Epilepsy Research (2012) 101, 261—267

jou rn al h om epa ge: www.elsev ier .com/ locate /ep i lepsyres

Do gene polymorphism in IL-1�, TNF-� and IL-6influence therapeutic response in patients with drugrefractory epilepsy?

Prabhakar Tiwari a, Rekha Dwivedib, Nasim Mansoori c, Rizwan Alamc,Ugam Kumari Chauhana, Manjari Tripathib,∗, Asok Kumar Mukhopadhyayc

a Center for Biotechnology, School of Environmental Biology, Awahdesh Pratap Singh University, Rewa 486003, Indiab Department of Neurology, All India Institute of Medical Sciences (AIIMS), Ansari Nagar, New Delhi 110029, Indiac Laboratory Medicine, All India Institute of Medical Sciences, Ansari Nagar, New Delhi 110029, India

Received 31 January 2012; received in revised form 27 March 2012; accepted 16 April 2012Available online 11 May 2012

KEYWORDSDrug RefractoryEpilepsy;SNPs;Polymorphism;Seizures;Therapeutic response

SummaryPurpose: Pro-inflammatory cytokines may play an important pathophysiological role in patientswith epilepsy. To understand the role of genes encoding pro-inflammatory cytokines in epilepsy,this study aimed to evaluate the polymorphisms of the promoter regions of IL-1�-511C > T(rs16944), TNF-�—308G > A (rs1800629) and IL-6-174G > C (rs1800795) genes and to look intothe interaction between these genes in influencing seizure susceptibility, seizure frequencyand response to therapy.Methods: The comparative frequency of polymorphism was determined in rs16944, rs1800629and rs1800795 using PCR-RFLP in a group of 120 persons with epilepsy (PWE) and 110 ethnicallymatched healthy subjects of comparable age and sex in the North Indian population.Results: Alleles and genotypes of rs16944, rs1800629 and rs1800795 were not found to influencethe odds ratio of having susceptibility to epilepsy. Also gene—gene interaction of possible ninecombinations of these genes did not show any positive association with epilepsy. The geno-type and allelic frequency of rs1800795 showed a significant association (p < 0.05) in seizurefrequency (number of seizures/6-months) and drug refractory epilepsy. However, the genotypeand allelic frequency of rs16944 and rs1800629 were not found to have such effect.Conclusion: This study demonstrates that the rs16944, rs1800629 and rs1800795 polymorphism

does not act as a strong susceptibility factor for epilepsy in North Indian population. The geno-typic association of rs1800795 with seizure frequency and drug-refractory epilepsy raises theissue that a specific set of polymin epilepsy.© 2012 Elsevier B.V. All rights re

∗ Corresponding author. Tel.: +91 11 26594494; fax: +91 11 26588248/2E-mail address: manjari.tripathi1@gmail.com (M. Tripathi).

0920-1211/$ — see front matter © 2012 Elsevier B.V. All rights reserved.http://dx.doi.org/10.1016/j.eplepsyres.2012.04.013

orphic genes can influence seizures and therapeutic response

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his study was undertaken to determine whether the pro-oter regions of Interleukin (IL)-1�-511C > T (rs16944),NF-�—308G > A (rs1800629) and IL-6-174G > C (rs1800795)enes have any effect on susceptibility, seizure frequencynumber of seizures/6-months) and response to therapy inpilepsy. Polymorphisms of cytokine inflammatory gene maynfluence the amount of cytokine production and susceptibil-ty to several diseases including epilepsy (Chou et al., 2010).everal studies have been done on the polymorphism of theegulatory promoter region of rs16944, and a limited studyas done for rs1800629 and rs1800795 polymorphism in tem-oral lobe epilepsy and hippocampal sclerosis or febrileeizure but they failed to demonstrate any significant asso-iation (Heils et al., 2000; Buono et al., 2001; Ozkara et al.,006; Kanemoto et al., 2003; Chou et al., 2010). In con-rast, some studies have shown a positive association forhe polymorphism of rs16944 in febrile seizures (Kanemotot al., 2000; Virta et al., 2002). IL-6 has been implicatedor the normal CNS development. The influence of serumevel of IL-6 has been found in generalized seizures and focaleizures types of epilepsy (Lehtimaki et al., 2007; Li et al.,011). There is one study from India (Sinha et al., 2008),hich measured the serum cytokine (IL-1�, TNF-alpha and

L-6) level on PWE (n = 100). They have found highly sig-ificant increased serum cytokine levels in patients withpilepsy PWE as compared to the control group. The rea-on of this increased level was not clear. However, the studyid not look into genetic polymorphism within these geneshich may influence seizure frequency and epilepsy. There-

ore, the present study targeted cytokines pro-inflammatoryenes in epilepsy. Our aims were to determine the poly-orphism association of rs16944, rs1800629 and rs1800795ro-inflammatory marker genes in epilepsy as compared toealthy controls. Further, we examined the interaction ofhese genes in altering the susceptibility to epilepsy or itsourse in terms of seizure frequency or any variation inesponse to therapy.

ethods

tudy subjects

total of 120 persons with epilepsy (PWE) were recruitedrom the outpatient department of neurology, All India Insti-ute of Medical Sciences (AIIMS), The age group of PWEnd healthy controls were between 7 to 65 years, and bothroups were from North India. A written informed con-ent was taken from all study subjects to participate inhe study. This study was approved by the Institute’s Ethicsommittee. Diagnosis of epilepsy was made according to

LAE (1989), which includes a clinical history, electroen-ephalography (EEG) and magnetic resonance imaging (MRI).fter diagnosis, demographic and clinical information wasecorded for all patients. All PWE had generalized or focaleizures. 52% of patients with controlled seizures and 78%

f drug refractory epilepsy subjects had an abnormal EEG.n MRI 95% of seizure controlled patients with epilepsy PWEad localized post traumatic gliosis/encephalomalacia. Inrug refractory epilepsy, patients were observed to have

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P. Tiwari et al.

eriventricular, parieto-occipital white matter changes, ule-yria, hippocampal sclerosis and cortical dysplasia. Patientsith controlled seizures were using one anti-epilepticrug (phenytoin or carbamazepine or phenobarbital or val-roate) or two antiepileptic drugs (phenytoin + valproater valproate + lamotrigene or carbamazepine + clobazam).owever, drug refractory epilepsy subjects were taking twor more antiepileptic drugs. Patients with febrile seizure<7 years) or with evidence of intracranial infection werexcluded. All the controls subjects (n = 110) were unre-ated healthy individuals (gender matched and comparablege ± 3 years). Any subject, who had a history of diseasesike stroke, tuberculosis, diabetes and AIDS, was excluded.

ample collection and DNA isolation:

lood samples (1 ml) were collected in EDTA vial andenomic DNA was extracted by using the QIAgen GenomicNA isolation kit (QIAgen, USA) as per manufacturer’s

nstructions.

enotyping of rs16944 SNP

he rs16944 genotypes were determined by PCR-RFLPs previously described in literature (Kira et al., 2010).riefly, PCR was performed using primer pair (for-ard: 5′-TGGCATTGATCTGGTTCATC-3′ and reverse: 5′-TTTAGGAATCTTCCCACTT-3′) with an initial denaturation at4 ◦C for 3 min, followed by 27 cycles of 94 ◦C for 1 min, 58 ◦Cor 30 s and 72 ◦C for 60 s, and a final extension at 72 ◦Cor 7 min. The reaction mixture (25 �l) contained 100 ngf genomic DNA, 10X reaction buffer, 0.2 mM of deoxynu-leotide triphosphates (dNTPs), 20 pmol of each primer and.0 units of Taq DNA polymerase (New England Biolabs, MA,SA). The PCR products were digested with AvaI restrictionnzyme (Fermentas, USA), separated on a 2% agarose gel,tained with ethidium bromide and then visualized underltraviolet light. Wild type (CC) produced two fragments189 and 116 bp) whereas heterozygous CT produced threeands (305, 189 and 116 bp) and homozygus variant (TT)emained intact as 305 bp. The major visible bands were05 and 189 bp.

enotyping of rs1800629 SNP

he extracted genomic DNA was used to check thes1800629 polymorphism by polymerase chain reaction-FLP (Wieser et al., 2002). The primers were usedforward) 5′-AGGCAATAGGTTTTGAGGGCCAT-3′ and (reverse)′-TCCTCCCTGCTCCGATTCCG-3′. The PCR conditions wereith an initial denaturation at 94 ◦C for 3 min followed by7 cycles at 94 ◦C for 30 s, 55 ◦C for 30 s, and 72 ◦C for 30 s,ith a subsequent final extension step at 72 ◦C for 7 min. The

eaction mixture (25 �l) contains genomic DNA, 10X reactionuffer, dNTPs, primers and Taq DNA polymerase (New Eng-and Biolabs, MA, USA) concentration as described above.

esultant PCR product digested with NcoI restriction enzymeNew England Biolabs, MA, USA), electrophoresed and ana-yzed as above. Wild type (GG) produced two fragments (87nd 20 bp) whereas heterozygous GA produced three bands

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Do gene polymorphism in IL-1�, TNF-� and IL-6 influence th

(107, 87 and 20 bp) and homozygus variant (AA) remainedintact as 107 bp. The major visible bands were 107 and 87 bp.

Genotyping of rs1800795 SNP

Following extraction of DNA, amplification by PCR andRFLP study of rs1800795 was done using the primers:forward 5′-TGACTTCAGCTTTACTCTTTGT-3′ and reverse 5′-CTGATTGGAAACCTTATTAAG-3 and the same protocol wasfollowed as in our earlier published article (Mansooriet al., 2010). Wild type (CC) remained undigested (198 bp),whereas heterozygous CT produced three bands (198, 175and 23 bp) and homozygous variant produced two bands (175and 23 bp). The major visible bands were 198 and 175 bp.

Statistical analysis

The statistical analysis was carried out using STATA 9.0 (Col-lege Station, TX, USA). Clinical data were summarized as thenumber (percentage) or mean ± SD. Demographic data, alle-les and genotypes of rs16944, rs1800629 and rs1800795 wereused as independent variables in PWE and healthy controls.These variables were compared by Student’s t-test/�2 tests.The effect of alleles, genotypes and gene—gene interactionof rs16944, rs1800629 and rs1800795 on epilepsy againsthealthy controls was tested using binary logistic regres-sion analysis, and same analysis was used to evaluate thegenotype effect of these genes with seizure frequency andseizure control (patients with controlled seizures vs. drug-refractory epilepsy). The results were reported as OR (95%CI). The p-value <0.05 was considered as statistical signifi-cance.

Results

In this study of 230 subjects, which included 120 PWE and110 unrelated healthy controls. They were studied for poly-morphism of rs16944, rs1800629 and rs1800795 and theirinteractions.

Demographic and clinical characteristics of thestudy subjects

The main demographic and clinical characteristics of dataare summarized in Table 1. The mean age of epilepsypatients (26.1 ± 12.7) as compared to healthy controls(26.5 ± 13.1) was not statistically significant (p = 0.71). Thepercentages of male (76.7%) were higher than female(23.3%) in the PWE. This was not found statistically signif-icant (p = 0.95) as compared to the healthy control group(Male: 76.4% Female: 23.6%). In epilepsy patients, themean ± SD of the age of onset of seizures and of the seizurefrequency were 18.2 ± 13.4 and 20.0 ± 39.3 respectively.The types of seizures were generalized seizures (GS-54.2%)and focal seizures (FS-45.8%). Mono-therapy, bi-therapy andpoly-therapy anti-epileptic drugs were used in 26.7%, 40.0%

and 33.3% of patients respectively. Out of 120 PWE, seizureswas controlled in 50 patients (41.7%) with one/two drugregime, whereas 70 patients (58.3%) had drug-refractoryepilepsy.

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eutic response in patients 263

llelic frequency of rs16944, rs1800629 ands1800795 in epilepsy and healthy controls

able 2 shows the distribution of allele frequency of rs16944,s1800629 and rs1800795 in PWE and healthy controls. The

allele (variant) frequency of rs16944 in PWE was 34.5%nd in healthy control was 33.3%, which did not show statis-ically significant (p = 0.78) association. The allele G (wild)nd A (variant) frequency of rs1800629 was 75% and 76.8%n PWE and 25% and 23.2% in healthy controls respectively,hich did not show statistically significant (p = 0.64) associ-tion. The G (wild) allele frequency of rs1800795 was 87.5%nd 85.5% while C (variant) allele frequency was 12.5% and4.5% in PWE and healthy controls respectively, which alsoid not show a significant association (p = 0.52) as comparedo healthy controls.

enotypic frequency of rs16944, rs1800629 ands1800795 in epilepsy and healthy controls

he genotypic distribution of rs16944, rs1800629 ands180079 were shown in Table 3. Analysis of genotype vari-tion in rs16944 showed heterozygous CT variant as mostrequent (108 out of total 230 subjects), and homozygous TTs least frequent (24 out of total 230 subjects). The rs16944

allele carrier genotypes (CT + TT) vs. CC, did not show anyignificant (p = 0.44) association in PWE (55.0% vs. 45.0%) asompared to healthy control subjects (60.0% vs. 40.0%).

The wild homozygous genotype (GG) in rs1800629 wasost frequent. It was almost equally present in both groups

121 out of 230 subjects). The homozygous AA variant waseast frequent (only 2 variants out of 230 subjects, and thatoo in PWE). For rs1800629, allele career genotype (GA + AA)s. GG did not show any significant (p = 0.77) association withpilepsy (48.3% vs. 46.4%) as compared to healthy controls51.7% vs. 53.6%).

For genotype of rs1800795, C allele carrier (GC + CC) vs.G did not show any significant (p = 0.49) association withpilepsy (23.3% vs. 76.7%) as compared to healthy controls27.3% vs. 72.7%). The wild homozygous genotype (GG) ofs1800795 was most frequent. It was almost equally presentn both groups (172 out of 230 subjects), and variant homozy-ous CC was least frequent (only four variants out of 230ubjects in which two variants was in PWE out of 120 andwo variants were in healthy controls out of 110). The abovellelic and genotypic data analyzed adjusting for age andender, but no impact of individual polymorphism on causingpilepsy was found with gender and age.

robability of gene—gene interaction of rs16944,s1800629 and rs1800795 in PWE as compared toealthy controls

able 4 shows the gene—gene interaction of rs16944,s1800629 and rs1800795 in epilepsy. To assess all possi-le interactions of these genes, nine possible combinations

s shown in the table were chosen. Logistic regressionnalysis was done to see whether the presence or absencef either variant allele of rs16944, rs1800629 and rs1800795lone or in combinations could alter odds ratio of having

264 P. Tiwari et al.

Table 1 Demographic and clinical data of the PWE and healthy controls.

Demographic/clinical variables Epilepsyn = 120

Healthy controlsn = 110

p value

Age (in years) 26.1 ± 12.7 26.5 ± 13.1 0.71GenderFemale 28 (23.3%) 26 (23.6%) 0.95

Male 92 (76.7%) 84 (76.4%)Age of onset of seizure (in years) 18.2 ± 13.4 —Seizure frequency (in 6 months) 20.0 ± 39.3 —Seizure control (in 6 months)Patients with controlled seizures 50 (41.7%) —Refractory drug epilepsy 70 (58.3%)Type of seizure —

GS 65 (54.2%)FS 55 (45.8%)

Anti-epileptic drugs (AEDs) —Mono-therapy 32 (26.7%)Bi-therapy 48 (40.0%)Poly-therapy 40 (33.3%)

Data presented as mean ± SD (range), GS — generalized seizure, FS — focal seizure.

Table 2 Allelic frequencies of rs16944, rs1800629 and rs1800795 in persons with epilepsy and healthy controls.

SNPs Allele Epilepsyn = 120

Healthy controlsn = 110

OR (95% CI) p value

rs16944 511*C 160 (66.7) 144 (65.5) 1.05 (0.70—1.58) 0.78511*T 80 (33.3) 76 (34.5)

rs1800629 308*G 180 (75.0) 169 (76.8) 0.90 (0.57—1.42) 0.64308*A 60 (25.0) 51 (23.2)

rs1800795 174*G 210 (87.5) 188 (85.5) 1.19 (0.67—2.11) 0.52174*C 30 (12.5) 32 (14.5)

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Data presented as number of alleles (percentage).

pilepsy. The result did not show any significant (p > 0.05)ssociation. The presence of all three variant career alle-es (T, A and C) also did not increase the odds of having

pilepsy. In the two combinations of genes (one combina-ion; only rs1800795 was present and another combination;s16944 and rs1800795 were present), we had found odds

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Table 3 Genotypic frequencies of rs16944, rs1800629 and rs1800

SNPs Genotype Epilepsyn = 120

Hen =

rs16944 C/C 54 (45.0) 44

C/T 52 (43.3) 56

T/T 14 (11.7) 10

rs1800629 G/G 62 (51.7) 59

G/A 56 (46.7) 51

A/A 2 (1.6) 0

rs1800795 G/G 92 (76.7) 80

G/C 26 (21.7) 28

C/C 2 (1.6) 2

Data presented as number of subject (percentage).

atio with 3.5 and 2.9 folds respectively. The explanationor these increased odds cannot be taken seriously due tomall numbers of cases in combinations. These data were

lso analyzed adjusting for gender and age. Little differ-nce was found between the adjusted and unadjusted datahich was not statistically significant (data not shown).

795 in PWE and healthy controls.

althy controls 110

OR (95% CI) p value

(40.0)(51.0) 1.10 (0.85—1.43) 0.44(9.0)(53.6)(46.4) 0.96 (0.74—1.24) 0.77(0.0)(72.7)(25.5) 1.11 (0.82—1.49) 0.49(1.8)

Do gene polymorphism in IL-1�, TNF-� and IL-6 influence therapeutic response in patients 265

Table 4 Regression analysis of genotypic interaction of rs16944, rs1800629 and rs1800795 between persons with epilepsy (PWE)and healthy controls (HCs).

rs16944(CT + TT)

rs1800629(GA + AA)

rs1800795(GC + CC)

Epilepsyn = 120

Healthy controlsn = 110

OR (95% CI) p value

Absent Absent Absent 26 (21.7) 15 (13.6) 1.0 (reference)Absent Absent Present 4 (3.3) 8 (7.3) 3.46 (0.89—13.48) 0.07Absent Present Absent 16 (13.3) 15 (13.6) 1.62 (0.63—4.20) 0.32Absent Present Present 8 (6.7) 6 (5.5) 1.30 (0.38—4.47) 0.68Present Absent Absent 26 (21.7) 26 (23.6) 1.73 (0.75—4.00) 0.20Present Absent Present 6 (5.0) 10 (9.1) 2.89 (0.87—9.54) 0.08Present Present Absent 24 (20.0) 24 (21.8) 1.73 (0.74—4.05) 0.20Present Present Present 10 (8.3). 6 (5.5) 1.04 (0.34—3.43) 0.95

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Data presented as number of subject (percentage)

Genotypic interaction of rs16944, rs1800629 andrs1800795 in determining the seizure frequency

Table 5a and b shows the genotypic and allelic interaction ofrs16944, rs1800629 and rs1800795 with seizure frequency.Genetic polymorphisms were grouped into two categoriesby having the presence of at least one variant form ofeach gene (rs16944: (CT + TT), rs1800629: (GA + AA) andrs1800795 (GC + CC)) against the wild genotype (rs16944CC, rs1800629 AA and rs180096 GG). The genotype andallelic frequency of rs1800795 showed significant association(p = 0.03 and 0.002) with seizure frequency. No statisticallysignificant (p = 0.72, 0.53 and 0.61, 0.37 respectively) differ-ence was found in the median value of seizure frequency inany of the genotypes and alleles of rs16944 and rs1800629.

Genotypic interaction of rs16944, rs1800629 andrs1800795 in determining the seizure control

Table 5a and b shows the genotypic and allelic interactionof rs16944, rs1800629 and rs1800795 with seizure con-trol (patients with controlled seizures vs. drug-refractoryepilepsy). The GG genotype in rs1800795 was found mostfrequent in (85.7%) in drug-refractory epilepsy as comparedto patients with controlled seizures (64%), with the p-valueof 0.01. The allelic frequency of rs1800795, as expectedshowed much more significant association (p = 0.001) withdrug refractory epilepsy. However, this was not seen forgenotypes and alleles of rs16944 and rs1800629 (p = 0.58,0.95 and 0.42, 0.93). This result shows that the G variantmay play a major role in drug-refractory epilepsy.

Discussion

Whether the genetic polymorphisms of pro-inflammatorycytokine genes have any role in susceptibility, seizure fre-quency and in response to therapy in PWE is not clearlyknown. This study was undertaken precisely to explorethis issue. The polymorphism of rs16944, rs1800629 and

rs1800795 did not show any association with susceptibilityto epilepsy. Seizure frequency was significantly influencedby presence of G allele in rs1800795. The gene—gene inter-action in possible nine combinations also did not show any

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ignificant alteration of odds ratio of having epilepsy. How-ver, the GG genotype and allelic frequency in rs1800795as been found to be most frequent in a significant man-er in cases of seizure frequency and drug-refractorypilepsy.

In literature, several studies reported on polymorphismf rs16944 in febrile seizures. In concurrence with our result,he result of the studies in Taiwanese, Turkish, German,nd Japanese population was found to have no associationetween genetic polymorphism and epilepsy (Chou et al.,010, 2003; Haspolat et al., 2005; Matsuo et al., 2006). Inne Japanese study, T allele of rs16944 was found to have

positive association with sporadic simple FSs in contrasto above-described studies (Kira et al., 2005). Similarly, theesult of the present study on association of rs1800629 withpilepsy is consistent with a recent polymorphism study ofaiwanese population in febrile seizures (Chou et al., 2010).ther important diseases, like schizophrenia in Korean,apanese and Chinese population have shown the lack ofssociation of the disease with rs1800629 (Pae et al., 2003;ashimoto et al., 2004; Duan et al., 2004). In the poly-orphism of rs1800795, the literature reports on Taiwaneseopulation in febrile seizures and some other diseases likelzheimer’s among Indians, Germans and Italians show noisease association as like ours (Chou et al., 2010; Mansoorit al., 2010; Bagli et al., 2000; Capurso et al., 2010). Inontrast, some other studies on Japanese, Spanish and Ital-an populations, polymorphism of rs1800795 in diseases likelzheimer’s, has shown a positive association with G alleleShibata et al., 2002; Mateo et al., 2006; Pola et al., 2002).

few studies (Nur et al., 2012; Chou et al., 2010) concern-ng association of human interleukin-6 gene polymorphismsith febrile seizures observed that the presence of G alleler GG genotype at −174 and GG genotype at −572 posi-ions of the promoter regions constituted risk factors foreveloping febrile seizures. Moreover, the GG genotype at174 was observed to be higher in drug refractory epilepsy.his rs1800729 polymorphism has been found to influencehe transcriptional regulation of IL-6 and plasma IL-6 lev-ls (Fishman et al., 1998; Sinha et al., 2008; Lehtimäkit al., 2011), which makes this polymorphism potentially

nteresting with regard to the development of seizures.hese elevated IL6 expression at pharmacological level may

ncrease both seizure frequency and drug refractoriness inC and GG genotype subjects.

266 P. Tiwari et al.

Table 5 Association of rs16944, rs1800629 and rs1800795 in seizure frequency and seizure control [patients with controlledseizures (PCS) vs. drug-refractory epilepsy (DRE)].

a. Genotypic associations

SNPs Genotype Epilepsyn = 120

Median of seizurefrequency (range)

p value Frequency of genotypes p value

PCS DRE

rs16944 CC 54 (45.0) 4 (0—240) 0.72 24 (48.0) 30 (42.0) 0.58CT + TT 66 (55.0) 6 (0—60) 26 (52.0) 40 (58.0)

rs1800629 GG 62 (51.7) 6 (0—240) 0.53 26 (52.0) 36 (51.4) 0.95GA + AA 58 (49.3) 5 (0—144) 24 (48.0) 34 (48.6)

rs1800795 GG 92 (76.7) 6 (0—240) 0.03* 32 (64.0) 60 (85.7) 0.01*GC + CC 28 (23.3) 0 (0—144) 18 (36.0) 10 (14.3)

b. Allelic associations

SNPs Alleles Epilepsyn = 120

Median of seizurefrequency (range)

p value Frequency of alleles p value

PCS DRE

rs16944 C 108 (45.0) 4 (0—240) 0.61 48 (48.0) 60 (42.0) 0.42T 132 (55.0) 6 (0—60) 52 (52.0) 80 (58.0)

rs1800629 G 124 (51.7) 6 (0—240) 0.37 52 (52.0) 72 (51.4) 0.93A 116 (49.3) 5 (0—144) 48 (48.0) 68 (48.6)

rs1800795 G 184 (76.7) 6 (0—240) 0.002* 64 (64.0) 120 (85.7) 0.001*

C 56 (23.3) 0 (0—144) 36 (36.0) 20 (14.3)

Data presented as number of patients (percentage).

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We did not find any positive association of rs16944,s1800629 and rs1800795 in terms of susceptibility whereashe significant association of G allele in rs1800795 witheizure frequency and response to therapy reveal that pres-nce of G allele of rs1800795 is related to seizures andefractoriness to first or second line of antiepileptic drugs.n the other hand, the patients with C allele responded wellith first or second line of antiepileptic drugs. The exact

eason of the variations of these responses for genotypesnd alleles of rs1800795 remains unclear. The findings ofhe our study would be helpful in understanding the variant

allele association of rs1800795 in seizure frequency andrug refractory epilepsy which may be a risk factor for induc-ion of seizures and drug refractoriness and may play anmportant role in etio-pathogenesis of epilepsy although thexact mechanism is still unknown. Till date, to our knowl-dge there is no report on genotypic association of rs1800795ith seizure frequency and drug refractory epilepsy.

Besides, most of the published reports included patientsf limited age group of 4 months to 5 years and that tooith febrile seizures and the patients were recruited fromefinite ethnic background (Chou et al., 2010; Tilgen et al.,002). In contrast, the range of age (in years) of patientsn our study is 7—65 and there is no specificity for febrile

eizure. The North Indian population contributes a mixturef different ethnic group of Indians.

To conclude, in this study we document the polymor-hism study of promoter regions of IL-1�-511C > T (rs16944),

meCo

NF-�—308G > A (rs1800629) and IL-6-174G > C (rs1800795)enes in 120 PWE along with 110 healthy controls (HCs)eporting to the epilepsy clinic of AIIMS. Gene—gene inter-ction was also studied in all possible combinations. Noositive association of these genes was found with suscepti-ility of epilepsy. However, the significant association of Gllele in rs1800795 with seizure frequency and drug refrac-ory epilepsy showed the polymorphism association and theirnteraction with epilepsy. Further studies with larger sam-le size from different ethnic backgrounds are required totrengthen our findings.

ompeting interests and disclosures

he authors declare that they have no competing interestsnd there is nothing conflicting for disclosure.

cknowledgements

e are thankful to all of the subjects who were agreed toarticipate in this study. We thank Mrs. M. Kalaivani, Depart-

ent of Biostatistics, AIIMS, for her help in the statistical

valuations of the result. Authors also acknowledge Indianouncil of Medical research (ICMR) as the study was a partf ICMR funded project.

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Do gene polymorphism in IL-1�, TNF-� and IL-6 influence th

References

Bagli, M., Papassotiropoulos, A., Knapp, M., et al., 2000. Associa-tion between an interleukin-6 promoter and 3 flanking regionhaplotype and reduced Alzheimer’s disease risk in a Germanpopulation. Neurosci. Lett. 283, 109—112.

Buono, R.J., Ferraro, T.N., O’Connor, M.J., et al., 2001. Lackof association between an interleukin 1 beta (IL-1b) genevariation and refractory temporal lobe epilepsy. Epilepsia 42,782—784.

Capurso, C., Solfrizzi, V., Colacicco, A.M., et al., 2010. Interleukin-6-174 G/C promoter and variable number of tandem repeats(VNTR) gene polymorphisms in sporadic Alzheimer’s disease.Prog. Neuropsychopharmacol. Biol. Psychiatry 34, 177—182.

Chou, I.C., Lin, W.D., Wang, C.H., et al., 2010. Interleukin (IL)-1�,IL-1 receptor antagonist, IL-6, IL-8, IL-10, and tumor necrosisfactor-� gene polymorphism in patients with febrile seizures. J.Clin. Lab. Anal. 24, 154—159.

Chou, I.C., Tsai, C.H., Hsieh, Y.Y., Peng, C.T., Tsai, F.J., 2003.Association between polymorphism of interleukin -1 beta-511promoter and susceptibility to febrile convulsions in Taiwanesechildren. Acta Paediatr. 92, 1356.

Duan, S., Xu, Y., Chen, W., 2004. No association between the pro-moter variants of tumor necrosis factor alpha (TNF-alpha) andschizophrenia in Chinese Han population. Neurosci. Lett. 366,139—143.

Fishman, D., Faulds, G., Jeffery, R., et al., 1998. The effect ofnovel polymorphisms in the interleukin-6 (IL-6) gene on IL-6transcription and plasma IL-6 levels, and an association withsystemic-onset juvenile chronic arthritis. J. Clin. Invest. 102 (7),1369—1376.

Hashimoto, R., Yoshida, M., Ozaki, N., 2004. Association analysis ofthe −308GA promoter polymorphism of the tumor necrosis factoralpha (TNF-alpha) gene in Japanese patients with schizophrenia.J. Neural. Transm. 111, 217—221.

Haspolat, S., Baysal, Y., Duman, O., Coskun, M., Tosun, O., Yegin,O., 2005. Interleukin-1alpha, interleukin-1beta, and interleukin-1Ra polymorphisms in febrile seizures. J. Child Neurol. 20,565—568.

Heils, A., Haug, K., Kunz, W.S., et al., 2000. Interleukin-1-betapolymorphism and susceptibility to temporal lobe epilepsy withhippocampal sclerosis. Ann. Neurol. 48, 948—950.

Kanemoto, K., Kawasaki, J., Miyamoto, T., Obayashi, H., Nishimura,M., 2000. Interleukin (IL) 1beta, IL-1alpha, and IL-1 receptorantagonist gene polymorphisms in patients with temporal lobeepilepsy. Ann. Neurol. 47, 571—574.

Kanemoto, K., Kawasaki, J., Yuasa, S., et al., 2003. Increased fre-quency of interleukin-1�-511 T allele in patients with temporallobe epilepsy, hippocampal sclerosis, and prolonged febrile con-

vulsion. Epilepsia 44, 796—799.

Kira, R., Ishizaki, Y., Torisu, H., et al., 2010. Genetic susceptibilityto febrile seizures: case—control association studies. Brain Dev.32, 57—63.

W

eutic response in patients 267

ira, R., Torisu, H., Takemoto, M., et al., 2005. Genetic suscep-tibility to simple febrile seizures: interleukin-1beta promoterpolymorphisms are associated with sporadic cases. Neurosci.Lett. 384, 239—244.

ehtimaki, K.A., Keranen, T., Palmio, J., Makinen, R., Hurme,M., Honkaniemi, J., et al., 2007. Increased plasma levels ofcytokines after seizures in localization-related epilepsy. ActaNeurol. Scand. 116, 226—230.

ehtimäki, K.A., Liimatainen, S., Peltola, J., et al., 2011. The serumlevel of interleukin-6 in patients with intellectual disability andrefractory epilepsy. Epilepsy Res. 95, 184—187.

i, G., Bauer, S., Nowak, M., et al., 2011. Cytokines and epilepsy.Seizure 20, 249—256.

ansoori, N., Tripathi, M., Alam, R., et al., 2010. IL-6-174 G/C andApoE gene polymorphism in Alzhemer’s and vascular dermentiapatients attending the cognitive disorder clinic of the All IndiaInstitute of Medical Sciences, New Delhi. Dement. Geriatr. Cogn.Disord. 30, 461—468.

ateo, I., Infante, J., Rodríguez, E., et al., 2006. Interactionbetween dopamine beta-hydroxylase and interleukin genesincreases Alzheimer’s disease risk. J. Neurol. Neurosurg. Psy-chiatry 77, 278—279.

atsuo, M., Sasaki, K., Ichimaru, T., Nakazato, S., Hamasaki,Y., 2006. Increased IL1beta production from dsRNA-stimulatedleukocytes in febrile seizures. Pediatr. Neurol. 35, 102—106.

ur, B.G., Kahramaner, Z., Duman, O., et al., 2012. Inrerleukin-6 gene polymorphism in febrile seizures. Pediatr. Neurol. 46(January (1)), 36—38.

zkara, C., Uzan, M., Tanriverdi, T., et al., 2006. Lack of associationbetween IL-1beta/alpha gene polymorphisms and temporal lobeepilepsy with hippocampal sclerosis. Seizure 15, 288—291.

ae, C.U., Chae, J.H., Bahk, W.M., 2003. Tumor necrosis factor-alpha gene polymorphism at position 308 and schizophrenia inthe Korean population. Psychiatry Clin. Neurosci. 57, 399—403.

ola, R., Flex, A., Gaetani, E., 2002. The −174 G/C polymorphism ofthe interleukin-6 gene promoter is associated with Alzheimer’sdisease in an Italian population.

hibata, N., Ohnuma, T., Takahashi, T., et al., 2002. Effect of IL-6polymorphism on risk of Alzheimer disease: genotype-phenotypeassociation study in Japanese cases. Am. J. Med. Genet. 114,436—439.

inha, S., Patil, S.A., Jayalekshmy, V., Satishchandra, P., 2008. Docytokines have any role in epilepsy? Epilepsy Res. 82, 171—176.

ilgen, N., Pfeiffer, H., Cobilanschi, J., et al., 2002. Associationanalysis between the human interleukin-1beta (−511) gene poly-morphism and susceptibility to febrile convulsions. Neurosci.Lett. 334, 68—70.

irta, M., Hurme, M., Helminen, M., 2002. Increased plasma levelsof pro- and anti-inflammatory cytokines in patients with febrile

seizures. Epilepsia 43, 920—923.

ieser, F., Fabjani, G., Tempfer, C., et al., 2002. Tumor necrosisfactor-alpha promoter polymorphisms and endometriosis. J. Soc.Gynecol. Investig. 9, 313—318.