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Association of serotonin transporter promoter (5-HTTLPR) and intron 2 (VNTR-2) polymorphisms with treatment response in temporal lobe epilepsy
Epilepsy Research (2010) 91, 35—38
journal homepage: www.elsevier.com/locate/epilepsyres
Association of serotonin transporter promoter (5-HTTLPR) and intron 2 (VNTR-2) polymorphisms with treatment response in temporal lobe epilepsy Hecimovic Hrvoje a,∗, Stefulj Jasminka b, Cicin-Sain Lipa b, Demarin Vida a, Jernej Branimir b,1 a b
Zagreb Epilepsy Center, Department of Neurology, University Hospital, Vinogradska 29, HR-10000 Zagreb, Croatia Laboratory of Neurochemistry and Molecular Neurobiology, Rudjer Boskovic Institute, HR-10000 Zagreb, Croatia
Received 8 March 2010; received in revised form 25 May 2010; accepted 20 June 2010 Available online 23 July 2010
KEYWORDS Temporal lobe epilepsy; Serotonin; 5-HTTLPR; VNTR-2; Limbic system
Summary Purpose: Temporal lobe epilepsy (TLE) is the most common epilepsy and about 30% of patients have poorly controlled seizures. Neurobiology underlying responsiveness to medical treatment in TLE patients is unclear and there are currently no biological tests to predict course of the disease. Animal and human studies repeatedly suggested serotonergic dysfunction in subjects with TLE. We investigated association of serotonin transporter (5-HTT) gene polymorphisms with medical treatment response in patients with TLE. Methods: We analyzed 5-HTT gene linked polymorphic region (5-HTTLPR) in promoter and variable number of tandem repeats in the second intron of the 5-HTT gene (VNTR-2) in 101 consecutive subjects with TLE. Results: TLE patients with the combination of transcriptionally more efficient genotypes, i.e. 5HTTLPR L/L and VNTR-2 12/12, had increased seizure refractoriness to antiepileptic medication therapy and shorter periods of seizure freedom, than subjects with other combinations of the 5HTT genotypes. There were no other clinical or demographic differences among patient groups based on the 5-HTT genotypes. Conclusion: Combination of the 5-HTT genotypes linked with higher 5-HTT gene expression was found to be associated with worse response to optimal drug therapy. Further studies should determine potential role of this 5-HTT genotype polymorphism in epileptogenesis. © 2010 Elsevier B.V. All rights reserved.
Introduction ∗ 1
Corresponding author. Tel.: +385 1 3787731; fax: +385 1 3787731. E-mail address: [email protected] (H. Hrvoje). Deceased.
Epilepsy is common chronic neurological disorder. The majority of patients suffer from temporal lobe epilepsy (TLE). Neurobiological mechanisms that contribute to TLE
0920-1211/$ — see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.eplepsyres.2010.06.008
36 remain unclear. Animal and human studies investigated contribution of the serotonergic (5-HT) system in the neurobiology of TLE (Theodore, 2003). Earlier studies showed that genetic epilepsy-prone rats have reduced hippocampal 5-HT receptor density (Dailey et al., 1992; De Sarro et al., 1996; Statnick et al., 1996; Salgado-Commissariat and Alkadhi, 1997) and that impairment of serotonergic activity may enhance seizure severity (Jobe et al., 1999). Serotonininduced anticonvulsant effect was mostly mediated by hippocampal 5-HT1A receptors (Salgado-Commissariat and Alkadhi, 1997; Lu and Gean, 1998). Recent human studies demonstrated decreased 5-HT1A receptor binding in the ictal onset and ictal spreading temporal lobe regions (Merlet et al., 2004), midbrain raphe and thalamus (Toczek et al., 2003; Giovacchini et al., 2005; Hasler et al., 2007), in mesial and neocortical TLE (Fedi et al., 2001). Decreased availability of 5-HT1A receptors highly correlates with the degree of epileptogenicity of cortical areas, as shown by intracranial recordings (Merlet et al., 2004). Thus, serotonergic system dysfunction may have an important role in neurobiology of epileptogenicity and seizure severity in TLE subjects. 5-HT transporter (5-HTT) is a transmembrane protein responsible for re-uptake of 5-HT from the synaptic cleft (Lesch and Mossner, 1998). 5-HTT is encoded by a single copy gene (SLC6A4) which contains two common polymorphisms: a 44 base pair (bp) insertion/deletion in the promoter region (5-HTT gene linked polymorphic region, 5-HTTLPR) and a 17 bp variable number of tandem repeats in the second intron (VNTR-2). It was demonstrated that long (L) and short (S) variants of the 5-HTTLPR differentially modulate transcription of the 5-HTT gene, with the L variant being more efficient in a recessive manner (Lesch et al., 1996). Similarly, 12-repeat allele of the VNTR-2 intron was shown to have stronger enhancer-like properties than shorter alleles (MacKenzie and Quinn, 1999). We have previously shown a combined effect of 5-HTTLPR and VNTR-2 polymorphisms on the level of 5-HTT gene expression, with the highest expression in the group with two ‘‘high-expressing’’ genotypes (L/L 12/12), approximately 20% lower in the group with one ‘‘low-expressing’’ genotype and 50% lower in the group with ‘‘low-expressing’’ genotypes at both loci (Hranilovic et al., 2004). 5-HTT genetic variations have been found to affect basal cerebral metabolic activity in limbic structures in normal population (Graff-Guerrero et al., 2005) and was observed to be associated with depression (Caspi et al., 2003), schizophrenia (Hranilovic et al., 2000), and suicidal behavior (Hranilovic et al., 2003). This is the first study to our knowledge that showed association of the combination of high-expressing genotypes of 5-HTT gene and seizure factors in the patients with TLE.
Methods Patients We recruited 101 consecutive patients with video/EEG confirmed TLE at the Zagreb Epilepsy Center, during 2007—2009. Clinical data included age, gender, seizure factors (age of epilepsy onset, seizure frequency, epilepsy duration, time to initial diagnosis, epileptic region lateralization) and number of antiepileptic drugs (AEDs), education, employment and marital status. Subjects with hip-
H. Hrvoje et al. pocampal sclerosis, but no other focal structural lesion shown on the epilepsy protocol with 1.5 T MRI in the temporal lobe, were also included in this study. Seizure frequency was self-reported by the patients and their family members and then averaged for each month for the previous 6 months. The study was approved by the local ethics committee and written informed consent was obtained from each patient.
Genotyping Genomic DNA was extracted from the peripheral blood according to the standard procedures. Polymerase chain reaction (PCR) conditions and primer sequences were in detail described in our previous report (Hranilovic et al., 2004). PCR products were separated on agarose gels and visualized with ultraviolet light after ethidium bromide staining. We had four subjects with 912 5-HTT genotype that were not included in statistical analysis.
Statistical analysis We used Student’s t-test and analysis of variance to test for differences in demographic and clinical variables. Alpha level <0.05, two-tailed, was set as significant. All data were analyzed with the SPSS (Version 11.0.1, SPSS Inc., Chicago, IL) statistical package.
Results We found that TLE patients with a combination of homozygotic L and homozygotic 12-repeat 5-HTT genotypes have significantly higher seizure frequency. The mean seizure frequency in TLE patients carrying L/L and 12/12 genotypes (group A, n = 7, in Fig. 1) was significantly higher than in the group with L/L 10/10, L/L 10/12, S/S 12/12 and S/L 12/12
Figure 1 Seizure frequency (shown as mean ± SD) was significantly higher in the group A, than in the group B (p < 0.005) or in the group C (p < 0.006). Group A presents L/L and 12/12 5-HTT genotypes (n = 7), group B presents L/L 10/10, L/L 10/12, S/S 12/12 and S/L 12/12 genotypes (n = 56), and group C presents ‘‘no L/L and no 12/12’’ 5-HTT genotypes that consist of S/L 10/10, S/L 10/12, S/S 10/10 and S/S 10/12 combinations, n = 34. Seizure frequency presents average number of complex partial and secondarily generalized seizures per month for the previous 6 months.
Association of serotonin transporter promoter (5-HTTLPR) and intron 2 (VNTR-2) polymorphisms in TLE Table 1
37
Demographic and clinical variables of 101 TLE patients, based on the 5-HTT gene polymorphism.
Age (years) Gender (M/F in %) Age of onset (years) Seizure Frequency/6 Mo Seizure duration (years) Current number of AEDs History of secondarily generalized seizures or status epilepticus Presence of mesial temporal sclerosis (%) Education (years, %) 8 12 16 Employed (Y, %) Married (Y, %)
L/L 12/12, n =7
L/L 10/10, L/L 10/12, S/S 12/12, S/L 12/12, n =56
No L/L, no 12/12, n =34
32 ± 14 42.9/57.1 19 ± 13 11.0 ± 8.5 15 ± 12 1.8 ± 0.9 71%
33 ± 14 23.2 ± 76.8 22 ± 15 2.0 ± 5.1 11 ± 10 1.5 ± 0.8 36%
32 ± 13 38.9 ± 61.1 20 ± 12 1.6 ± 3.5 13 ± 11 1.5 ± 0.8 44%
25
31
19
28.6 42.9 28.6 57.1 28.6
3.6 67.9 63.9 71.4 37.5
5.6 63.9 25.0 77.8 50
M: male; F: female; Mo: months; TLE: temporal lobe epilepsy; AED: antiepileptic drug.
genotypes, p < 0.005 (group B, n = 56) or in the group with ‘‘no L/L, no 12/12’’ genotypes, p < 0.006 (group C, n = 34), or when the L/L 12/12 group was compared with the rest of the patients (p < 0.0001, n = 90). Increased seizure frequency was significant both for the previous month (p < 0.001) and averaged for the previous 6 months (p < 0.0001). The group A had also shorter seizure-free periods (p < 0.01). Clinical and demographic variables of the three groups of TLE patients show that patients from the L/L 12/12 group had more secondarily generalized seizures or history of status epilepticus (Table 1). There was a similar distribution of the subjects with mesial temporal sclerosis in all groups, and only two of them had fully controlled seizures. There was no difference in age, gender, age of epilepsy onset, duration of epilepsy or number of antiepileptic drugs among the groups. Interestingly, there was a greater tendency for persons from the L/L 12/12 group to have lower level of education, less were married or employed (Table 1).
Discussion In this study we found association of the combination of transcriptionally more efficient (homozygotic L and homozygotic 12-repeat) 5-HTT genotype with poorer response to optimal medical treatment in TLE patients. These patients also have shorter periods of seizure freedom. Previous studies suggested a role of the serotonin transporter gene in the etiology of TLE using association analysis of the 5-HTT gene in TLE patients (Manna et al., 2007) or 5-HTT gene variation in refractory mesial TLE with evidence of hippocampal sclerosis (Kauffman et al., 2009). Kauffman et al. (2009) observed that homozygous carriers of the 12repeat allele of the VNTR-2 5-HTT gene polymorphism had poorer response to medical treatment in the presence of hippocampal sclerosis. Our study evaluated broader range of TLE patients, including subjects with hippocampal sclerosis. TLE may have
very variable clinical expression, from mild and medically treatable in some, to more resistant to antiepileptic therapy in others, suggesting a combination of environmental and genetic factors in etiology of the disease. Animal and, more recently, human studies pointed to critical importance of 5-HT availability in seizure regulation in epilepsy-prone rats and reduced serotonin binding to mainly 5-HT1A receptors in patients with chronic epilepsy (Statnick et al., 1996; Giovacchini et al., 2005). Biochemical studies showed that increased transcription of 5-HTT, due to genotypes with higher expression of the 5-HTT gene, may lead to decreased 5-HT concentration in the synaptic cleft (Fiskerstrand et al., 1999; Kauffman et al., 2009). Lower extracellular levels of serotonin seem to be pro-convulsant. This may potentially be a trigger for aggravation of seizure frequency in patients with TLE. In our previous study we showed that combination of homozygotic L and homozygotic 12repeat 5-HTT genotypes is linked with higher levels of the 5-HTT mRNA expression (Hranilovic et al., 2004). Our results may indicate that enhancement of the 5-HTT expression leads to seizure refractoriness and poorer response to medical treatment. This may potentially provide a new diagnostic tool for seizure refractoriness and introduce other modalities of epilepsy treatment at earlier stage of the disease. There are important limitations to this study. Association studies have limits and subtle biases and they need to be repeated with larger sample sizes. Additional genetic studies of the 5-HT receptors are required to fully understand the potential role of the 5-HTT gene in the etiopathogenesis of TLE. We also hypothesized earlier that drugs that inhibit 5-HT re-uptake can act in synergy with antiepileptic medications (Gilliam et al., 2004; Mainardi et al., 2008). In summary, we demonstrated association between a combination of 5-HTTLPR and VNTR-2 genotypes of the 5HTT gene and poorer treatment response in patients with TLE. We suggest that higher expression of the 5-HTT results
38 in serotonin network changes and increased severity of TLE. These findings may contribute to better understanding of serotonergic mechanisms in epileptogenesis.
Acknowledgements This work was supported by Epilepsy Foundation grant to HH and Croatian Ministry of Science, Education and Sport project to BJ.
References Caspi, A., Sugden, K., Moffitt, T.E., Taylor, A., Craig, I.W., Harrington, H., McClay, J., Mill, J., Martin, J., Braithwaite, A., Poulton, R., 2003. Influence of life stress on depression: moderation by a polymorphism in the 5-HTT gene. Science 301, 386—389. Dailey, J.W., Mishra, P.K., Ko, K.H., Penny, J.E., Jobe, P.C., 1992. Serotonergic abnormalities in the central nervous system of seizure-naive genetically epilepsy-prone rats. Life Sci. 50, 319—326. De Sarro, G., Di Paola, E.D., Aguglia, U., de Sarro,.A., 1996. Tolerance to anticonvulsant effects of some benzodiazepines in genetically epilepsy prone rats. Pharmacol. Biochem. Behav. 55, 39—48. Fedi, M., Reutens, D., Okazawa, H., Andermann, F., Boling, W., Dubeau, F., White, C., Nakai, A., Gross, D.W., Andermann, E., Diksic, M., 2001. Localizing value of alpha-methyl-L-tryptophan PET in intractable epilepsy of neocortical origin. Neurology 57, 1629—1636. Fiskerstrand, C.E., Lovejoy, E.A., Quinn, J.P., 1999. An inotropic polymorphic domain often associated with susceptability to affective disorders has allele dependent differential enhancer activity in embryonic stem cells. FEBS Lett. 458, 171—174. Gilliam, F., Santos, J., Vahle, V., Carter, J., Brown, K., Hecimovic, H., 2004. Depression in epilepsy: ignoring clinical expression of neuronal network dysfunction? Epilepsia 45 (Suppl. 2), 28—33. Giovacchini, G., Toczek, M.T., Bonwetsch, R., Bagic, A., Lang, L., Fraser, C., Reeves-Tyer, P., Herscovitch, P., Eckelman, W.C., Carson, R.E., Theodore, W.H., 2005. 5-HT 1A receptors are reduced in temporal lobe epilepsy after partial-volume correction. J. Nucl. Med. 46, 1128—1135. Graff-Guerrero, A., de la Fuente-Sandoval, S., Camarena, B., Gomez-Martin, D., Apiquian, R., Fresan, A., Aguilar, A., MendezNunez, J.C., Escalona-Huerta, C., Drucker-Colin, R., Nicolini, H., 2005. Frontal and limbic metabolic differences in subjects selected according to genetic variation of the SLC6A4 gene polymorphism. Neuroimage 25, 1197—1204. Hasler, G., Bonwetsch, R., Giovacchini, G., Toczek, M.T., Bagic, A., Luckenbaugh, D.A., Drevets, W.C., Theodore, W.H., 2007. 5-HT1A receptor binding in temporal lobe epilepsy patients with and without major depression. Biol. Psychiatry 62, 1258—1264. Hranilovic, D., Schwab, S.G., Jernej, B., Knapp, M., Lerer, B., Albus, M., Rietschel, M., Kanyas, K., Borrmann, M., Lichtermann, D., Maier, W., Wildenauer, D.B., 2000. Serotonin transporter gene and schizophrenia: evidence for association/linkage disequilibrium in families with affected siblings. Mol. Psychiatry 5, 91—95.
H. Hrvoje et al. Hranilovic, D., Stefulj, J., Furac, I., Kubat, M., Balija, M., Jernej, B., 2003. Serotonin transporter gene promoter (5-HTTLPR) and intron 2 (VNTR) polymorphisms in Croatian suicide victims. Biol. Psychiatry 54, 884—889. Hranilovic, D., Stefulj, J., Schwab, S., Borrmann-Hassenbach, M., Albus, M., Jernej, B., Wildenauer, D., 2004. Serotonin transporter promoter and intron 2 polymorphisms: relationship between allelic variants and gene expression. Biol. Psychiatry 55, 1090—1094. Jobe, P.C., Dailey, J.W., Wernicke, J.F., 1999. A noradrenergic and serotonergic hypothesis of the linkage between epilepsy and affective disorders. Crit. Rev. Neurobiol. 13, 317—356. Kauffman, M.A., Consalvo, D., Gonzalez-Moron, D., Aguirre, F., D’Alessio, L., Kochen, S., 2009. Serotonin transporter gene variation and refractory mesial temporal epilepsy with hippocampal sclerosis. Epilepsy Res. 85, 231—234. Lesch, K.P., Bengel, D., Heils, A., Sabol, S.Z., Greenberg, B.D., Petri, S., Benjamin, J., Muller, C.R., Hamer, D.H., Murphy, D.L., 1996. Association of anxiety-related traits with a polymorphism in the serotonin transporter gene regulatory region. Science 274, 1527—1531. Lesch, K.P., Mossner, R., 1998. Genetically driven variation in serotonin uptake: is there a link to affective spectrum, neurodevelopmental, and neurodegenerative disorders? Biol. Psychiatry 44, 179—192. Lu, K.T., Gean, P.W., 1998. Endogenous serotonin inhibits epileptiform activity in rat hippocampal CA1 neurons via 5hydroxytryptamine1A receptor activation. Neuroscience 86, 729—737. MacKenzie, A., Quinn, J., 1999. A serotonin transporter gene intron 2 polymorphic region, correlated with affective disorders, has allele-dependent differential enhancer-like properties in the mouse embryo. Proc. Natl. Acad. Sci. U.S.A. 96, 15251—15255. Mainardi, P., Leonardi, A., Albano, C., 2008. Potentiation of brain serotonin activity may inhibit seizures, especially in drugresistant epilepsy. Med. Hypotheses 70, 876—879. Manna, I., Labate, A., Gambardella, A., Forabosco, P., La Russa, A., Le Piane, E., Aguglia, U., Quattrone, A., 2007. Serotonin transporter gene (5-Htt): association analysis with temporal lobe epilepsy. Neurosci. Lett. 421, 52—56. Merlet, I., Ostrowsky, K., Costes, N., Ryvlin, P., Isnard, J., Faillenot, I., Lavenne, F., Dufournel, D., Le, B.D., Mauguiere, F., 2004. 5-HT1A receptor binding and intracerebral activity in temporal lobe epilepsy: an [18F]MPPF-PET study. Brain 127, 900— 913. Salgado-Commissariat, D., Alkadhi, K.A., 1997. Serotonin inhibits epileptiform discharge by activation of 5-HT1A receptors in CA1 pyramidal neurons. Neuropharmacology 36, 1705—1712. Statnick, M.A., Dailey, J.W., Jobe, P.C., Browning, R.A., 1996. Abnormalities in brain serotonin concentration, high-affinity uptake, and tryptophan hydroxylase activity in severe-seizure genetically epilepsy-prone rats. Epilepsia 37, 311—321. Theodore, W.H., 2003. Does serotonin play a role in epilepsy? Epilepsy Curr. 3, 173—177. Toczek, M.T., Carson, R.E., Lang, L., Ma, Y., Spanaki, M.V., Der, M.G., Fazilat, S., Kopylev, L., Herscovitch, P., Eckelman, W.C., Theodore, W.H., 2003. PET imaging of 5-HT1A receptor binding in patients with temporal lobe epilepsy. Neurology 60, 749—756.
journal homepage: www.elsevier.com/locate/epilepsyres
Association of serotonin transporter promoter (5-HTTLPR) and intron 2 (VNTR-2) polymorphisms with treatment response in temporal lobe epilepsy Hecimovic Hrvoje a,∗, Stefulj Jasminka b, Cicin-Sain Lipa b, Demarin Vida a, Jernej Branimir b,1 a b
Zagreb Epilepsy Center, Department of Neurology, University Hospital, Vinogradska 29, HR-10000 Zagreb, Croatia Laboratory of Neurochemistry and Molecular Neurobiology, Rudjer Boskovic Institute, HR-10000 Zagreb, Croatia
Received 8 March 2010; received in revised form 25 May 2010; accepted 20 June 2010 Available online 23 July 2010
KEYWORDS Temporal lobe epilepsy; Serotonin; 5-HTTLPR; VNTR-2; Limbic system
Summary Purpose: Temporal lobe epilepsy (TLE) is the most common epilepsy and about 30% of patients have poorly controlled seizures. Neurobiology underlying responsiveness to medical treatment in TLE patients is unclear and there are currently no biological tests to predict course of the disease. Animal and human studies repeatedly suggested serotonergic dysfunction in subjects with TLE. We investigated association of serotonin transporter (5-HTT) gene polymorphisms with medical treatment response in patients with TLE. Methods: We analyzed 5-HTT gene linked polymorphic region (5-HTTLPR) in promoter and variable number of tandem repeats in the second intron of the 5-HTT gene (VNTR-2) in 101 consecutive subjects with TLE. Results: TLE patients with the combination of transcriptionally more efficient genotypes, i.e. 5HTTLPR L/L and VNTR-2 12/12, had increased seizure refractoriness to antiepileptic medication therapy and shorter periods of seizure freedom, than subjects with other combinations of the 5HTT genotypes. There were no other clinical or demographic differences among patient groups based on the 5-HTT genotypes. Conclusion: Combination of the 5-HTT genotypes linked with higher 5-HTT gene expression was found to be associated with worse response to optimal drug therapy. Further studies should determine potential role of this 5-HTT genotype polymorphism in epileptogenesis. © 2010 Elsevier B.V. All rights reserved.
Introduction ∗ 1
Corresponding author. Tel.: +385 1 3787731; fax: +385 1 3787731. E-mail address: [email protected] (H. Hrvoje). Deceased.
Epilepsy is common chronic neurological disorder. The majority of patients suffer from temporal lobe epilepsy (TLE). Neurobiological mechanisms that contribute to TLE
0920-1211/$ — see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.eplepsyres.2010.06.008
36 remain unclear. Animal and human studies investigated contribution of the serotonergic (5-HT) system in the neurobiology of TLE (Theodore, 2003). Earlier studies showed that genetic epilepsy-prone rats have reduced hippocampal 5-HT receptor density (Dailey et al., 1992; De Sarro et al., 1996; Statnick et al., 1996; Salgado-Commissariat and Alkadhi, 1997) and that impairment of serotonergic activity may enhance seizure severity (Jobe et al., 1999). Serotonininduced anticonvulsant effect was mostly mediated by hippocampal 5-HT1A receptors (Salgado-Commissariat and Alkadhi, 1997; Lu and Gean, 1998). Recent human studies demonstrated decreased 5-HT1A receptor binding in the ictal onset and ictal spreading temporal lobe regions (Merlet et al., 2004), midbrain raphe and thalamus (Toczek et al., 2003; Giovacchini et al., 2005; Hasler et al., 2007), in mesial and neocortical TLE (Fedi et al., 2001). Decreased availability of 5-HT1A receptors highly correlates with the degree of epileptogenicity of cortical areas, as shown by intracranial recordings (Merlet et al., 2004). Thus, serotonergic system dysfunction may have an important role in neurobiology of epileptogenicity and seizure severity in TLE subjects. 5-HT transporter (5-HTT) is a transmembrane protein responsible for re-uptake of 5-HT from the synaptic cleft (Lesch and Mossner, 1998). 5-HTT is encoded by a single copy gene (SLC6A4) which contains two common polymorphisms: a 44 base pair (bp) insertion/deletion in the promoter region (5-HTT gene linked polymorphic region, 5-HTTLPR) and a 17 bp variable number of tandem repeats in the second intron (VNTR-2). It was demonstrated that long (L) and short (S) variants of the 5-HTTLPR differentially modulate transcription of the 5-HTT gene, with the L variant being more efficient in a recessive manner (Lesch et al., 1996). Similarly, 12-repeat allele of the VNTR-2 intron was shown to have stronger enhancer-like properties than shorter alleles (MacKenzie and Quinn, 1999). We have previously shown a combined effect of 5-HTTLPR and VNTR-2 polymorphisms on the level of 5-HTT gene expression, with the highest expression in the group with two ‘‘high-expressing’’ genotypes (L/L 12/12), approximately 20% lower in the group with one ‘‘low-expressing’’ genotype and 50% lower in the group with ‘‘low-expressing’’ genotypes at both loci (Hranilovic et al., 2004). 5-HTT genetic variations have been found to affect basal cerebral metabolic activity in limbic structures in normal population (Graff-Guerrero et al., 2005) and was observed to be associated with depression (Caspi et al., 2003), schizophrenia (Hranilovic et al., 2000), and suicidal behavior (Hranilovic et al., 2003). This is the first study to our knowledge that showed association of the combination of high-expressing genotypes of 5-HTT gene and seizure factors in the patients with TLE.
Methods Patients We recruited 101 consecutive patients with video/EEG confirmed TLE at the Zagreb Epilepsy Center, during 2007—2009. Clinical data included age, gender, seizure factors (age of epilepsy onset, seizure frequency, epilepsy duration, time to initial diagnosis, epileptic region lateralization) and number of antiepileptic drugs (AEDs), education, employment and marital status. Subjects with hip-
H. Hrvoje et al. pocampal sclerosis, but no other focal structural lesion shown on the epilepsy protocol with 1.5 T MRI in the temporal lobe, were also included in this study. Seizure frequency was self-reported by the patients and their family members and then averaged for each month for the previous 6 months. The study was approved by the local ethics committee and written informed consent was obtained from each patient.
Genotyping Genomic DNA was extracted from the peripheral blood according to the standard procedures. Polymerase chain reaction (PCR) conditions and primer sequences were in detail described in our previous report (Hranilovic et al., 2004). PCR products were separated on agarose gels and visualized with ultraviolet light after ethidium bromide staining. We had four subjects with 912 5-HTT genotype that were not included in statistical analysis.
Statistical analysis We used Student’s t-test and analysis of variance to test for differences in demographic and clinical variables. Alpha level <0.05, two-tailed, was set as significant. All data were analyzed with the SPSS (Version 11.0.1, SPSS Inc., Chicago, IL) statistical package.
Results We found that TLE patients with a combination of homozygotic L and homozygotic 12-repeat 5-HTT genotypes have significantly higher seizure frequency. The mean seizure frequency in TLE patients carrying L/L and 12/12 genotypes (group A, n = 7, in Fig. 1) was significantly higher than in the group with L/L 10/10, L/L 10/12, S/S 12/12 and S/L 12/12
Figure 1 Seizure frequency (shown as mean ± SD) was significantly higher in the group A, than in the group B (p < 0.005) or in the group C (p < 0.006). Group A presents L/L and 12/12 5-HTT genotypes (n = 7), group B presents L/L 10/10, L/L 10/12, S/S 12/12 and S/L 12/12 genotypes (n = 56), and group C presents ‘‘no L/L and no 12/12’’ 5-HTT genotypes that consist of S/L 10/10, S/L 10/12, S/S 10/10 and S/S 10/12 combinations, n = 34. Seizure frequency presents average number of complex partial and secondarily generalized seizures per month for the previous 6 months.
Association of serotonin transporter promoter (5-HTTLPR) and intron 2 (VNTR-2) polymorphisms in TLE Table 1
37
Demographic and clinical variables of 101 TLE patients, based on the 5-HTT gene polymorphism.
Age (years) Gender (M/F in %) Age of onset (years) Seizure Frequency/6 Mo Seizure duration (years) Current number of AEDs History of secondarily generalized seizures or status epilepticus Presence of mesial temporal sclerosis (%) Education (years, %) 8 12 16 Employed (Y, %) Married (Y, %)
L/L 12/12, n =7
L/L 10/10, L/L 10/12, S/S 12/12, S/L 12/12, n =56
No L/L, no 12/12, n =34
32 ± 14 42.9/57.1 19 ± 13 11.0 ± 8.5 15 ± 12 1.8 ± 0.9 71%
33 ± 14 23.2 ± 76.8 22 ± 15 2.0 ± 5.1 11 ± 10 1.5 ± 0.8 36%
32 ± 13 38.9 ± 61.1 20 ± 12 1.6 ± 3.5 13 ± 11 1.5 ± 0.8 44%
25
31
19
28.6 42.9 28.6 57.1 28.6
3.6 67.9 63.9 71.4 37.5
5.6 63.9 25.0 77.8 50
M: male; F: female; Mo: months; TLE: temporal lobe epilepsy; AED: antiepileptic drug.
genotypes, p < 0.005 (group B, n = 56) or in the group with ‘‘no L/L, no 12/12’’ genotypes, p < 0.006 (group C, n = 34), or when the L/L 12/12 group was compared with the rest of the patients (p < 0.0001, n = 90). Increased seizure frequency was significant both for the previous month (p < 0.001) and averaged for the previous 6 months (p < 0.0001). The group A had also shorter seizure-free periods (p < 0.01). Clinical and demographic variables of the three groups of TLE patients show that patients from the L/L 12/12 group had more secondarily generalized seizures or history of status epilepticus (Table 1). There was a similar distribution of the subjects with mesial temporal sclerosis in all groups, and only two of them had fully controlled seizures. There was no difference in age, gender, age of epilepsy onset, duration of epilepsy or number of antiepileptic drugs among the groups. Interestingly, there was a greater tendency for persons from the L/L 12/12 group to have lower level of education, less were married or employed (Table 1).
Discussion In this study we found association of the combination of transcriptionally more efficient (homozygotic L and homozygotic 12-repeat) 5-HTT genotype with poorer response to optimal medical treatment in TLE patients. These patients also have shorter periods of seizure freedom. Previous studies suggested a role of the serotonin transporter gene in the etiology of TLE using association analysis of the 5-HTT gene in TLE patients (Manna et al., 2007) or 5-HTT gene variation in refractory mesial TLE with evidence of hippocampal sclerosis (Kauffman et al., 2009). Kauffman et al. (2009) observed that homozygous carriers of the 12repeat allele of the VNTR-2 5-HTT gene polymorphism had poorer response to medical treatment in the presence of hippocampal sclerosis. Our study evaluated broader range of TLE patients, including subjects with hippocampal sclerosis. TLE may have
very variable clinical expression, from mild and medically treatable in some, to more resistant to antiepileptic therapy in others, suggesting a combination of environmental and genetic factors in etiology of the disease. Animal and, more recently, human studies pointed to critical importance of 5-HT availability in seizure regulation in epilepsy-prone rats and reduced serotonin binding to mainly 5-HT1A receptors in patients with chronic epilepsy (Statnick et al., 1996; Giovacchini et al., 2005). Biochemical studies showed that increased transcription of 5-HTT, due to genotypes with higher expression of the 5-HTT gene, may lead to decreased 5-HT concentration in the synaptic cleft (Fiskerstrand et al., 1999; Kauffman et al., 2009). Lower extracellular levels of serotonin seem to be pro-convulsant. This may potentially be a trigger for aggravation of seizure frequency in patients with TLE. In our previous study we showed that combination of homozygotic L and homozygotic 12repeat 5-HTT genotypes is linked with higher levels of the 5-HTT mRNA expression (Hranilovic et al., 2004). Our results may indicate that enhancement of the 5-HTT expression leads to seizure refractoriness and poorer response to medical treatment. This may potentially provide a new diagnostic tool for seizure refractoriness and introduce other modalities of epilepsy treatment at earlier stage of the disease. There are important limitations to this study. Association studies have limits and subtle biases and they need to be repeated with larger sample sizes. Additional genetic studies of the 5-HT receptors are required to fully understand the potential role of the 5-HTT gene in the etiopathogenesis of TLE. We also hypothesized earlier that drugs that inhibit 5-HT re-uptake can act in synergy with antiepileptic medications (Gilliam et al., 2004; Mainardi et al., 2008). In summary, we demonstrated association between a combination of 5-HTTLPR and VNTR-2 genotypes of the 5HTT gene and poorer treatment response in patients with TLE. We suggest that higher expression of the 5-HTT results
38 in serotonin network changes and increased severity of TLE. These findings may contribute to better understanding of serotonergic mechanisms in epileptogenesis.
Acknowledgements This work was supported by Epilepsy Foundation grant to HH and Croatian Ministry of Science, Education and Sport project to BJ.
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