INTRODUCTION

 

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 epilepsy¹. The relatively high prevalence of neuropsychiatric comorbidities in TLE patients suggests shared mechanisms and/or substrates between these two conditions².

The main psychiatric disorders involved in TLE, in order of frequency, are depression, anxiety and psychosis with prevalence ranging from 11 to 44%, 15-25% and 2-8%, respectively³. Psychiatric disorders in epilepsy can be classified into ictal (the psychiatric symptoms are a clinical manifesta­tion of the seizure), peri-ictal (symptoms precede and/or follow the seizure occurrence) and interictal (symptoms occur independently of the seizure occurrence) disorders⁴.

Serotonin (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. Genes coding for proteins related to serotonin neurotransmission can regulate 5-HT availability. Of particular interest are the gene encoding the serotonin transporter (5-HTT), responsible for the clearance of 5-HT from the synaptic cleft, and the gene encoding the serotonin receptor 1A (5-HT1A), that acts as an autoreceptor presynaptically and mediates the action of serotonin postsynaptically⁵.

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 exons⁶. 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) ⁷. 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 uptake⁸. The second polymorphism is a 17 bp variable number of tandem repeats in the second intron (5-HTTVNTR)⁶. 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 allele⁹. The 5-HT1A gene contains a single nucleotide polymorphism (SNP) in the promoter region (C-1019G) that seems to regulate gene expression^10,11. The G allele of C-1019G has been postulated to up-regulate autoreceptor expression, but to decrease postsynaptic 5-HT1A expression¹².

The 5-HTTLPR and 5-HTTVNTR polymorphisms in the 5- HTT gene and the C-1019G polymorphism in the 5-HT1A gene have been studied in various psychiatric diseases, among them depression^13,14, suicidal behavior^15,16, anxiety^17,18, personality disorder¹⁹, bipolar disorder and schizophrenia²⁰. Recently, some authors have evaluated the association between 5- HTT variants and epilepsy^21,22. Most of these studies have shown that modulation of the 5-HTT gene might influence epileptogenesis or the clinical characteristics of epilepsy. Also, Savic et al. found a reduced serotonin receptor (5-HT1A) binding potential in limbic structures in patients with mesial TLE, supporting the hypothesis of an involvement of serotonin in the neurobiology of TLE, perhaps suggesting a mechanism underlying affective symptoms in these patients²³.

Thus, it is biologically possible that alterations in serotonin-related genes may be one of the biological mechanisms involved in higher susceptibility to neuropsychiatric comorbidities in TLE.

Aim of work: To investigate the possible effect of 5-HTTLPR and 5-HTTVNTR polymorphisms in the 5-HTT gene and the C-1019G polymorphism in the 5-HT1A gene in psychiatric comorbidities in Egyptian patients with temporal lobe epilepsy.

 

PATIENTS AND METHODS

 

Study Design and Population

This is a case-control study carried out on 50 patients with non-lesional TLE. They were recruited in the period between September 2012 and September 2013 from the Epilepsy Outpatient Clinic of Kasr El Aini Hospital (Cairo university hospitals). According to the results of the Present State Examination 10th revision (PSE-10) of the Schedules of Clinical Assessment in Neuropsychiatry (SCAN)²⁴, patients were subdivided into two groups: 35 TLE patients with psychiatric disorder(s) we termed them TLE-PSYCH group and 15 patients in whom PSE-10 was negative. These two groups of patients were compared for clinical and genotypic differences.

Inclusion criteria: 50 epilepsy patients, their diagnosis were based on the 1989 ILAE’s electroclinical classification (Commission on Classification and Terminology of the International League Against Epilepsy, 1989)²⁵ 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 using Nihon Kohden 14-channel EEG machine; electrodes were arranged according to 10-20 international system of electrode placement.  It 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.

All patients were submitted to the Arabic version of the Present State Examination 10th revision (PSE-10) of the Schedules of Clinical Assessment in Neuropsychiatry (SCAN)²⁴ The PSE-10 of the SCAN has been translated into Egyptian colloquial Arabic by Hamdi and colleagues²⁶. An abridged form of the Arabic version of the PSE-10 was employed in the survey based upon the short English form of the PSE-10 that was translated and expanded by further questions from the Arabic version in the domains of anxiety, phobias and obsessions, psychotic disorders, somatoform and dissociative disorders, and mood disorders²⁷. The final version was back translated, tested during training of the field researchers, modified according to feedback and tested for reliability²⁸.

Patients then submitted to further evaluated by Hamilton Anxiety Rating Scale (HAM-A)²⁹, Hamilton Depression Rating Scale (HAM-D)³⁰ and Young Mania Rating Scale (YMRS)³¹ to assess severity of anxiety, depression and manic symptoms respectively.

 

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, 5-HTTVNTR and C-1019G. 5-HTTLPR: The amplification reaction (PCR) for the 5-HTTLPR polymorphism was carried out using primers described by Heils et al.⁷. The PCR amplified products were digested with MspI restriction enzyme (New England Biolabs) which identify the biallelic polymorphism (L and S variants)⁷. The digestion products were visualized by 3% agarose gel electrophoresis stained with ethidium bromide under UV light. For 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.³². 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. For C-1019G, An allele-specific polymerase chain reaction (ARMSPCR) analysis was performed according to Parsey et al.³³. Each sample was amplified twice using a specific primer for the G allele and another primer for the C allele 10. Electrophoresis was performed on 1.5% agarose gel and the amplification product was visualized under UV light.

 

Statistical Methods

The clinical variables and genotypes of the polymorphisms, 5-HTTLPR biallelic model, and 5-HTTVNTR in the 5-HTT gene and C-1019 G in the 5-HT1A gene were compared between TLE patients with psychiatric comorbidities and those without it. Variables were analysed using “Statistical Package of Social Science Software program” version 21 (SPSS). Data were summarized using mean and standard deviation (parametric variables), frequency, and percentage for (non-parametric variables).  Comparison between groups non-parametric nominal variables Chi square test was preformed. Comparison between numerical variables for more than 2 groups ANOVA and Post Hoc Tukey test was preformed. P values less than 0.05 were considered statistically significant, and less than 0.01 were considered highly significant.

 

RESULTS

 

The study included 50 temporal lobe patients with age ranged from 17 to 50 years with a mean of 30.38 and SD 8.14, 25 of them were females (50%). In our study, the most frequent psychiatric comorbidities were mood disorder (56%), anxiety disorder (42%), drug abuse (42%) followed by somatoform disorder (24%) and lastly psychosis (12%). The main drug of abuse was the tramadol. More than one psychiatric co morbidity can occur in the same patients and these concomitant occurrences were identified by PSE-10. In our study, mood and anxiety disorders occurred together in nine patients (26% of the total).

Comparing TLE-PSYCH group with TLE-ONLY group we found a significant lower age of the patients and lower age of onset of epilepsy in TLE-PSYCH group (p<0.01) . Most of the patients with psychiatric disorders (TLE-PSYCH group) were women (23(65.7%)), while only 34.3% were men, whereas in TLE-ONLY group 13.3% were women and 86.7% were men, a significant difference for sex (p=0.001). The history of positive consanguinity and history of status epilepticus were significantly higher in patients with psychiatric disorders (TLE-PSYCH group).

Mean time of epilepsy, family history of epilepsy, family history of psychiatric disease and mesial temporal sclerosis in brain MRI did not differ between TLE patients with and without psychiatric comorbidities in our cohort. Clinical and demographic characteristics of the sample are presented in Table (1).

When we compared the genotype distribution of 5-HTTLPR, 5-HTTVNTR and C-1019G polymorphisms in TLE-PSYCH group with TLE-ONLY group we found a higher significant frequency of CC homozygous repeat in C-1019G polymorphism in TLE-PSYCH group (p<0.05). There was no significant difference as regards different genotype frequencies for 5-HTTLPR or 5-HTTVNTR polymorphism between both groups as in Table (2).

Clinical and genotype distribution characteristics of TLE patients according to presence of anxiety disorders or mood disorders are summarized in Table 3. In comparing TLE with anxiety disorders with those without it, we found significant lower age of the patients with anxiety disorders (p=0.048). In addition, Women with TLE had more frequently anxiety disorders than men (p=0.002). The frequency of positive consanguinity and family history of psychiatric disease was significantly higher in TLE with anxiety disorders (p=0.03 and0.008 respectively).The frequency of SS repeat in 5-HTTLPR polymorphism was significantly higher in patients with anxiety disorders (p=0.039). On the other hand, when we compared TLE with mood disorders with those without them, we did not observe significant differences between clinical and demographic characteristics of TLE patients regarding the presence of mood disorders, except for significant lower age of patient and predominance of female gender in TLE with mood disorders (p=0.039 and 0.004 respectively). Also, the frequency of 12/12 repeat in HTTVNTR polymorphism was significantly higher in patients with mood disorders than those without them.

Observing the relation between the severity of the anxiety and the mood disorder in different genotypes for 5-HTTLPR, 5-HTTVNTR and C-1019G polymorphism in patients with psychiatric disorders (TLE-PSYCH group) we found significant higher score of HAM-A in SS genotype for 5-HTTLPR than SL and LL genotypes (p<0.05). On the other hand, 12/12 genotype patients had a significant higher score of HAM-D than other genotypes for 5-HTTVNTR polymorphism (p<0.05). There was no significant difference in means of scores of HAM-A, HAM-D and YMRS between different genotypes for both C-1019G polymorphism as in Table (4).

 

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

 

Variable	TLE-ONLY group (n=15)	TLE-PSYCH group (n=35)	P-value
Mean age (years, SD)	36.13±7.93	27.91±6.98	0.001**
Mean age of epilepsy onset (years, SD)	19.13±8.42	11.59±5.63	0.005**
Mean time of epilepsy (years, SD)	16.33±8.68	16.33±4.36	0.998
Female sex (%)	2(13.3%)	23(65.7%)	0.001**
Consanguinity (%)	3(20%	19(54.28%)	0.025*
Family history of epilepsy (%)	2(13.3%)	10(28.57%)	0.248
Family history of psychiatric disease (%)	4(26.6%)	14(40%)	0.368
History of status epilepticus (%)	3(20%)	16(45.71%)	0.041*
Mesial temporal sclerosis in brain MRI (%)	2(13.3%)	12 (34.28%)	0.135

Values are presented as frequency (percentage) or mean (SD)

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

 

Table 2. Comparison between TLE patients with and without neuropsychiatric comorbities as regards frequency of genotype distribution in 5-HTTLPR, 5-HTTVNTR and C-1019G polymorphism.

 

Data		TLE-ONLY group (n=15)		TLE-PSYCH group (n=35)		Chi-square value	P-value
		Frequency	Percent	Frequency	Percent
5-HTTLPR	SS	4	27	15	43	4.12	0.128
	SL	6	40	5	14
	LL	5	33	15	43
5-HTTVNTR	10/10	6	40	5	14	4.59	0.100
	10/12	4	27	9	26
	12/12	5	33	21	60
C-1019G	CC	0	0	11	46	6.08	0.048*
	CG	4	27	7	20
	GG	11	73	17	34

*Significant at P<0.05

 

Table 3. Clinical and genotype distribution characteristics of TLE patients according to presence of anxiety disorders or mood disorders.

 

Variable		No anxiety disorders (n =29 )	Anxiety disorders (n = 21)	P-value	No mood disorders (n=22)	Mood disorders (n=28)	P-value
Mean age (years, SD)		32.31±8.661	27.71±6.68	0.048*	33.05±8.51	28.29±7.33	0.039*
Mean age of epilepsy onset (years, SD)		15.36 ±8.47	11.76±4.99	0.089	16.20±8.56	12.00±5.81	0.052
Mean time of disease (years, SD)		16.60±7.12	15.95±3.70	0.704	16.39±7.29	16.29±4.65	0.953
Female sex		9(31.0%)	16(76.2%)	0.002**	6	19	0.004**
Consanguinity		9	13	0.03*	8	14	0.335
Family history of epilepsy		7	7	0.475	8	6	0.243
Family history of psychiatric disease		6	12	0.008**	7	11	0.585
History of status epilepticus		9	10	0.233	5	14	0.049*
Mesial temporal sclerosis in MRI		9	5	0.574	5	9	0.462
5-HTTLPR	SS	5	9	0.039*	10	9	0.264
	SL	6	1		6	5
	LL	18	11		6	14
5-HTTVNTR	10/10	9	3	0.207	9	2	0.015*
	10/12	9	4		5	8
	12/12	12	14		8	18
C-1019G	CC	4	7	0.258	3	8	0.075
	CG	7	4		8	3
	GG	18	10		11	17

Values are presented as frequency (percentage) or mean (SD).

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

 

Table 5. Comparison of score of HAM-A, HAM-D and YMARS between genotypes for 5-HTTLPR and 5-HTTVNTR and C-1019G polymorphism in patients.

 

Data		HAM-A				HAM-D				YMARS
		Mean	±SD	F-value	P-value	Mean	±SD	F-value	P-value	Mean	±SD	F-value	P-value
5-HTTLPR	SS	20.67	5.85	1.75	0.019*	12.80	6.68	0.46	0.64	10.07	7.32	0.32	0.731
	SL	11.20	5.54			10.00	4.80			6.96	3.11
	LL	10.80	7.24			12.87	5.94			7.75	2.00
5-HTTVNTR	10/10	17.60	8.36	0.09	0.919	9.40	6.66	2.73	0.040*	9.40	6.66	0.43	0.657
	10/12	16.11	7.42			9.78	4.58			12.89	8.42
	12/12	16.19	6.90			18.29	5.99			10.67	7.18
C-1019G	CC	17.55	7.49	0.25	0.807	9.73	6.26	2.47	0.100	13.09	7.661	1.39	0.263
	CG	16.00	7.59			11.43	6.02			7.29	6.130
	GG	15.76	6.85			14.59	5.41			11.29	7.346

HAM-A Hamilton Anxiety Rating Scale, HAM-D Hamilton Depression Rating Scale, YMARS Young Mania Rating Scale, *Significant at P<0.05

DISCUSSION

 

Prevalence of the association of epilepsy and psychiatric disorders ranges from 20 to 50%, reaching 80% in selected populations like individuals with TLE, and medically intractable patients, candidates to surgical treatment. These indices are far superior to those found in general population (10-20%). Differences in methods of investigation and in populations studied are the main contributory factors for variable results. Also distinct epidemiological definitions (punctual prevalence, cumulative prevalence, lifelong prevalence), with their proper meanings, may contribute equally to variability of results³⁴.

In our study, we observed a high rate of psychiatric disorders in our TLE patients. Psychiatric comorbidities were present in 70% of them.  The most frequent psychiatric comorbidities were mood disorder (56%), anxiety disorder (42%), drug abuse (42%) followed by somatoform disorder (24%) and lastly psychosis (12%). The main drug of abuse was the tramadol. These results are matches with those of Lambert and Robertson, Gaitatzis et al, and Bragatti et al.^{35-37. In Bragatti series, 63.9% of TLE patient had psychiatric disorders, 48.2% had mood disor­ders followed by anxiety disorders occurred in 30.7% of patients. Psy­chotic disorders and substance abuse were observed in 8.4% and 4.8% of patients, respectively37}. The high prevalence of drug abuse in our patients was mainly due to notable widespread tramadol addiction in our community³⁸.

In our study, mood and anxiety disorders occurred together in 13 patients (26% of the total). This association has been recognized since an­cient times, but its pathophysiologic mechanisms are still poorly understood. Studies with adults and children suffering from epilepsy have shown a high prevalence of this comorbidity in association with epilepsy, some­times up to 70%. Depression, anxiety and epilepsy seem to share some biological and structural mechanisms re­lated to limbic system dysfunctions. This is an interesting topic which has been intensely investigated over the last few years³⁹.

We found a significant lower age of the patients and lower age of onset of epilepsy in TLE-PSYCH group compared to TLE-ONLY group. This is in accordance with the results of Davies et al.; Gaitatzis et al.; Pellock and Swinkels et al.^40,36,41,42. In contrast, Schenkel et al. did not find significant difference between TLE patients with and without psychiatric co morbidities regarding the age of the patients and age of onset of epilepsy⁴³.

Most of our patients with psychiatric disorders (TLE-PSYCH group) were women. These results are in agreement with Bragatti et al. and Swinkels et al.^37,42.

The history of positive consanguinity was significantly higher in patients with psychiatric disorders (TLE-PSYCH group). Our finding might suggest that genetic predisposing factors might be   important   in the pathogenesis of psychiatric disorders in epilepsy. This is in line with the results of Bragatti et al.³⁷.

History of status epilepticus was significantly higher in patients with psychiatric disorders especially those with mood disorders. This is matched with the results of many others who concluded that the severity of epilepsy is one of the most important risk factors for development of psychiatric disorders in epilepsy time^44-46.

Mean time of epilepsy, family history of epilepsy, family history of psychiatric disease and mesial temporal sclerosis in brain MRI did not differ between TLE patients with and without psychiatric comorbidities in our study. This is in accordance with the results of Schenkel et al.⁴³.

When we compared TLE with mood disorders with those without it, we did not observe significant differences between clinical and demographic characteristics of TLE patients regarding the presence of mood disorders, except for significant lower age of patient and predominance of female gender in TLE with mood disorders. This is in accordance with Bragatti et al., who stated that women with TLE were significantly had mood disorder  in comparison to men but they found that  positive family history of psychiatric disorder is significantly associated  with mood disorder³⁷.

In comparing the genotype distribution of 5-HTTLPR, 5-HTTVNTR and C-1019G polymorphisms in TLE-PSYCH group with TLE-ONLY group ,we found a higher significant frequency of CC homozygous repeat in C-1019G polymorphism in TLE-PSYCH group. There was no significant difference as regards different genotype frequencies for 5-HTTLPR or 5-HTTVNTR polymorphism between both groups. These results are partially matched with those of Schenkel et al., who found that there was no significant association between these polymorphisms in the 5-HT1A and 5-HTT genes and the combined presence of neuropsychiatric disorders in patients with TLE (TLE-ONLY group versus TLE-PSYCH group)⁴³. In contrast, other studies have found that the G allele of C-1019G is a risk factor for the development of neuropsychiatric diseases, among them depression and panic disorder¹⁸.

When we analyzed each psychiatric disorder individually (mood disorder, anxiety disorder, psychosis, drug abuse and somatoform disorder), the frequency of 5-HTTLPR polymorphism was different between TLE patients with and without anxiety disorder. The frequency of SS genotype of 5-HTTLPR polymorphism was higher in patients with anxiety disorder than in patients without anxiety. Caspi et al. research on the association between 5-HTTLPR alleles and personality traits found that carriers of S alleles (S/S or S/L) were reported to have higher neuroticism (considered a vulnerability factor for anxiety and depression) scores than L homozygotes (L/L)⁴⁷. Hariri et al. demonstrated that S carriers had a significantly greater amygdala response than participants with the L/L alleles, suggesting that the S allele is associated with a greater degree of amygdala excitability⁴⁸.

Also, the frequency of 5-HTTVNTR polymorphism was different between TLE patients with and without mood disorder. This is supported by the results of Lothe et al. who found that changes of the serotoninergic pathway are associated with depressive symptoms in TLE patients⁴⁹.

On the other hand, we found that the frequency of C-1019G polymorphism did not differ significantly between TLE patients with and without anxiety disorder. This is unlike Schenkel et al. who found that temporal lobe epileptic patients with the C allele of C-1019G polymorphism in the 5-HT1A gene had higher frequency of anxiety disorder⁴³.

Our study might help to beginning understand the molecular basis of psychiatric comorbidities in epilepsy. In fact, this might be an initial work in a field that remains almost unexplored. Further studies are necessary to clarify these matters.

 

Conclusion

Psychiatric comorbidities were present in 70% of our TLE patients .The most frequent psychiatric comorbidities were mood disorders (56%). Most of our patients with psychiatric disorders were women (65.7%). There was a higher significant frequency of CC homozygous repeat in C-1019G polymorphism in TLE-PSYCH group. There could be a possible role 5-HT gene polymorphisms in molecular mechanisms involved in psychiatric comorbidities in TLE. Further large scale studies are necessary to confirm our findings.

 

[Disclosure: Authors report no conflict of interest]

 

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