Serotonin transporter gene (5HTT) polymorphisms and temporal lobe epilepsy

Results We observed that less efficient transcriptional genotypes for 5-HTT polymorphisms were more frequent in epileptic patients (O.R. = 3.24; 95% C.I. = 1.08–9.73; p = 0.036). Our results suggest that less efficient transcriptional genotypes for serotonin transporter gene are associated with TLE. Conclusion In this study we observed an association between the presence of 5HTTLPR and 5-HTTVNTR less transcriptional efficient combined genotypes and TLE. Our results suggest that modulation of the serotoninergic system might be implied in epileptogenesis in TLE. Keywords Epileptogenesis Serotoninergic system 5HTTLPR 5-HTTVNTR Introduction Epilepsy is the second most frequent cause of neurological disorders in young adults. The annual incidence of epilepsy is estimated at 70/100,000 individuals in the general population, whereas in developing countries epilepsy rates are two times higher ( Sander, 2003 ). The majority of patients, approximately 40%, suffer from temporal lobe epilepsy (TLE). Since the serotonin neurotransmitter (5-HT) contributes to the neurodevelopment, functionality and plasticity of the adult brain, serotoninergic system may also contribute to the etiology of epilepsy ( Catalano, 2001; Lesch, 2001 ). Preclinical and clinical studies have shown that reduction of serotonin concentration in the brain can enhance susceptibility to seizures ( Lazarova et al., 1983; Cavalheiro et al., 2004; Savic et al., 2004 ). The impairment of serotoninergic activity may facilitate seizure onset or enhance seizure severity. Agents that increase extracellular 5-HT levels, such as 5-hydroxytryptophan and selective serotonin reuptake inhibitors (SSRI), might inhibit focal and generalized seizures ( Bagdy et al., 2007 ). Conversely, antiepileptic drugs might enhance basal serotonin levels as part of their mechanism of action ( Ahmad et al., 2005 ). More recently it was observed that SSRIs are associated with a reduced likelihood of ictal oxygen desaturation in patients with partial seizures ( Bateman et al., 2010 ). Thus serotoninergic neurotransmission may have an important role in the neurobiology of epilepsy. The serotonin transporter (5-HTT) is an integral membrane protein responsible for the reuptake of 5-HT from the synaptic cleft, modulating the serotoninergic neurotransmission. The human 5-HTT gene (SLC6A4) has been mapped to chromosome 17q11.1–q12 ( Lesch et al., 1994 ). Two polymorphisms of the 5-HTT gene have shown functional consequences. A 44 bp insertion/deletion polymorphism in the 5′ flanking region of this gene (5-HTTLPR) originates two alleles (L – long and S – short). The S allele has been reported to be associated with lower transcriptional efficiency of the 5-HTT gene, resulting in lower serotonin uptake activity when compared with the L allele. ( Lesh et al., 1996; Heils et al., 1996 ). This polymorphism with L and S alleles has been proposed to be biallelic for years. However, Nakamu et al., 2000 described a functional SNP A>G within the 5-HTTLPR polymorphism which generates a triallelic model with an La allele conferring gain-of function and Lg and S alleles being low-function variants. The second polymorphism in the 5-HTT gene is a variable number of tandem repeats (VNTR) containing 9, 10 and 12 repeats of a 17 bp sequence in intron 2 (5-HTTVNTR) ( Olgivie et al., 1996 ). The 5-HTTVNTR domain might act as a transcriptional regulator and the 12 allele has been associated with higher transcriptional activity of the 5 -HTT gene when compared to the 10 allele ( Lovejoy et al., 2003 ). Hranilovic et al. (2004) demonstrated a dominant role of low-expressing alleles of these polymorphisms (S allele for 5-HTTLPR and 10 allele for 5-HTTVNTR) and showed the combined effect of 5-HTTLPR-biallelic and 5-HTTVNTR polymorphisms on 5-HTT gene expression. Mean 5-HTT gene expression was the highest in the group with two high-expression genotypes (L/L and 12/12), 20% lower in the group with one low-expression genotype (L/L 10/10, L/L 10/12, S/S 12/12 or S/L 12/12) and 50% lower in the group with both low-expression genotypes (no L/L and no 12/12). In this study, we evaluated a plausible association between 5HTTLPR and 5HTTVNTR allele variants and epileptogenesis. For this purpose, we compared the frequency of 5HTTLPR and 5HTTVNTR allele variants between TLE patients and a control group of healthy individuals. Methods Patients This is a case–control candidate gene study. The cases were 175 patients with TLE, selected from the Epilepsy Outpatient Clinic of Hospital de Clínicas de Porto Alegre (HCPA). Inclusion criteria were based on the 1989 ILAE's electroclinical classification (Commission on Classification Terminology of the International League Against Epilepsy, 1989) and neuroimaging results. Patients with extratemporal epilepsies, mental retardation, and those with systemic diseases were excluded. The control group consisted of 155 healthy unrelated subjects selected within the institutional staff (HCPA). The study was approved by the an Ethics Committee, in accordance with the Declaration of Helsinki, and all subjects provided written informed consent to participate in this study. Genotyping DNA was extracted from peripheral leukocytes according to the salt precipitation method ( Miller et al., 1988 ). Subjects were genotyped for the 5-HTTLPR and 5-HTTVNTR. 5-HTTLPR: The amplification reaction (PCR) for the 5-HTTLPR polymorphism was carried out using primers described by Heils et al. (1996) . The amplified product was digested with Msp I restriction enzyme (New England Biolabs) which allows the detection of the A/G SNP, identifying the triallelic polymorphism (La, Lg and S variants). The digestion products were visualized by 3% agarose gel electrophoresis stained with ethidium bromide under UV light. 5-HTTVNTR: The intron 2 region of the 5-HTT gene containing the VNTR polymorphism was amplified using primers described by Weese-Mayer et al. (2003) . The PCR product was visualized by 3% agarose gel electrophoresis stained with ethidium bromide. The rare allele of 9 repeats was grouped with the 10 allele (both short alleles) and compared to the 12 variant (long allele). Statistical analysis The clinical variables and genotypes of the polymorphisms, 5-HTTLPR bi- and triallelic model and 5-HTTVNTR, were compared between TLE patients and a control group. In a second analysis we further compared the frequency of combined genotypes grouped according Hranilovic et al. (2004) between cases and controls. Categorical variables were compared by the two-tailed Chi-squared test and Fisher's exact test if necessary. Numerical variables were compared by the independent Student t -test, with the Levene test for equality of analysis of variance. As the age of patients differs from controls, we used a binary logistic regression model for controlling the effect of age. All statistical analyses were carried out using the SPSS 14.0 statistical package for Windows (SPSS Inc., Chicago, IL, USA). Results are expressed as odds ratio (95% confidence interval) and the level of significance was set at p < 0.05. Results Of the 175 patients and 155 controls, 114 (65.1%) and 116 (74.8%) were women and 61 (34.9%) and 39 (25.2%) were men, respectively, with no significant differences between cases and controls ( p = 0.072). The mean age of patients and controls was 44.0 years and 32.6 years ( p < 0.01), respectively. The distributions of genotype frequency were in Hardy-Weinberg equilibrium for both cases and controls. Table 1 shows the genotype distribution of the 5-HTTLPR bi- and triallelic model and 5-HTTVNTR in TLE patients and controls. We found no significant difference in the genotype frequencies of these polymorphisms between epileptic patients and controls. Considering the dominant role of low-expressing alleles, the genotypes containing at least one low efficient allele (10 for 5-HTTVNTR and S for 5-HTTLPR-biallelic) were observed in 64.6% and 72.1% of TLE patients and in 56.8% and 65.2% of controls, a nonsignificant difference ( p = 0.17 and p = 0.23, respectively). However, we observe significant differences when 5-HTTVNTR and 5-HTTLPR biallelic genotypes were combined by transcriptional efficiency. The combination of low expressing genotypes (no L/L and no 12/12) was more frequently observed in TLE patients, whereas the high expressing genotypes (L/L and 12/12) were more frequently observed in the controls ( p = 0.028, see Table 2 ). Demographic, clinical, and radiological variables of patients, distributed according to grouped genotype of 5HTTLPR and 5HTTVNTR are present in Table 3 . There were no differences in these genotypes regarding mean age of patients, mean age of epilepsy onset, mean time of epilepsy, sex, seizure control, presence of aura, history of initial precipitating injury, number of antiepileptic drugs used, use of benzodiazepines, and neuroimaging findings. For a final analysis we divided the patients into two different groups, i.e., a group of more efficient transcriptional genotypes (L/L and 12/12) and a group of less efficient transcriptional genotypes, pooled together. A comparison between these two groups showed that more efficient transcriptional genotypes where less frequent in epileptic patients (O.R. = 3.38; 95% C.I. = 1.19–9.62; p = 0.016). This result remains significant after correction for age differences between patients and controls (O.R. = 3.24; 95% C.I. = 1.08–9.73; p = 0.036). Taken together, our results suggest that less efficient transcriptional genotypes are associated with TLE development. Discussion In this study we found an association between the combination of 5HTTLPR biallelic and 5-HTTVNTR less transcriptional efficient genotypes and TLE. Although, we did not find any association between TLE patients and separate genotype analysis of 5-HTTLPR and 5-HTTVNTR, we observed that TLE patients showed higher frequencies of combined low transcriptional activity 5-HTT genotypes compared to control subjects. As far as we now, only four studies have previously explored the potential roles of 5-HTT allele variants in epileptogenesis or epilepsy characteristics. Stefulj et al. (2010) failed to show any association between 5HTTLPR or 5HTTVNTR and TLE. However, Manna and collaborators found an association between TLE and 5-HTTVNTR allele 12, but not with 5-HTTLPR ( Manna et al., 2007 ). The allele 12 of 5-HTTVNTR was also associated with risk for mesial temporal epilepsy not responding to medical treatment ( Kauffman et al., 2009 ). Similarly, Hecimovic et al. found an association between high expression of combined genotypes (L/L of 5-HTTLPR and 12/12 of 5-HTTVNTR) and seizure refractoriness to antiepileptic medication therapy and shorter periods of seizure freedom ( Hecimovic et al., 2010 ). Taken together, the last three studies contrast with our findings because they suggest that epilepsy, its severity or its response to medical treatment is associated with high, rather than low, transcriptional activity 5-HTT genotypes. These discrepant results need to be further discussed, especially if one considers the results of Manna et al. ( Manna et al., 2007 ) and Hecimovic et al. (2010) , as presented above. It is possible that these genetic association observed might be related to linkage disequilibrium with other genetic polymorphisms at the same gene or close genes. These possibilities could explain discrepant results found in association studies performed in different populations. Moreover there are possible mechanisms that could explain how increase or decrease of serotonin might be related with epileptogenesis. Considering the neurobiology of epilepsy, we are tempted do hypothesize that a tide modulation of serotoninergic system during brain development or during response to brain insults might be important for brain homeostasis and for preventing epilepsy. Variability in this system that increase or decrease serotonin availability due to more efficient or less efficient transcriptional genotypes for serotonin transporter gene increase would turn neural network more prone to epileptogenesis. This view might be supported by all theses studies that are showing that modulation of the 5-HTT gene might influence epileptogenesis or the clinical characteristics of epilepsy. These are interesting questions that certainly deserve further investigations. In agreement with our observations, there are several studies showing that low transcriptional activity 5-HTT genotypes are associated with neuropsychiatric disorders, among them depression ( Holmes et al., 2010 ), suicidal behavior ( Anguelova et al., 2003 ), attention deficit hyperactivity disorder ( Kent et al., 2002 ), and personality disorder ( Garcia et al., 2010 ). Curiously, a bi-directional relationship has been well established between temporal lobe epilepsy and neuropsychiatric disorders, especially mood disorders. In this venue some authors have suggested that common neurobiological substrates like neural networks or genetic factors might explain the association between epilepsy and neuropsychiatric disorders ( Kanner, 2009; Bragatti et al., 2009 ). Thus, based on our results, we are tempted to suggest that the 5HTT gene might be a candidate gene involved in the genesis or clinical variability of both epilepsy or neuropsychiatric disorders. There may be several possible mechanisms for this association. For example, the excessive serotonin levels during brain development could have consequences for the size and capacity of the serotoninergic system and associated neural network. This could well explain the paradoxical relation between genetic variants associated with higher levels of serotonin (such as low-function alleles of 5-HTT polymorphisms) and the neurological disorders associated with lower serotoninergic levels ( Nordquist and Oreland, 2010 ). Thus, in our view, it is plausible that low transcriptional function genotypes of 5-HTTLPR and 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 possibly associated with risk for neuropsychiatric disorders or limbic epilepsy later in life. Further studies will be conducted in this venue to clarify these matters. We recognize that our study might have some bias. The ethnic admixture of the Brazilian sample may be a bias in genetic association studies. However, the population of Rio Grande do Sul State is mainly composed of European descendants (82% of the population) ( Cordeiro et al., 2008 ). Moreover, the fact that the present sample is in Hardy–Weinberg equilibrium indicates that there may not be important problems of population stratification. In addition to the ethnic stratification limitation discussed above, sample size is an important limitation of this study. Thus, negative results need to be interpreted with caution due to lack of statistical power. On the other hand, significant results are less problematic in this scenario. Even so, it is necessary to consider also the possibility of false positive associations because of cofounders such as etiology of epilepsy and the presence of limbic lesions sclerosis in our sample. Although no patients with tumors or arteriovenous malformations were included in this study, 19% of our epileptic patients had mesial temporal lobe epilepsy associated with hippocampal sclerosis. If possible that a study using a more homogeneous sample (e.g. only patients with hippocampal sclerosis) would produce a different result. Nevertheless it is important to observe that our sample better represents the full spectrum of temporal lobe epilepsy and that might be an advantage. Thus, considering interesting preliminary findings, we believe that further studies conducted on large populations are needed to establish the real impact of 5-HTT polymorphisms in epileptogenesis or in the clinical characteristics of epilepsy. In this study we observed an association between the presence of 5HTTLPR biallelic and 5-HTTVNTR less transcriptional efficient combined genotypes and TLE. In our opinion, this finding is important because it might help to elucidate epileptogenesis, especially regarding TLE. Moreover, implication of the serotoninergic system in epileptogenesis might offer an interesting link to better understand the association between TLE and frequent neuropsychiatric comorbidities observed in these patients. Further studies are necessary to confirm our findings. Acknowledgements This work was supported by Brazilian Government Research Funds , FIPE-HCPA and CNPq (#551902/2009-4; #306644/2010-0; #483108/2010-3). References Ahmad et al., 2005 S. Ahmad L.J. 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