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 higher¹. Temporal lobe epilepsy (TLE), a common epileptic syndrome with adult onset, accounts for more than one third of partial epilepsy and more than half of intractable epilepsy². TLE has a wide heterogeneous clinical presentation from severe forms in some patients to very mild in others, suggesting that this syndrome might originate from a combination of environmental and genetic factors³.

Genes known to be involved in biologic mechanisms that can result in disease development are ideal candidates to target for genetic etiological involvement. 5-HT is one of the neurotransmitters influencing the cortical and subcortical excitatory/ inhibitory balance and participates in many physiological

and pathological processes of the brain, including epilepsies. Activation of 5-HT receptors by administration of 5-HT agonists or reuptake inhibitors can inhibit focal and generalized seizures, while destruction of serotonergic terminals and depletion of brain 5-HT results in reduction of seizure threshold and increased neuronal excitability in experimental models⁴. More recently, increased threshold to kainic acid-induced seizures was observed in mice with genetically increased 5-HT levels⁵. Conversely, antiepileptic drugs might enhance basal serotonin levels as part of their mechanism of action. Thus serotoninergic neurotransmission may have an important role in the neurobiology of epilepsy⁶.

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 is located on chromosome 17q11.1-q12, spans 31 kb and consists of 14 exons⁷. Two polymorphic regions of the 5-HTT gene, with supposed functional consequences, have been identified. The first polymorphism is a 44 bp insertion/deletion in the promoter region, the 5-HTT linked polymorphic region (5-HTTLPR)⁸. The short variant (S) indicates the presence of a deletion, resulting in a 484 bp allele, while the absence of this deletion yields a long variant (L) of 528 bp. The shorter allele impairs transcriptional activity of 5-HTT and lowers biological activity of the transporter. Furthermore, the L variant has been reported to be associated with greater m-RNA concentrations and serotonin uptake⁹. The second polymorphism is a 17 bp variable number of tandem repeats in the second intron (5-HTTVNTR)⁷. Common VNTR lengths are 9, 10, and 12 repeats. It has been suggested that the VNTR region may act as a transcriptional regulator of the 5-HTT gene in an allele dependent manner, with the 12 repeat allele having stronger enhancer-like properties than the 10 repeat allele¹⁰.

 

Aim of Work

This study was conducted to investigate the possible associations of the upstream promoter region polymorphism as well as the intron 2-VNTR polymorphism of the 5-HTT gene with temporal lobe epilepsy (TLE) Egyptian patients.

 

PATIENTS AND METHODS

 

Study Design and Population

This is a case-control study carried out on 20 patients with non-lesional TLE and 20 age and sex matched healthy control subjects. They were recruited in the period between September 2012 to May 2013 from the Epilepsy Outpatient Clinic of Kasr El Aini Hospital (Cairo university hospitals). 

Inclusion criteria: 20 epilepsy patients were based on the 1989 ILAE’s electroclinical classification (Commission on Classification and Terminology of the International League Against Epilepsy, 1989)¹² and neuroimaging results. Non-Lesional temporal lobe epilepsy and temporal lobe epilepsy with mesial temporal sclerosis patients were included.

Exclusion criteria: patients with extratemporal epilepsies, mental retardation and those with systemic diseases were excluded. Lesional temporal lobe epilepsy patients were excluded.

All patients were subjected to EEG recording. The diagnosis of TLE was mainly based on typical temporal auras and/or interictal EEG discharges with a maximum over the temporal lobes using Nihon Kohden 14-channel EEG machine, electrodes were arranged according to 10-20 international system of electrode placement. Mono and bi-polar montages were used. All EEGs were carried out under normal standard conditions i.e. with the patient lying supine, completely relaxed in a quiet room. Hyperventilation for 3 minutes and intermittent photic stimulation were used as provocative techniques. The EEG tracing was analyzed carefully regarding; the background activity as well as the presence of generalized or focal epileptogenic activity. Electroencephalography was performed to support the clinical diagnosis of temporal lobe epilepsy. A daytime EEG compliant to the 10-20 international system was performed to all patients under standard conditions in the Neurophysiology unit, Neurology department, Cairo University.

In all patients, brain MR images obtained by using a 1.5 Tesla Philips Gyroscan machine (Philips Medical Systems, Best, the Netherlands) in diagnostic radiology department, Cairo university hospitals images were obtained using sequences and slices to optimize visual detection of mesial temporal structures. Based on the MR study, the TLE was classified as non-lesional if no focal mass lesions such as cerebral tumors, cortical dysgenesis, vascular lesions or malformations, or post-traumatic scars were detected. TLE patients with neuroimaging evidence of mesial temporal sclerosis were included.

 

Genotyping

DNA was extracted from peripheral leukocytes by genomic DNA High Pure PCR template extraction kit (Roche Diagnostics, Germany) according to the manufacturers’ protocol.  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.⁸. The PCR amplified products were digested with MspI restriction enzyme (New England Biolabs) which allows the detection of the A/G SNP, identifying the triallelic polymorphism (La, Lg and S variants)⁸. 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.¹³. The PCR product was visualized by 3% agarose gel electrophoresis stained with ethidium bromide. The 5-HTTVNTR gene had three alleles: Stin2.9 (250 bp), Stin2.10 (267 bp) and Stin2.12 (300 bp). Stin2.9 (250 bp) was not detected, but three genotypes (10/12, 10/10, 12/12) were determined in this study.

 

Statistical Methods

The clinical variables and genotypes of the polymorphisms, 5-HTTLPR biallelic model and 5-HTTVNTR, were compared between TLE patients and a control group. Variables were analysed using “Statistical Package of Social Science Software program” version 21 (SPSS). Data were summarized using mean and standard deviation (parametric variables) and frequency and percentage for (non-parametric variables). Comparison between groups non parametric nominal variables Chi square test was performed. Comparison between numerical variables for more than 2 groups ANOVA and Post Hoc Tukey test was performed. P values less than 0.05 were considered statistically significant, and less than 0.01 were considered highly significant.

 

RESULTS

 

The study included a healthy control group that included 7 male (35 %) and 13 female (65%). The patient group included 9 male patients (45%) and 11 female patients (55%) with no significant difference in sex distribution among both groups (p>0.05) in (Table 1). Control group age ranged from 15 to 48 with a mean of 32.95 and SD 10.55, while patient's age ranged from 17 to 50 with a mean of 31.35 and SD 9.6 with no significant difference between both groups (p>0.05). Age of onset of epilepsy ranged from age 6 months to 29 years with a mean of 14.13 years with a SD of 8.2 years, Duration of epilepsy since onset to date of inclusion in this study ranged from 7 to 34 years with a mean of 17.23 and SD of 7.58 years. Frequency of seizures in patient ranged from no seizure to 5 seizures/year with a mean of 2.15 and SD 1.53.

Comparing TLE patients with healthy control we found a higher significant frequency of 12/12 homozygous repeat in 5-HTTVNTR polymorphism in TLE patients (p<0.05). There was no significant difference as regards different genotype frequencies for 5-HTTLPR polymorphism between patients and control group as in Table (2).

Observing age of onset of epilepsy, duration of epilepsy and frequency of seizure/year in different genotypes for 5-HTTLPR and 5-HTTVNTR polymorphism in patients, we found significant lower age of epilepsy onset in LL genotype for 5-HTTLPR than SL genotype (p<0.05), 12/12 genotype patients had a significant lower age of epilepsy onset than other genotypes for 5-HTTVNTR polymorphism (p<0.05). Frequency of seizures was significantly higher in LL genotype for 5-HTTLPR than in SS genotype, while frequency of seizures in 12/12 genotype for 5-HTTLPR polymorphism was highly significant higher and significantly higher than in both 10/10 and 10/12 genotypes respectively. There was no significant difference in means of duration of epilepsy between different genotypes for both 5-HTTVNTR and 5-HTTLPR polymorphism as in Table (3).

Patients need for antiepileptics was influenced by their genotypes; Frequency of polytherapy treatment with antiepileptics was significantly higher in LL genotype for 5-HTTLPR polymorphism than other genotypes and in 12/12 genotype for 5-HTTVNTR polymorphism than other genotypes as in Table (4).

 

Table 1. Study parameters in control and patients.

 

Group		Control group (n=20)		Patient group (n=20)
		Frequency	Percent	Frequency	Percent
Gender	Male	7	35	9	45
	Female	13	65	11	55
Consanguinity	-ve consanguinity	18	90	13	65
	+ve consanguinity	2	10	7	35
Family history	-ve Family history	--	--	18	90
	+ve Family history	--	--	2	10
History of status epilepticus	-ve	--	--	14	70
	+ve	--	--	6	30
MRI brain	Mesial temporal sclerosis	--	--	3	15
	Normal MRI	--	--	17	85
Treatment	Monotherapy	--	--	10	50
	Polytherapy	--	--	10	50
5-HTTLPR	SS	12	60	9	45
	SL	3	15	4	20
	LL	5	25	7	35
5-HTTVNTR	10/10	13	65	5	25
	10/12	2	10	5	25
	12/12	5	25	10	50

-ve negative, +ve positive

Table 2. Comparison between healthy control and temporal lobe epilepsy patients as regards frequency of genotype distribution in 5-HTTLPR and 5-HTTVNTR polymorphism.

 

Data		Control group		patient group		Chi-square value	P-value
		Frequency	Percent	Frequency	Percent
5-HTTLPR	SS	12	60	9	45	0.9	0.64
	SL	3	15	4	20
	LL	5	25	7	35
5-HTTVNTR	10/10	13	65	5	25	6.5	0.04*
	10/12	2	10	5	25
	12/12	5	25	10	50

* Significant at P<0.05

 

Table 3. Comparison of age of epilepsy onset, duration of epilepsy and frequency of seizures/year between genotypes for 5-HTTLPR and 5-HTTVNTR polymorphism in patients.

 

Data		Age of onset				Duration of epilepsy				Frequency
		Mean	SD	F-value	P-value	Mean	SD	F-value	P-value	Mean	SD	F-value	P-value
5-HTTLPR	SS	16.28	7.81	5.32	0.016*	19.39	6.99	1.16	0.34	1.33	0.87	5.81	0.012*
	SL	20.5	9.57			12.5	7.14			1.75	1.5
	LL	7.7	1.98			17.14	8.34			3.43	1.51
5-HTTVNTR	10/10	20.2	7.43	7.12	0.006**	19.4	11.17	1.08	0.36	0.6	0.55	10.43	0.001**
	10/12	18.8	6.76			13	4.3			1.6	0.55
	12/12	8.75	5.69			18.25	6.63			3.2	1.39

* Significant at P<0.05 ** Significant at P<0.01

 

Table 4. Comparison between frequency of monotherapy and polytherapy antiepileptic medications between genotypes for 5-HTTLPR and 5-HTTVNTR polymorphism.

 

Data		Monotherapy		Polytherapy		Chi-square value	P-value
		Frequency	Percent	Frequency	Percent
5-HTTLPR	SS	7	70	2	20	6.35	0.04*
	SL	2	20	2	20
	LL	1	10	6	60
5-HTTVNTR	10/10	4	40	1	10	7.2	0.027*
	10/12	4	40	1	10
	12/12	2	20	8	80

*Significant at P<0.05

 

 

DISCUSSION

 

There is a potential a role for serotoninergic neuro-transmission in the pathophysiology of the epilepsies. A reduced serotoninergic tone can reduce the threshold of audiogenic, chemical, and electrical-induced epilepsy¹⁴. Antiepileptic drugs such as sodium valproate, lamotrigine, carbamazepine, and phenytoin sodium can increase basal ganglia 5-HT levels or contribute to 5-HT release⁶. 5-HTT is encoded by a single gene (SLC6A4) on chromosome 17q12. The human 5-HTT gene contains many polymorphic loci, including 5-HTTVNTR and 5-HTTLPR¹⁵.

In this study, we found significant higher 12/12 frequencies in the TLE group than the normal control group and  we did not find any significant difference as regards different genotype frequencies for 5-HTTLPR polymorphism between patients and control group. Our results are in line with Li et al.¹⁶, who reported higher frequencies of 5-HTTVNTR 12/12 genotype in the patients with non-lesional temporal lobe epilepsy than normal controls and did not find a significant difference in the frequencies of genotypes in the 5-HTTLPR in those patients. On the other hand, Manna et al., ¹⁷ found that frequencies of the 10/10 genotype were significantly lower in TLE patients than in healthy controls. Racial differences could account for these differences, as 12/12 levels are higher in Asian populations than Europeans, with the 9-copy allele not detected¹⁸.

These data suggest that 12/12 genotype may be a risk gene or genetic marker for TLE. Hranilovic et al.¹¹, have confirmed that Stin2.12/12 genotype increases 5-HTT mRNA expression. Therefore, Stin2.12/12 genotype probably decreases brain 5-HT levels, thereby decreasing the threshold of epilepsy onset. There are several possible mechanisms by which variations in the intron 2-VNTR polymorphism of the 5-HT transporter gene might influence susceptibility to TLE. Variations in the VNTR region might have a role in regulating transcription of the gene, possibly through an adjacent Activator Protein-1 (AP-1) motif. Alternatively, a smaller number of VNTR repeats may influence the stability of transcription complexes and steady-state concentrations of messenger RNA⁸.

Many studies have previously explored the potential roles of 5-HTT allele variants in epileptogenesis or epilepsy characteristics. Stefulj et al.¹⁹, failed to show any association between 5HTTLPR or 5HTTVNTR and TLE. On the other side, Schenkel et al.²⁰, reported an association between the presence of 5HTTLPR and 5-HTTVNTR less transcriptional efficient combined genotypes and TLE.

Reason of the mentioned discrepancy is not clear at the present, but it is possible that this genetic association observed might be related to linkage disequilibrium with other genetic polymorphisms at the same gene or close genes. These possibilities, in addition to racial differences could explain discrepant results found in association studies performed in different populations. Also, there are possible mechanisms that could explain how increase or decrease of serotonin might be related with epileptogenesis. Moreover, different types of TLE (medial temporal lobe epilepsy, lateral temporal lobe epilepsy, or temporal lobe epilepsy combined with hippocampal sclerosis) may have different predisposing genes.

In this study, we found that the frequency of seizures was significantly higher in LL genotype for 5-HTTLPR than in SS genotype, while frequency of seizures in 12/12 genotype for 5-HTTLPR polymorphism was highly significant higher and significantly higher than in both 10/10 and 10/12 genotypes respectively. Also, we found that the frequency of polytherapy treatment with antiepileptics was significantly higher in LL genotype for 5-HTTLPR polymorphism than other genotypes and in 12/12 genotype for 5-HTTVNTR polymorphism than other genotypes. This is in agreement with Hecimovic et al.²¹, who 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. Similarly, Kauffman et al.²², demonstrated that the allele 12 of 5-HTTVNTR was associated with risk for mesial temporal epilepsy not responding to medical treatment.

 

Conclusion

5-HTT polymorphism is associated with increased susceptibility to temporal lobe epilepsy, it also associated with earlier age of onset of seizures, increased frequency of seizures and polytherapy treatment with antiepileptics. Our results suggest that modulation of the serotoninergic system may play a role in the etiology of TLE. Further large scale studies are necessary to confirm our findings.

 

[Disclosure: Authors report no conflict of interest]

 

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