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pilepsy Research (2012) 99, 260—266

j ourna l ho me pag e: www.elsev ier .com/ locate /ep i lepsyres

erotonin gene polymorphisms and psychiatryomorbidities in temporal lobe epilepsy

aila Cigana Schenkela,b,c, José Augusto Bragatti b,c,d,uliana Allebrand Beckera, Carolina Machado Torresb,c,d,elin Cristine Martinc,d, Ana Claudia de Souzac,d, Gisele Gus Manfroc,e,andra Leistner-Segala,c, Marino Muxfeldt Bianchinb,c,d,∗

Medical Genetics Service, Hospital de Clinicas de Porto Alegre, BrazilPost-Graduation Course in Medicine, Medical Sciences, Universidade Federal do Rio Grande do Sul, BrazilBasic Research and Advanced Investigations in Neurology, Experimental Research Centre, Hospital de Clínicas de Porto Alegre,razilDivision of Neurology, Hospital de Clínicas de Porto Alegre, BrazilAnxiety Disorder Program, Hospital de Clínicas de PortoAlegre, Universidade Federal do Rio Grande do Sul, Brazil

eceived 22 March 2011; received in revised form 31 October 2011; accepted 11 December 2011vailable online 4 January 2012

KEYWORDSEpilepsy;Serotoninergicsystem;5-HTT;5-HT1A;Mood disorders

SummaryObjective: Neuropsychiatric comorbidities are frequent in temporal lobe epilepsy (TLE). It isbiologically plausible that alterations in serotonin-related genes may be involved in highersusceptibility to psychiatric disease in these individuals. Here we report results of an associationstudy of serotonin gene polymorphisms and psychiatry comorbidities in TLE.Methods: Case-control study of 155 patients with temporal lobe epilepsy. We evaluate theinfluence of 5-HTTLPR and 5-HTTVNTR polymorphisms in the 5-HTT gene and the C-1019Gpolymorphism in the 5-HT1A gene in psychiatric comorbidities of TLE.Results: After logistic regression, female sex (OR = 2.34; 95% CI 1.06—5.17; p = 0.035) and thepresence of C allele of 5-HT1A C-1019G polymorphism (OR = 2.77; 95% CI 1.01—7.63; p = 0.048)

remained independent risk factors for anxiety disorders in temporal lobe epilepsy.Conclusion: C allele of 5-HT1A C-1019G polymorphism might be an independent risk factor foranxiety disorders in temporal lobe epilepsy. We believe that other studies in this venue willshade some light on molecular mechanisms involved in psychiatric comorbidities in epilepsy.© 2011 Elsevier B.V. All rights re

∗ Corresponding author at: Division of Neurology and B.R.A.I.N. HospitS 90035-903, Brazil. Tel.: +55 51 96 37 69 69.

E-mail address: mmbianchin@hotmail.com (M.M. Bianchin).

920-1211/$ — see front matter © 2011 Elsevier B.V. All rights reserved.oi:10.1016/j.eplepsyres.2011.12.005

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Serotonin gene polymorphisms and psychiatry comorbidities

Introduction

Epilepsy is a chronic disorder that affects people of allages and social levels. Neuropsychiatric comorbidities aremore frequent in these patients than in the general popu-lation. Among epileptic patients, those with temporal lobeepilepsy (TLE) are especially at risk to develop psychiatricdisorders. The main psychiatric disorders involved in TLE,in order of frequency, are depression, anxiety and psychosiswith prevalence ranging from 11 to 44%, 15—25% and 2—8%,respectively (Schmitz and Wolff, 1995; Schmitz, 2006). Evi-dence suggests that the association of psychiatric disorderswith epilepsy might be related to common biological sub-strates (Torta and Keller, 1999; Dudra-Jastrzebska et al.,2007).

Dysfunction of serotoninergic neurotransmission has beenlong associated with psychiatric diseases. Recent studiessupport that serotonin (5-HT) may also contribute to apredisposition to epilepsy. Serotoninergic activation maychange ionic conductance, resulting in de- or hyperpolar-ization of neurons, and as a consequence can cause asignificant shift in the neuroexcitability involved in epilepsy(Bagdy et al., 2007). There is evidence that anticonvulsantscan cause increased 5-HT levels as part of their mecha-nisms of action (Ahmad et al., 2005; Grosso et al., 2008).Furthermore, agents that elevate extracellular serotoninlevels inhibit both focal and generalized seizures (Bagdyet al., 2007). On this basis, Savic et al. (2004) found areduced serotonin receptor (5-HT1A) binding potential inlimbic structures in patients with mesial TLE, supporting thehypothesis of an involvement of serotonin in the neurobi-ology of TLE, perhaps suggesting a mechanism underlyingaffective symptoms in these patients.

Genes coding for proteins related to serotonin neuro-transmission can regulate 5-HT availability. Of particularinterest are the gene encoding the serotonin transporter(5-HTT), responsible for the clearance of 5-HT from thesynaptic cleft, and the gene encoding the serotonin recep-tor 1A (5-HT1A), that acts as an autoreceptor presynapticallyand mediates the action of serotonin postsynaptically. Pre-vious studies have shown two functional polymorphisms inthe 5-HTT gene, in the promoter (5-HTTLPR) and intron2 (5-HTTVNTR) regions. 5-HTTLPR is a 44 base pair inser-tion/deletion polymorphism in the 5′ flanking region of thisgene, that gives origin to two alleles (L — long and S —short). The S allele of 5-HTTLPR has been reported to beassociated with lower transcriptional efficiency of the 5-HTT gene, resulting in lower serotonin uptake activity whencompared with the L allele (Lesch et al., 1994; Heils et al.,1996). 5-HTTVNTR consists of a variable number of tan-dem repeats containing 9, 10 and 12 repeats of a 17 bpsequence in intron 2 (5-HTTVNTR) (Ogilvie et al., 1996). The5-HTTVNTR domain might act as a transcriptional regulatorand the 12 allele has been associated with higher transcrip-tional activity of the 5-HTT gene when compared to the 10allele (Lovejoy et al., 2003). The 5-HT1A gene contains a sin-gle nucleotide polymorphism (SNP) in the promoter region(C-1019G) that seems to regulate gene expression (Wu and

Comings, 1999; Czesak et al., 2006). The G allele of C-1019Ghas been postulated to up-regulate autoreceptor expres-sion, but to decrease postsynaptic 5-HT1A expression (Savitzet al., 2009).

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261

The 5-HTTLPR and 5-HTTVNTR polymorphisms in the 5-TT gene and the C-1019G polymorphism in the 5-HT1A geneave been studied in various psychiatric diseases, amonghem depression (Holmes et al., 2010; Parsey et al., 2006;ishi et al., 2009), suicidal behavior (Anguelova et al., 2003;erretti et al., 2007), anxiety (Freitag et al., 2006; Hettemat al., 2008), personality disorder (Garcia et al., 2010), bipo-ar disorder and schizophrenia (Kishi et al., 2011). Recently,ome authors have evaluated the association between 5-TT variants and epilepsy (Kauffman et al., 2009; Hecimovict al., 2010; Manna et al., 2007; Stefulj et al., 2010).ost of these studies have shown that modulation of the-HTT gene might influence epileptogenesis or the clinicalharacteristics of epilepsy. Thus, it is biologically plausiblehat alterations in serotonin-related genes may be one ofhe biological mechanisms involved in higher susceptibil-ty to psychiatric disease in patients with TLE. Based onhese previous investigations and in a biologically plausi-le hypothesis, we study the contribution of 5-HTTLPR and-HTTVNTR and C-1019G polymorphisms in the genesis ofsychiatric comorbidities in a cohort of 155 patients withLE. As far as we know, this is the first study evaluating a pos-ible genetic involvement in the development of psychiatricomorbidities in epilepsy.

ethods

atients

his is a retrospective cohort study of 155 patients with TLE of thepilepsy Outpatient Clinic of Hospital de Clínicas de Porto AlegreHCPA). Inclusion criteria were based on the 1989 ILAE’s electroclin-cal classification (Commission on Classification and Terminology ofhe International League Against Epilepsy, 1989) and neuroimagingesults. Patients with extratemporal epilepsies, mental retardation,nd those with systemic diseases were excluded. All patients wereubmitted to the Structured Clinical Interview for DSM-IV (SCID)First et al., 2001). SCID is divided into six modules, for the detec-ion of one or more life-long diagnoses of the Axis I Diagnostic andtatistical Manual, fourth edition (DSM-IV) (American Psychiatricssociation, 2000). SCID detected Axis I Psychiatric Diagnostic in9 (63.9%) of the 155 patients with TLE (TLE-PSYCH group). In6 (36.1%) of the 155 TLE patients SCID was negative (TLE-ONLYroup). These two groups of patients (TLE-PSYCH group and TLE-NLY group) were compared for clinical and genotypic differences.he study was approved by the Ethics Committee of our institution,omitê de Ética em Pesquisa do Hospital de Clínicas de Porto Alegre,nd it is in accordance with the Declaration of Helsinki. All subjectsncluded provided written informed consent to participate in thistudy.

enotyping

NA was extracted from peripheral leukocytes by the salt precip-tation method (Miller et al., 1988). Subjects were genotyped forhe 5-HTTLPR and 5-HTTVNTR polymorphisms in the 5-HTT gene andhe C-1019G polymorphism in the 5-HT1A gene.

5-HTTLPR: The amplification reaction (PCR) for the 5-HTTLPRolymorphism was carried out using primers described by Heils et al.1996). The amplified product was digested with MspI restriction

nzyme (New England Biolabs) which allows the detection of the/G SNP, identifying the triallelic polymorphism (La, Lg and S vari-nts). The digestion products were visualized by 3% agarose gellectrophoresis with ethidium bromide staining under UV light.

262 L.C. Schenkel et al.

Table 1 Characteristics of TLE patients with and without neuropsychiatric comorbities.

Variable All (n = 155) TLE-ONLY group (n = 56) TLE-PSYCH group (n = 99) p

Mean age (yrs, SD) 44.6 (12.5) 45.2 (12.2) 44.0 (12.8) 0.55Mean age of epilepsy onset (yrs, SD) 19.0 (14.8) 19.0 (15.0) 19.1 (14.6) 0.96Mean time of disease (yrs, SD) 25.6 (14.0) 26.2 (13.4) 24.9 (14.5) 0.57Female sex 100 (64.5) 29 (51.8) 71 (71.7) 0.015*

Family history of epilepsy 52 (33.5) 18 (32.1) 34 (34.3) 0.86Family history of psychiatric disease 52 (33.5) 13 (23.2) 39 (39.4) 0.05Seizure control 71 (45.8) 27 (48.2) 44 (44.4) 0.74Presence of aura 75 (48.4) 28 (50.0) 47 (47.5) 0.87EEG interictal unilateral 90 (58.1) 34 (60.7) 56 (56.6) 0.73Initial precipitating injury 38 (24.5) 14 (25.0) 24 (24.2) 1.0Benzodiazepine use 27 (17.4) 8 (14.3) 19 (19.2) 0.51

Values are presented as frequency (percentage) or mean (SD). yrs = years. TLE-PSYCH and TLE-ONLY groups = temporal lobe epilepsy

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patients with and without psychiatric comorbidities, respectively.* Significant.

5-HTTVNTR: The intron 2 region of the 5-HTT gene containinghe VNTR polymorphism was amplified using primers described byeese-Mayer et al. (2003). The PCR product was visualized by 3%

garose gel electrophoresis and ethidium bromide staining. The rare repeat was grouped with the 10 allele (both short alleles) andompared to the 12 variant (long allele).

C-1019G: An allele-specific polymerase chain reaction (ARMS-CR) analysis was performed according to Parsey et al. (2006). Eachample was amplified twice using a specific primer for the G allelend another primer for the C allele (Wu and Comings, 1999). Elec-rophoresis was performed on 1.5% agarose gel and the amplificationroduct was visualized under UV light.

tatistical analysis

ategorical variables were compared by the two-tailed Chi-squareest and Fisher’s exact test. Numerical variables were compared byhe independent Student’s t-test, with the Levene test for equalityf analysis of variance. All statistical analyses were carried out usinghe SPSS 14.0 statistical package for Windows (SPSS Inc., Chicago,L, USA). In order to examine the independent effect of each vari-ble we used an unconditional binary logistic regression model. Toetermine the number of independent variables to be included inur logistic regression model we used the parameters suggestedStevens, 1996; Hosmer and Lemeshow, 1989; Tabachnick and Fidell,996). Results are reported as odds ratio (95% confidence interval)nd were considered significant if p was lower than 0.05.

esults

f the 155 TLE patients, 100 (64.5%) were women and5 (35.5%) were men. Psychiatric disorders were observedn 99 (63.9%) of the patients studied. In our sample, theost frequent psychiatric comorbidities were mood disor-er (47.1%) and anxiety disorder (31.0%). More than onesychiatric comorbidity can occur in the same patients andhese concomitant occurrences were identified by SCID. Inur study, mood and anxiety disorders occurred together in1 patients (20% of the total). The presence of any mood or

nxiety disorder or any alcohol/substance abuse occurred in3 (60%) of the patients. The mean age of epileptic patientsith psychiatric disorders (TLE-PSYCH group) and withoutsychiatric disorders (TLE-ONLY group) was 44.0 and 45.2

tw(a

ears, respectively, with no significant difference betweenhem (p = 0.55). Most of the patients with psychiatric dis-rders (TLE-PSYCH group) were women (71.7%), while only8.3% were men, whereas in TLE-ONLY group 51.8% wereomen and 48.2% were men, a significant difference for sex

p = 0.015). The age of onset, time of disease, family historyor both epilepsy and psychiatric disorders, seizure control,resence of aura, EEG interictal activity, presence of ini-ial precipitating injury and use of benzodiazepine (BZD) didot differ between TLE patients with and without psychi-tric comorbidities in our cohort. Clinical and demographicharacteristics of the sample are presented in Table 1.

The genotype distribution of C-1019G, 5-HTTVNTR and-HTTLPR polymorphisms according to psychiatric diseasen TLE patients is summarized in Table 2. We found no sig-ificant association between these polymorphisms in the-HT1A and 5-HTT genes and the combined presence ofeuropsychiatric disorders in patients with TLE (TLE-ONLYroup versus TLE-PSYCH group). However, when we ana-yzed each psychiatric disorder individually (mood disorder,nxiety disorder, psychosis and drug/alcohol abuse), the fre-uency of C-1019G polymorphism was different betweenLE patients with and without anxiety disorder. The fre-uency of GG genotype of C-1019G polymorphism was lowern patients with anxiety disorder than in patients withoutnxiety, observed in 6 (12.5%) of 48 patients with anxietyisorders but 27 (25.2%) of 107 patients without anxiety dis-rders. This difference approached statistical significancep = 0.058). As the relative limited number of patient pre-luded detailed analysis of specific sub-types of humor ornxiety disorders, only results for major groups are showedn Table 2. However, because of observations regardingnxiety comorbidities, we further studied the clinical andemographics variables of our cohort regarding the presencer absence of anxiety disorder in TLE patients. The resultsf this analysis are presented in Table 3. In this analysis,e did not observe significant differences between clinicalnd demographics characteristics of TLE patients regarding

he presence of anxiety disorder, except for gender. Womenith TLE had more frequently anxiety disorders than men

p = 0.031). In order to study the independence of variabless risk factor for anxiety disorders in TLE, we included sex,

Serotonin gene polymorphisms and psychiatry comorbidities 263

Table 2 Genotype distribution of C-1019G in 5-HT1A gene, 5-HTTVNTR and 5-HTTLPR in 5-HTT gene according to neuropsychi-atric disorders.

TLE-ONLYgroup

TLE-PSYCHgroup

Mooddisorder

Anxietydisorder

Psychosis Alchool/drugabuse

Mood andanxietydisorders

Any mood oranxiety disorder orsubstance abuse

All (n = 155) 56 (36.1) 99 (63.9) 73 (47.1) 48 (31.0) 14 (9.0) 08 (5.2) 31 (20.0) 93 (60.0)C-1019G

CC 13 (23.2) 27 (27.3) 21 (28.8) 10 (20.8) 05 (35.7) 03 (37.5) 08 (25.8) 25 (26.9)CG 28 (50.0) 54 (54.5) 38 (52.1) 32 (66.7) 08 (57.1) 03 (37.5) 21 (67.7) 50 (53.8)GG 15 (26.8) 18 (18.2) 14 (19.2) 6 (12.5) 01 (7.1) 02 (25.0) 02 (6.5) 18 (19.4)p 0.447 0.678 0.058 0.353 0.644 0.060 0.761

5-HTTVNTR10/10 06 (10.7) 22 (22.2) 16 (21.9) 10 (20.8) 02 (14.3) 03 (37.5) 07 (22.6) 21 (22.6)10/12 28 (50.0) 43 (43.4) 32 (43.8) 19 (39.6) 09 (64.3) 03 (37.5) 11 (35.5) 32 (34.4)12/12 22 (39.3) 34 (34.3) 25 (34.2) 19 (39.6) 03 (21.4) 02 (25.0) 13 (41.9) 40 (43.0)p 0.202 0.500 0.573 0.335 0.336 0.427 0.201

5-HTTLPRSS 14 (25.0) 19 (19.2) 15 (20.5) 10 (20.8) 01 (7.1) 00 (0.0) 07 (22.6) 18 (19.4)SL 26 (46.4) 53 (53.5) 37 (50.7) 26 (54.2) 10 (71.4) 05 (62.5) 17 (54.8) 49 (52.7)LL 16 (28.6) 27 (27.3) 21 (28.8) 12 (25.0) 03 (21.4) 03 (37.5) 07 (22.6) 26 (28.0)p 0.624 0.956 0.846 0.231 0.316 0.846 0.760

Values are presented as frequency (percentage). TLE-PSYCH and TLE-ONLY groups = temporal lobe epilepsy patients with and withoutpsychiatric comorbidities, respectively.

Table 3 Clinical and demographic characteristics of TLE patients according with anxiety disorders.

Variables All patients (n = 155) No anxietydisorders (n = 107)

Anxiety disorders(n = 48)

p

Mean age (yrs, SD) 44.6 (12.5) 12.3 (1.18) 13.4 (1.93) 0.56Mean age of epilepsy onset (yrs, SD) 19.0 (14.8) 14.2 (1.37) 15.8 (2.28) 0.18Mean time of epilepsy (yrs, SD) 25.6 (14.0) 13.3 (1.28) 15.4 (2.23) 0.63Female sex 100 (64.5) 63 (58.9) 37 (77.1) 0.03*

Family history of epilepsy 52 (33.5) 36 (33.6) 16 (33.3) 1.00Family history of psychiatric disease 52 (33.5) 36 (33.6) 16 (33.3) 1.00Seizure control 71 (45.8) 51 (47.7) 20 (41.7) 0.60Presence of aura 75 (48.4) 53 (49.5) 22 (45.8) 0.73EEG interictal unilateral 90 (58.1) 63 (58.9) 27 (56.3) 0.86Initial precipitating injury 38 (24.5) 26 (24.3) 12 (25.0) 1.00Benzodiazepine use 27 (17.4) 20 (18.7) 7 (14.6) 0.65C allele of C-1019G 122 (78.7) 80 (74.8) 42 (87.5) 0.07G allele of C-1019G 115 (74.2) 77 (72.0) 38 (79.2) 0.34

Values are presented as frequency (percentage) or mean (SD). yrs = years.* Significant.

Table 4 Risk factors for anxiety disorders after logistic regression.

Variables No anxietydisorders n = 107

Anxiety dsordersn = 48

OR (95% CI) p

SexFemale 63 (58.9) 37 (77.1)Male 44 (41.1) 11 (22.9) 2.34 (1.06—5.17) 0.035*

Mean age of epilepsy onset (yrs, SD) 14.2 (1.37) 15.8 (2.28) 1.01 (0.98—1.03) 0.364C-1019G

Presence of C allele 80 (74.8) 42 (87.5) 2.77 (1.01—7.63) 0.048*

Presence of G allele 77 (72.0) 38 (79.2) 1.78 (0.74—4.16) 0.20

Values are presented frequency (percentage) or mean (SD). yrs = years.* Significant.

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ime of epilepsy, age of epilepsy onset and C-1019G polymor-hism in a binary logistic regression model. After logisticegression, presence of C allele of 5-HT1A C-1019G poly-orphism and sex remained significantly and independently

ssociated with anxiety disorder in TLE patients. The finalesults of logistic regression are presented in Table 4.

iscussion

n the present study we investigated the influence of 5-TTVNTR and 5-HTTLPR in the 5-HTT gene and of C-1019G

n the 5-HT1A gene on psychiatric comorbidities in TLE.e observed no significant differences in these genotype

requencies among TLE patients regarding the presence ofsychiatric comorbidities. As expected, women had a higherrevalence of psychiatric comorbidities than men, exceptor substance abuse. These results are in agreement withhe literature (Andrade et al., 2006; Bragatti et al., 2009,010). Interesting, we observed that the presence of C alleleas an isolated predictor of anxiety disorder in TLE.

5-HT1A is highly expressed in limbic structures and sero-onin activation of this receptor might have antiepilepticffect (Bagdy et al., 2007). PET studies found a reductionn 5-HT1A receptor binding in depressed patients (Drevetst al., 1999) as well as in epileptic patients (Savic et al.,004). Interestingly, individuals with epilepsy plus depres-ion have lower 5-HT1A activity than those with epilepsylone (Theodore, 2003) and changes of the serotoninergicathway are associated with depressive symptoms in TLEatients (Lothe et al., 2008). Taken together, these studiesuggest that abnormalities in the 5-HT1A receptor might be

common mechanism linking epilepsy and depression. Thus,t is plausible that genetic changes in the 5-HT1A gene alter-ng its expression may be associated with a risk for epilepsyr for psychiatric comorbidites in epileptic patients. Heree observed that patients with the C allele of C-1019Golymorphism in the 5-HT1A gene had higher frequencyf anxiety disorder. Some studies indicate that C-1019Golymorphism impact transcriptional regulation of the genehrough altered biding of the transcription factors humanuclear deformed epidermal autoregulatory factor-1 (DEAF-) and Hairy/enhancer of split-5 (Hes5). Specifically, G allelef C-1019G polymorphism abolishes repression of the 5-HT1Aene by DEAF-1 and partially impairs Hes5-mediated repres-ion (Lemonde et al., 2003). As a consequence it wouldead to the increase of 5-HT1A autoreceptor expression and,ccording with Fakar et al. it is associated with decrease ofhe amygdala reactivity (Fakra et al., 2009). Several studiesave correlating amygdala reactivity with anxiety disordersSomerville et al., 2004; Stein et al., 2002; Schwartz et al.,003). Taken together, these evidences are in line with ouresults, once we observed that patients with C allele showedncreased frequency of anxiety disorders, and it might beue to increased amygdala activation. In contrast, sometudies have found that the G allele of C-1019G is a riskactor for the development of neuropsychiatric diseases,mong them depression and panic disorder (Freitag et al.,

006; Hettema et al., 2008; Le Francois et al., 2008). As fars we know, only one study has evaluated the influence ofhe 5-HT1A gene C-1019G polymorphism on epileptogenesis,howing no significant results (Stefulj et al., 2010). However,

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L.C. Schenkel et al.

o one before has evaluated the effect of 5-HT1A gene-1019G polymorphism on the development of neuropsychi-tric disorders in TLE. Here we observed for the first timen association between 5-HT1A gene C-1019G polymorphismnd anxiety disorders in TLE.

Regarding the 5-HTT polymorphisms (5-HTTVNTR and-HTTLPR), several studies have shown that low tran-criptional activity 5-HTT genotypes are associated witheuropsychiatric disorders, among them depression (Holmest al., 2010), suicidal behavior (Anguelova et al., 2003),ttention deficit hyperactivity disorder (Kent et al., 2002),nd personality disorder (Garcia et al., 2010). Moreover,ecent studies have suggested that epilepsy, its severityr its response to medical treatment is associated with 5-TTLPR and 5-HTTVNTR polymorphism (Kauffman et al.,009; Hecimovic et al., 2010; Manna et al., 2007). Since-HTT polymorphisms seem to be associated with bothpilepsy and psychopathologies, we investigated the asso-iation of these genetic variants in psychiatric comorbidityn patients with TLE. However, despite strong biologicallausibility, we were unable to demonstrate any associationf 5-HTTLPR and 5-HTTVNTR with mood disorder, anxietyisorder, psychosis or drug/alcohol abuse in TLE patients.owever, because of the impact of serotonin gene poly-orphisms on several psychiatric diseases and the evidence

f the influence of the serotoninergic system on epilepsy,urther studies with larger samples evaluating possible influ-nces of serotoninergic gene polymorphisms in psychiatricomorbidities in epilepsy are still necessary before reachingnal conclusions.

We recognize that our work has limitations. The eth-ic admixture of the Brazilian sample may be a bias inenetic studies. However, the population of Rio Grandeo Sul State, where this study was conduced, is mainlyomposed of Caucasian European descendants (82% of theopulation) (Cordeiro et al., 2009). In addition to the eth-ic stratification limitation discussed above, sample size isn important limitation of this study. In fact, due to theelatively limited number of patients included in our studye were unable to provide detailed analysis of possibleenetic polymorphism impact in specific subtypes of neu-opsychiatric comorbidities (e.g. humor or anxiety subtypeisorders). Also, negative results need to be interpretedith caution due to lack of statistical power (error type II)

n our study. Moreover, we recognize that due to multiplenalysis we might had incurred also in error type I. Oneould argue that error type I could be avoided by the usef Bonferroni correction, for example. However, the use ofonferroni correction might not be appropriated in studiess ours (Perneger, 1998). Moreover, we used univariate testsignificance only as a screening step before a logistic regres-ion model, and our conclusions are based on one singleodel of logistic regression. Finally, but not less impor-

ant, we need to consider that we are observing an effectf a functional polymorphisms and thus our conclusions areased on biologically plausibility. In this venue our observa-ions might help to beginning understand the molecular basisf anxiety in epilepsy. In fact, this might be an initial work

n a field that remains almost unexplored. Further studiesre necessary to clarify these matters.

In summary, we observed in our study that presencef C allele of 5-HT1A C-1019G polymorphism might be an

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Serotonin gene polymorphisms and psychiatry comorbidities

independent risk factor for anxiety disorders in TLE. As far aswe know, this is the first study showing a biological plausibleassociation between a functional gene polymorphism and apsychiatric comorbidity in TLE. We believe that other studiesin this venue will shade some light on molecular mechanismsinvolved in psychiatric comorbidities in epilepsy.

Acknowledgments

This work was supported by Brazilian GovernmentResearch Funds, FIPE-HCPA and CNPq (#551902/2009-4;#306644/2010-0; #483108/2010-3) and FAPERGS/PRONEM.The authors have declared that no competing interestsexist. The funders had no role in study design, data collec-tion and analysis, decision to publish, or preparation of themanuscript.

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