Frequency of Serotonin Transporter Promote Gene Polymorphism in Opioid Addicts

¹International Campus, Shahid Sadoughi University of Medical Sciences, Iran

²Research and Clinical, Centre for Infertility, Shahid Sadoughi University of Medical Sciences, Iran

³Immunology department, Shahid Sadoughi University of Medical Science, Iran

Corresponding Author:

Amir Hossein Mansourabadi
Immunology Department
Shahid Sadoughi University of Medical Science, Iran
Tel: 09380097059
E-mail: a.mansourabadi.67@gmail.com

Received date: Jan 23, 2016; Accepted date: Apr 15, 2016; Published date: Apr 21, 2016

Citation: Nazari T, Ghasemi N, Mansourabadi A (2016) Frequency of Serotonin Transporter Promote Gene Polymorphism in Opioid Addicts. J Addict Res Ther 7: 278. doi:10.4172/2155-6105.1000278

Copyright: © 2016 Nazari T, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Visit for more related articles at Journal of Addiction Research & Therapy

Abstract

Background: The human serotonin transporter promoter (5-HTT) gene genotype was previously reported to be associated with temperament and personality traits at risk for substance abuse in the subjects with antisocial behaviour. Association studies of polymorphism in the promoter region of this gene in addiction have shown conflicting results. A common 44-base pair insertion/deletion polymorphism in the region of 5-HTT has been observed. This biallelic functional polymorphism designated long (L) and short (S) promotors, which affect 5-HTT gene expression since the short variant is associated with lower transcriptional activity. Since there is strong evidence of a disturbance in brain serotonergic transmission among antisocial, impulsive, anxiety, and different kinds of addiction, the purpose of present study was to evaluate the frequency of these genotypes among adolescents with opioid addiction.
Method: One hundred and forty opium dependent males, aged 20-50 years, entered the study, after signing informed written consent. They referred to the rehabilitation centre to treat their addiction. Three questionnaires, general, Minnesota Multiphasic Personality Inventory (MMPI), genetic and family pedigree) were filled out by experts. Information obtained from these questionnaires included their age, gender, consanguinity of parents, type of substance abuse, socioeconomic position, psychiatric disorder and family history of drug abuse and any other diseases. First, behavioural disorders were studied in addict cases. Then, polymorphic region of the human serotonin transporter gene was evaluated and compared between 31 addicts and 31 normal volunteer matched in age, sex and socioeconomic situation as controls. Whole blood sample (2ml) from each participant was collected on sterile tube and stored for subsequent extraction of DNA using phenol-chloroform method. Then DNA was PCR-amplified. The PCR products were resolved in 2.5% agarose gel. Data were analysed using chi-square test and P < 0.05 was considered significant.
Results: Short allele frequency in cases and controls were 0.532 and 0.387 respectively. Long allele frequency in cases and control were 0.468 and 0.613 respectively. Despite of OR =1.8 this study indicate no significant association between frequency of short allele and addiction to opioid.
Conclusions: Although previous findings indicated association of short allele with addiction, this study did not confirm these results. Several possibilities exist for these discrepant results, including small sample size, potential population stratification, and categorical phenotypes.

Keywords

Opioid addicts; Polymorphism; Serotonin transporter promoter

Introduction

Long term use of drugs leads to the brain deformation, followed by some abnormal behaviour [1]. The molecular bases of the addictions are complex and show no obvious pattern of Mendelian transmission [2]. Etiology and identification of involved factors in addiction are particularly important for different aspects. It is tempting to consider that addictions are polygenic [3,4], while these genes act as a complex network and may interact with each other. Furthermore, other factors such as environmental variants, factors that influence on gene expression [2-5], like methylation of promoter region of some genes, and incomplete penetrance is poorly understood.

Some specific alleles of opioidergic system gene may be involved in opioid addiction like the opioid receptor and prodynorphin genes. The 5HTT system has received considerable attention in attempts to understand the determinants of different kinds of addiction. Neurotransmitters are chemicals that communicate among nerve cells, Serotonin, also known as 5-hydroxytryptamine, is one of the important brain neurotransmitters used by many neurons [6,7].

Serotonin is a signaling molecule with a widespread effect in the CNS [5] and has a very important role in different aspects of mammalian life, like food intake, emotion, mood, respiration, pain sensitivity, cardiovascular regulation, sexual behaviour, learning and memory, circadian rhythm, sensorimotor activity, and cognition [8]. Apart from its role in development, there is evidence that disturbances in central serotonin (5-HTT) function have a specific role in neuropsychiatric disorders, and in autism [9-11] like, impulsive aggression, violence, and criminality and many different phenomena, including alcoholism, clinical depression, obsessive-compulsive disorders, and hypertension [1,12-15]. Therefore genetic polymorphism seems to be involved in the biological vulnerability for psychoactive drugs use and dependence [16-18].

The serotonin transporter is a monoamine transporter protein. It transports and directs reuptake neurotransmitter serotonin from synaptic spaces into presynaptic neurons [7,19]. Research has shown that the repeat length polymorphism in the promoter of this gene is involved in serotonin uptake. The 5-HTT gene is located on chromosome 17q11.2, the 44-base pair insertion/deletion polymorphism in the promoter re-join of human serotonin transporter sequence localized 1.2 kb upstream the gene. The most frequently polymorphisms observed in the 5′-HTTLPR alleles are the short and long variants; the short variation has 14 Repeats of a sequence and the long variation has 16 repeats [5,20-22]. The short allele found to be associated with a lower level expression of the gene [1,10,20,22], while the activity of the long variant “L” resulted in higher serotonin transporter (5-HTT) expression. The short allele is typically considered as the “risk” allele, while the findings related to the long allele are discussed in the studies of psychopathy. Some studies found the evidence for the role of serotonin in autism and addiction. In contrast with these findings, some other studies failed to demonstrate an association between serotonin transporter gene polymorphism and the personality traits at risk for substance abuse and autism [6,10,23,24]. Based on the above-mentioned reasons, the present study designed to investigate the hypothesis that the frequency of the “low activity” S allele might be higher in the opioid addiction, when compared with non-addiction people.

Materials and Methods

One hundred and forty, addict men, aged 20-50 years, entered the study and signed inform written consent. They were refereed to psychiatric clinic to treat their addiction. Three questionnaires (general, MMPI, genetic and family pedigree) were completed for all participants. The general questionnaire and MMPI were filled by the participants, after it was explained for them that how to do it and the genetic and family pedigree were filled by a genetic expert. Information obtained from these questionnaires included; age, gender, consanguinity of parents, type of substance abuse, socioeconomic position, psychiatric disorder and family history of drug abuse and any diseases. In the second part of investigation 2ml blood samples were taken from 31 addicts and 31 normal matched volunteers for genetic test.

Genetic evaluation

Two ml of peripheral blood from each patient was collected in sterile tubes and stored for DNA extraction using phenol-chloroform method. The 5-HTT promoter region was amplified by polymerase chain reaction (PCR). The PCR reaction was performed in a 25 ml volume containing approximately 20 ng of genomic DNA, 200 mM of each dATP, dTTP, dGTP and dCTP, 50 mMdITP (20-Deoxyinosine 50- triphospate), 0.25 mM of each primer, 50 mMtris/HCl (pH 8.8), 12.5 mM (NH₄)₂SO₄, 10% dimethyl sulfoxide, 6.25 mg BSA, 1 mM MgCl2, and 2.5 U of Taq Gold DNA polymerase. After an initial denaturation step at 94°C for 10 min, the cycling parameters were 45 cycles with denaturation at 94°C for 1 min, annealing at 55°C for 1 min, and extension at 72°C for 1 min. The PCR products were resolved in 2.5% agarose gel containing 50 mg/ml ethidium bromide in TAE buffer (40 mMTris-acetate, 1mM EDTA pH 8.0). Each gel contained one lane of 50 bp ladder to identify the 450 bp fragment designated as L and the 406 bp fragment designated as S. Data were analysed using chi-square test and P < 0.05 was considered significant.

Results

The obtained results showed that the mean age of the addict persons who had family history of addiction was lower than the addicts without family history (Table 1). Borderline personality disorder was the most common psychiatric problem (30.5%). However, for the others schizoid personality was the most common disorders (35.9%).

Table 1: Age of addicts with and without family history, Chi-square test (p-value <0.05).

Depression and anxiety were the most common psychiatric problems in the first group, while in the second group, affective disorder, passive aggressive and somatization were more common.

The average age of addicts with family history of addiction is significantly lower than addicts without family history.

Psychological disorders are significantly higher among addict individuals without family history in comparison with addict individuals with family history.

The 5-HTTLPR genotype frequencies are shown in Table 2. The frequency for LL, LS and SS for the total are 35.5%, 37.1% and 27.4%, respectively. The proportion of the SS genotype was not significantly more consistent in addicts' samples. The allele frequencies are shown in Table 2. The frequency of the low activity allele (S) in addicts was 53.2% and in controls it was 38.7%.

The frequency of the alleles and genotypes was not different in cases and controls. However, the odd ratio was 1.8 respectively and p-value > 0.05

In spite of the fact that OD = 1.8, the results of the present study suggested that the SS genotype of 5-HTTLPR might be involved in 5- HT reuptake derangement, associated with increased psychobiological vulnerability for substance abuse.

Discussion

Genetic factors have a non-specific but significant effect on the risk of drug dependence. Some family, twin and adoption studies support this genetic role [2,23,25]. It has also been stated that different drug dependencies may have unique genetic [26]. The relationship between the 5HTT gene and dependence has been widely studied and the short allele of 5HT system has received considerable attention in addiction. Although the findings remain equivocal in other studies, it showed no evidence for association [24,27,28], while in some studies a positive association was found [6,29]. Some recent evidences indicate that the association of addiction with short allele may be different in men and women, while it was observed to be homozygote for the short allele among males, this was heterozygote among women. This means that a genotype sex interaction was observed [4] while some other studies deny this relationship [10].

In the present study we found no significant difference in rate of short allele in opioid addicted people in comparison with control cases (Tables 3 and 4). More recent researchers have found evidences of psychological disorder as a factor that increase risk of dependence but according to data from present study (Table 2), consumption of drug is not associated to psychological distress and some other investigation confirmed this result. Our data indicate that the average onset age of addiction in people with family history of addiction is significantly lower than this age in addicts without family history (Table 1). Furthermore individuals with family history of addiction are more likely at risk for drug abuse in comparison with individuals without this family history. These findings highlight the importance of genetics and inheritance in opioid addiction. The present findings need to be interpreted with caution: some unmeasured confounds may have reduced the strength and the validity of the study. Several possibilities exist for these discrepant results, including small sample size, inter individual and cultural variability, potential population stratification, and categorical phenotypes. Several studies also illustrated the importance of others moderating factors such as environmental factors. Understanding of possible associations of these variants would bring progress in principles and treatment, prevention and identification risk of addiction. Therefore further studies are required to determine whether any association is mediated by factors such as personality and sex etc.

Table 2: 5-HTTLPR genotypes frequencies in addicts and controls.

Table 3: Frequency of behavioural disorder among addicts with and without family history.

Table 4: Frequencies of S and L alleles in addicts and controls.

Acknowledgements

The authors acknowledge with grateful appreciation the kind assistance and financial support provided by the Vice Chancellor for Research at the Shahidsadoghi University of Medical Sciences (Yazd, Iran).

Conflict of interest

There is no conflict of interest associated with this study.

References

1. Gerra G, Garofano L, Santoro G, Bosari S, Pellegrini C, et al. (2004) Association between low-activity serotonin transporter genotype and heroin dependence: Behavioural and personality correlates. American Journal of Medical Genetics Part B: Neuropsychiatric Genetics 126: 37-42.
2. Goldman D, Oroszi G, Ducci F (2005) The genetics of addictions: uncovering the genes.Nat Rev Genet 6: 521-532.
3. Rezvanfard M, Ekhtiari H, Mokri A, Djavid G, Kaviani H (2010) Psychological and behavioural traits in smokers and their relationship with nicotine dependence level.Arch Iran Med 13: 395-405.
4. Munafo MR, Lingford-Hughes AR, Johnstone EC, Walton RT (2005) Association between the serotonin transporter gene and alcohol consumption in social drinkers. American Journal of Medical Genetics Part B: Neuropsychiatric Genetics 135: 10-14.
5. Watanabe MA, Nunes SO, Amarante MK, Guembarovski RL, Oda JM, et al. (2011) Genetic polymorphism of serotonin transporter 5-HTTLPR: involvement in smoking behaviour.J Genet 90: 179-185.
6. Gerra G, Zaimovic A, Garofano L, Ciusa F, Moi G, et al. (2007) Perceived parenting behavior in the childhood of cocaine users: relationship with genotype and personality traits. American Journal of Medical Genetics Part B: Neuropsychiatric Genetics 144: 52-57.
7. Lovinger DM (1997) Serotonin's role in alcohol's effects on the brain.Alcohol Health Res World 21: 114-120.
8. Anderson BM, Schnetz-Boutaud NC, Bartlett J, Wotawa AM, Wright HH, et al. (2009) Examination of association of genes in the serotonin system to autism.Neurogenetics 10: 209-216.
9. Kranzler H, Lappalainen J, Nellissery M, Gelernter J (2002) Association study of alcoholism subtypes with a functional promoter polymorphism in the serotonin transporter protein gene. Alcoholism: Clinical and Experimental Research 26: 1330-1335.
10. Gerra G, Garofano L, Castaldini L, Rovetto F, Zaimovic A, et al. (2005) Serotonin transporter promoter polymorphism genotype is associated with temperament, personality traits and illegal drugs use among adolescents.J Neural Transm (Vienna) 112: 1397-1410.
11. Anderson GM, Gutknecht L, Cohen DJ, Brailly-Tabard S, Cohen JH, et al. (2002) Serotonin transporter promoter variants in autism: functional effects and relationship to platelet hyperserotonemia. Mol Psychiatry 7: 831-836.
12. Tordjman S, Gutknecht L, Carlier M, Spitz E, Antoine C, et al. (2001) Role of the serotonin transporter gene in the behavioural expression of autism.Mol Psychiatry 6: 434-439.
13. Longo D, Schüler-Faccini L, Brandalize AP, dos Santos-Riesgo R, Bau CH (2009) Influence of the 5-HTTLPR polymorphism and environmental risk factors in a Brazilian sample of patients with autism spectrum disorders. Brain research 1267: 9-17.
14. Huang CL (2010) The role of serotonin and possible interaction of serotonin-related genes with alcohol dehydrogenase and aldehyde dehydrogenase genes in alcohol dependence-a review. American journal of translational research 2:190.
15. Gunthert KC, Conner TS, Armeli S, Tennen H, Covault J, et al. (2007) Serotonin transporter gene polymorphism (5-HTTLPR) and anxiety reactivity in daily life: A daily process approach to gene-environment interaction. Psychosomatic medicine 69:762-768.
16. van der Zwaluw CS, Engels RC, Vermulst AA, Rose RJ, Verkes RJ, et al. (2010) A serotonin transporter polymorphism (5-HTTLPR) predicts the development of adolescent alcohol use.Drug Alcohol Depend 112: 134-139.
17. Nilsson KW, Sjöberg RL, Damberg M, Alm PO, Öhrvik J, et al. (2005) Role of the serotonin transporter gene and family function in adolescent alcohol consumption. Alcoholism: Clinical and Experimental Research 29: 564-570.
18. Kuzelova H, Ptacek R, Macek M (2010) The serotonin transporter gene (5-HTT) variant and psychiatric disorders: review of current literature.NeuroEndocrinolLett 31: 4-10.
19. Sen S, Villafuerte S, Nesse R, Stoltenberg SF, Hopcian J, et al. (2004) Serotonin transporter and GABAA alpha 6 receptor variants are associated with neuroticism.Biol Psychiatry 55: 244-249.
20. Betancur C, Corbex M, Spielewoy C, Philippe A, Laplanche JL, et al. (2002) Serotonin transporter gene polymorphisms and hyperserotonemia in autistic disorder.Mol Psychiatry 7: 67-71.
21. Golimbet VE, Alfimova MV, Shcherbatykh TV, Rogaev EI (2003) Gene insertion and deletion polymorphism in the serotonin transporter gene and personality traits measured by MMPI.Genetika 39: 534-539.
22. Greenberg BD, Li Q, Lucas FR, Hu S, Sirota LA, et al. (2000) Association between the serotonin transporter promoter polymorphism and personality traits in a primarily female population sample. American journal of medical genetics 96: 202-216.
23. Gerra G, Garofano L, Zaimovic A, Moi G, Branchi B, et al. (2005) Association of the serotonin transporter promoter polymorphism with smoking behaviour among adolescents. American Journal of Medical Genetics Part B: Neuropsychiatric Genetics 135: 73-78.
24. Sieminska A, Buczkowski K, Jassem E, Tkacz E (2008) Lack of association between serotonin transporter gene polymorphism 5-HTTLPR and smoking among Polish population: a case-control study.BMC Med Genet 9: 76.
25. Elkins IJ, McGue M, Malone S, Iacono WG (2004) The effect of parental alcohol and drug disorders on adolescent personality.Am J Psychiatry 161: 670-676.
26. Saiz PA, Garcia-Portilla MP, Florez G, Arango C, Corcoran P, et al. (2009) Differential role of serotonergic polymorphisms in alcohol and heroin dependence. Progress in Neuro-Psychopharmacology and Biological Psychiatry 33: 695-700.
27. Laucht M, Treutlein J, Schmid B, Blomeyer D, Becker K, et al. (2009) Impact of psychosocial adversity on alcohol intake in young adults: moderation by the LL genotype of the serotonin transporter polymorphism. Biological psychiatry 66: 102-109.
28. Stoltenberg SF, Twitchell GR, Hanna GL, Cook EH, Fitzgerald HE, et al. (2002) Serotonin transporter promoter polymorphism, peripheral indexes of serotonin function, and personality measures in families with alcoholism. American journal of medical genetics 114: 230-234.
29. Feinn R, Nellissery M, Kranzler HR (2005) Meta-analysis of the association of a functional serotonin transporter promoter polymorphism with alcohol dependence. American Journal of Medical Genetics Part B: Neuropsychiatric Genetics 133:79-84.