action may also account for its lowering of the seizure threshold (Rojas-Corrales et al., 1998; Hara et al., 2005; Barber, 2011). The serotoninergic pathway is widely distributed in the brain and has been implicated in the control of behavior, mood, impulsivity, and drug dependence, among others (Koob and Volkow, 2010).
The serotonin is released in the synaptic cleft, to act on a receptor, and is then rapidly taken up by the presynaptic neuron via the aid of a transporter protein (Thorn et al., 2013).
The serotonin receptor type 5-HT1A is distributed in the brain in the raphe nucleus, cerebral cortex, amygdala, and hippocampus. The transcription of this protein is controlled through the 5-HT1A receptor gene. A specific variant reported in the promotor region of this gene C/G −1019 (rs6295) has been studied in relation with mood disorders, anxiety, and depression (Strobel et al., 2003; Savitz and Drevets, 2009; Kishi et al., 2013) but not studied in relation to drug abuse predisposition. It is hypothesized that this promotor region variant can alter the binding of serotonin to the receptor and thus modulating the behavior toward a drug (David et al., 2005).
The serotonin transporter (5-HTT) is a solute carrier family 6 member 4 (SLC6A4) protein coded by a gene found on chromosome 17 (Sun et al., 2014). A variable number of tandem repeats (VNTR) in the second intron (STin2) may exhibit one of the three following alleles: 9, 10, and 12 repeats of 17-bp sequences (Hahn and Blakely, 2002). The 12-repeat alleles have higher transcriptional activity compared with the 10-repeat alleles. Increased transcription of 5-HTT gene hypothetically will lower the serotonin in the synaptic cleft (Lovejoy et al., 2003).
To our knowledge, no research has been performed tackling the 5-HT or 5-HTT gene in tramadol-dependent patients.
On the basis of these earlier findings, we hypothesize that the frequency of the allele 10 (of 5-HTT) and the G allele (of the 5-HTR), whether in homozygous or heterozygous patterns, is more in the tramadol-dependent patients than in control. A group of tramadol addicts with psychiatric comorbidity was added to control for the psychiatric diseases as a confounding factor.
| Patients and methods|| |
This is a descriptive cross-sectional study. Approval from the Ethical and Research Committee of the Departments of Psychiatry, School of Medicine, Cairo University was obtained in accordance with the provisions of the world medical association’s Declaration of Helsinki. A written consent of the participant was taken after describing the steps of assessment.
The study included 90 patients recruited from Cairo University Hospital, Egypt. Patients were divided into two groups: group 1 was the tramadol-dependence group, which included 30 tramadol-dependent patients with psychiatric comorbidity and group 2 had another 30 tramadol-dependent patients without psychiatric comorbidity. The control group included 30 age-matched and sex-matched individuals recruited from pre-employment testing programs, and a multidrug testing profile was performed to confirm no drug use (ABON multidrug one step screen test panel kit; ABON Biopharm, Hangzhou, China).
Psychiatric comorbidity was assessed using the Structured Clinical Interview for Diagnostic and Statistical Manual of Mental Disorders, 4th ed., Axis I disorders (SCID-I) scale (Association AP, 2000). Patients were excluded from the study if they refused to sign the consent form. All eligible participants (based on self-reports of drug use) were then subjected to a urinalysis for psychoactive substance assessments to confirm their use of tramadol. The controls (30), matched for age and sex with the patients, were free of history of psychiatric disease and had a multidrug testing profile to confirm no drug use. Participants were interviewed in quiet, private, and comfortable settings, and the nature and scope of the study was discussed with each participant. Written informed consent was obtained from all patients before each interview. Data collected from the patient s by way of a semistructured interview. The Addiction Severity Index (McLcllan et al., 1980) was used to assess all participants. The Addiction Severity Index is a semistructured interview designed to address seven potential problem areas in substance-using patients: medical status, employment and support, drug use, alcohol use, legal status, family/social status, and psychiatric status. The SCID-I scale is a semistructured interview for making the major Diagnostic and Statistical Manual of Mental Disorders, 4th ed., Axis I diagnoses (Association AP, 2000). The SCID is broken down into separate modules corresponding to categories of diagnoses. Most sections begin with an entry question that would allow interviewer to skip the associated questions if not met. For all diagnoses, symptoms are coded as present, subthreshold, or absent.
Sampling and DNA extraction
A sample of 2-ml blood in EDTA-containing tube was acquired under aseptic venipuncture. Genomic DNA of peripheral blood lymphocytes was extracted from peripheral blood lymphocytes using a standard column extraction protocol (GeneJET Nucleic acid extraction; Thermo-Fisher Scientific, Waltham, MA, USA).
Genotyping of 5-HTTVNTR intronic region
The intron 2 region of the 5-HTT gene containing the VNTR polymorphism was genotyped using SYBR Green-based real-time PCR, and the consequent melting curve analysis was performed on LightCycler 2.0 Instruments (Roche diagnostics, Indianapolis, IN, USA) using the primer sequence shown in [Table 1]. The 20-μl reaction mixture included 9 μl of PCR grade water, 1 μl of each of the primers ([Table 1]), and 4 μl of Master Mix, 5× concentration, to which 50 ng of DNA were added. The following conditions were used: initial denaturation at 95°C for 10 min, followed by 45 cycles of denaturation at 95°C for 10 s, annealing at 59°C for 20 s, and extension at 72°C for 25 s. After amplification, melting curve analysis was performed by heating the reaction mixture from 65 to 95°C at a rate of 0.1°C/s.
|Table 1 Primers used for amplification of 5-HTTVNTR and 5-HT1A C/G (−1019)|
Click here to view
Genotyping of 5-HT1A receptor gene promoter region polymorphism C/G (−1019) (rs6295)
The reactions were performed in a total volume of 25 μl, containing Dream Taq green buffer, 10 pmol of each primer ([Tables 1]), 0.5 U Taq (Dream Taq Green PCR Master Mix, 2×), (Thermo-fisher Scientific), and 100 ng of genomic DNA. After an initial 5 min at 95°C, each cycle consisted of 45 s at 95°C, 45 s at 56°C, and 45 s at 72°C, with final elongation of 10 min at 72°C. The amplicon was 182 bp long. DNA was digested with the restriction enzyme MaeII (Thermos-fisher Scientific), an isoschizomer to the HpyCH4IV restriction enzyme listed in the original method Choi et al. (2010), which cuts at the −1019G site, and the product was electrophoresed in 3% agarose gels and stained with ethidium bromide. Homozygous genotypes were identified by the presence of a single 182-bp band (C/C), or bands of 158 and 24 bp (G/G). The heterozygous genotype had three bands: 182, 158, and 24 bp (G/C).
The distribution of the genotypes in tramadol abuse group was compared with that of the control group by using the χ2-test (3×2 contingency table). To compare the allele frequencies of the two groups, a 2×2 contingency (χ2) test was used. Odds ratios and 95% confidence intervals were used to quantify any association between polymorphism and tramadol abuse. 3×3 contingency tables were used to determine linkage disequilibrium of the studied single-nucleotide polymorphisms. A P value of 0.05 was considered statistically significant. The SPSS, version 17.0 program (SPSS Inc., Delaware, Chicago, USA), was used for statistical analysis.
| Results|| |
This study included 90 participants: 30 tramadol abusers with no psychiatric comorbidity (28 were males and two were females), with mean age of 25.80±4.3 years; 30 tramadol abusers without psychiatric comorbidity (27 were males and three were females), with mean age of 26.50±5.0, and 30 control individuals. Collectively tramadol addicts grouped together were found to have a significantly higher frequency of the 5-HTTVNTR variant genotype 12/10 and the 10/10 genotypes, whereas no difference between groups was found for 5-HTR1A C/G (−1019) (rs6295) ([Table 2]). However, further subdivision of the groups to tramadol addicts with a comorbid psychiatric and tramadol addicts without a comorbid psychiatric condition showed a higher frequency of the variant VNTR in the group with comorbid psychiatric conditions compared with the controls and the same did not apply to the group of tramadol addicts with no associated comorbidity. These findings were confirmed using alleles for comparison. Additionally, a higher frequency of the variant allele 10 was seen between the tramadol dependent with comorbid psychiatric conditions and the group without psychiatric comorbidity. The most prevalent psychiatric disorders in group 1 were as follows: 40% bipolar versus substance-induced mood, 40% psychosis (10% schizophrenia and 30% substance-induced psychosis), and 20% major depressive disorder ([Table 3],[Table 4],[Table 5]).
|Table 2 Distribution of genotypes and allele frequencies in both healthy controls and patients|
Click here to view
|Table 3 Wild and mutant genotype frequencies of 5-HTTVNTR gene between tramadol addicts and control groups|
Click here to view
|Table 4 Genotype frequencies of 5-HTTVNTR gene between tramadol addicts with and without comorbid psychiatric conditions and control groups|
Click here to view
|Table 5 Genotype frequencies of 5-HTTVNTR genes among tramadol-dependent patients with and without comorbid psychiatric conditions and control group|
Click here to view
| Discussion|| |
In the current study, we found that tramadol dependence per se is not associated with the evaluated 5-HTTVNTR variant genotypes 12/10 and 10/10, instead our data suggest that it is the comorbid psychiatric disorders that are associated with these genotypes. To our knowledge, this is the first study of the association of this 5-HTTVNTR polymorphism to tramadol dependence. Previously, other studies were done to detect its relation to cocaine dependence (Patkar et al., 2002), methamphetamine (Hong et al., 2003), alcohol (Saiz et al., 2009), and heroin (Li et al., 2002). They failed to corroborate the previously reported association between the polymorphism in the 5-HTTVNTR gene and these substance dependencies, except for one study, where a group of researchers (Tan et al., 1999) found a significant excess of both 10/10 and 12/10 genotypes and mutant allele (10) in addicts when compared with their control group among Singaporeans of Chinese ancestry.
We have found that there was a significant difference between the tramadol with psychiatric comorbidity group and control group among mutant allele frequencies (53.3 and 15%, respectively) (odds ratio: 6.476; 95% confidence interval: 2.709–15.480; P<0.001), and mutant genotype frequencies (70 and 23.3%, respectively) (odds ratio: 7.667; 95% confidence interval: 2.424–24.245; P<0.001). Meanwhile, several studies have shown that low transcriptional activities of 5-HTTVNTR genotypes are associated with neuropsychiatric disorders, including depression (Anyanwu et al., 1994) and suicidal behavior (Anguelova et al., 2003).
The higher frequency of 12/10 genotype (which indicates lower transcription activity of the 5-HTT) and the genotype 10/10 (which indicates more severe abnormality in the transcription activity), might be acting as an attempt to increase the 5-HT in the synaptic cleft in these patients to overcome the already low 5-HT levels (Nordquist and Oreland 2010). Low transcriptional function genotypes of 5-HTTVNTR could be related to increased levels of serotonin during brain development, causing functional alterations and inhibiting the outgrowth of the serotonin system and thus being associated with risk for neuropsychiatric disorders later in life (Schenkel et al., 2011).
We proposed that the 5-HTTVNTR polymorphism is associated with tramadol and not other substances, which may be related to other neurotransmitter systems. However, this will need further extensive study on the transporter protein genes of these neurotransmitters.
However, we could not prove shared genetic vulnerability between tramadol and psychiatric disorders; meanwhile, a significant genetic correlation between addiction and psychiatric disorders was proved on alcoholism (Di Sclafani et al., 2008). This study opens a venue to further studies on the 5-HTT gene polymorphism in addiction and psychiatric disorders.
Surprisingly, we found no significant association between the C(×1019) G gene polymorphism (of 5-HT1A receptor) and the tramadol dependence whether with or without psychiatric disorders. Meanwhile, others (Huang et al., 2004) found that this polymorphism is significantly higher in polysubstance users with and without psychiatric comorbidity than in healthy controls and in postmortem cases (suicide victims than in nonsuicide controls). Psychiatric patients included those with substance use disorder, those with mood and psychotic disorder, and those with anxiety disorders. The difference between our results and other studies were because we studied tramadol dependence mainly. However, other studies included more psychiatric disorders. However tramadol dependence until now was only found linked to the polymorphism of the OPRM and the ABCB genes in the study by Enabah et al. (2014).
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
American Psychiatric Association (2000). Diagnostic and statistical manual of mental disorders, (4th ed., text revision). Washington, DC: American Psychiatric Association.
Anderson J (1987). Communication research: Issues and methods. New York: McGraw Hill.
Anguelova M, Benkelfat C, Turecki G (2003). A systematic review of association studies investigating genes coding for serotonin receptors and the serotonin transporter: I. Affective disorders. Mol Psychiatry 8:574–591.
Anyanwu E, Harding GFA, Edson A (1994). The involvement of serotonin (5-hydroxytryptamine) in photosensitive epilepsy. J Basic Clin Physiol Pharmacol 5:179–206.
Barber J (2011). Examining the use of tramadol hydrochloride as an antidepressant. Exp Clin Psychopharmacol 19:123.
Choi WS, Lee BH, Yang JC, Kim YK (2010). Association study between 5-HT1A receptor gene C (-1019) G polymorphism and panic disorder in a Korean population. Psychiatry Investig 7:141–146.
David SP, Murthy NV, Rabiner EA, Munafó MR, Johnstone EC, Jacob R et al.
(2005). A functional genetic variation of the serotonin (5-HT) transporter affects 5-HT1A receptor binding in humans. J Neurosci 25:2586–2590.
Di Sclafani V, Finn P, Fein G (2008). Treatment-naive active alcoholics have greater psychiatric comorbidity than normal controls but less than treated abstinent alcoholics. Drug Alcohol Depend 98:115–122.
Enabah D, El Baz H, Moselhy H (2014). Higher frequency of C. 3435 of the ABCB1 gene in patients with tramadol dependence disorder. Am J Drug Alcohol Abuse 40:317–320.
Hahn MK, Blakely RD (2002). Monoamine transporter gene structure and polymorphisms in relation to psychiatric and other complex disorders. Pharmacogenomics J 2:217–235.
Hamdi E, Gawad T, Khoweiled A, Sidrak AE, Amer D, Mamdouh R et al.
(2013). Lifetime prevalence of alcohol and substance use in Egypt: a community survey. Subst Abus 34:97–104.
Hara K, Minami K, Sata T (2005). The effects of tramadol and its metabolite on glycine, γ-aminobutyric acid A, and N
-methyl-d-aspartate receptors expressed in xenopus oocytes. Anesth Analg 100:1400–1405.
Hong CJ, Cheng CY, Shu LR, Yang CY, Tsai SJ (2003). Association study of the dopamine and serotonin transporter genetic polymorphisms and methamphetamine abuse in Chinese males. J Neural Transm 110:345–351.
Huang Y, Battistuzzi C, Oquendo MA, Harkavy-Friedman J, Greenhill L, Zalsman G et al.
(2004). Human 5-HT1A receptor C (−1019) G polymorphism and psychopathology. Int J Neuropsychopharmacol 7:441–451.
Kishi T, Yoshimura R, Fukuo Y, Okochi T (2013). The serotonin 1A receptor gene confer susceptibility to mood disorders: results from an extended meta-analysis of patients with major depression and bipolar disorder. Eur Arch Psychiatry Clin Neurosci 263:105–118.
Koob GF, Volkow ND (2010). Neurocircuitry of addiction. Neuropsychopharmacology 35:217–238.
Li T, Liu X, Zhao J, Hu X, Ball DM, Loh E-W et al.
(2002). Allelic association analysis of the dopamine D2, D3, 5‐HT2A, and GABAAγ2 receptors and serotonin transporter genes with heroin abuse in Chinese subjects. Am J Med Genet 114:329–335.
Lovejoy EA, Scott AC, Fiskerstrand CE, Bubb VJ, Quinn JP (2003). The serotonin transporter intronic VNTR enhancer correlated with a predisposition to affective disorders has distinct regulatory elements within the domain based on the primary DNA sequence of the repeat unit. Eur J Neurosci 17:417–420.
McLcllan AT, Luborsky S, Woody GE, O’Brien CP (1980). An improved diagnostic instrument for substance abuse patients: the Addiction Severity Index. J Nerv Ment Dis 21:28–33.
Nordquist N, Oreland L (2010). Serotonin, genetic variability, behaviour, and psychiatric disorders-a review. Ups J Med Sci 115:2–10.
Ogata J, Minami K, Uezono Y, Okamoto T, Shiraishi M, Shigematsu A, Ueta Y (2004). The inhibitory effects of tramadol on 5-hydroxytryptamine type 2C receptors expressed in xenopus oocytes. Anesth Analg 98:1401–1406.
Patkar AA, Berrettini WH, Hoehe M, Hill KP, Gottheil E, Thornton CC et al.
(2002). No association between polymorphisms in the serotonin transporter gene and susceptibility to cocaine dependence among African-American individuals. Psychiatr Genet 12:161–164.
Rojas-Corrales MO, Gibert-Rahola J, Micó JA (1998). Tramadol induces antidepressant-type effects in mice. Life Sci 63:PL175–PL180.
Saiz PA et al.
(2009). Differential role of serotonergic polymorphisms in alcohol and heroin dependence. Prog Neuropsychopharmacol Biol Psychiatry 33:695–700.
Savitz JB, Drevets WC (2009). Imaging phenotypes of major depressive disorder: genetic correlates. Neuroscience 164:300–330.
Schenkel LC, Bragatti JA, Torres CM, Martin KC, Gus-Manfro G, Leistner-Segal S, Bianchin MM (2011). Serotonin transporter gene (5HTT) polymorphisms and temporal lobe epilepsy. Epilepsy Res 95:152–157.
Strobel A, Gutknecht L, Rothe C (2003). Allelic variation in 5-HT1A receptor expression is associated with anxiety-and depression-related personality traits. J Neural Transm 110:1445–1453.
Sun S, Wang K, Lei H, Li L, Tu M, Zeng S et al.
(2014). Inhibition of organic cation transporter 2 and 3 may be involved in the mechanism of the antidepressant-like action of berberine. Prog Neuropsychopharmacol Biol Psychiatry 49:1–6.
Tan EC, Yeo BK, Ho BK, Tay AH, Tan CH (1999). Evidence for an association between heroin dependence and a VNTR polymorphism at the serotonin transporter locus. Mol Psychiatry 4:215.
Thorn CF, Klein TE, Altman RB (2013). PharmGKB: the pharmacogenomics knowledge base. Methods Mol Biol 1015:311–320.
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]