Association of Intronic Polymorphismrs3773364 A>G in Synapsin-2 Gene With

Idiopathic EpilepsyRAM LAKHAN,1 J. KALITA,2 U.K. MISRA,2 RITU KUMARI,1 AND BALRAJ MITTAL1*

1Department of Genetics, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow,Uttar Pradesh 226014, India

2Department of Neurology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow,Uttar Pradesh 226014, India

KEY WORDS seizure; single-nucleotide polymorphism; pharmacogenetics

ABSTRACT In epilepsy, there is a tendency towards recurrent unprovoked seiz-ures. Seizures result due to the excessive electrical misfiring in the brain betweenneurons and disturbance in neurotransmitter release. Several gene products affect thebehavior of these neurons by regulating neurotransmission via several mechanisms.One such gene, Synapsin-2 (SYN2), involved in synaptogenesis is also reported to reg-ulate the neurotransmitter release. We hypothesized that SYN2 gene and its polymor-phisms could affect the process of epileptogenesis and therapeutic response inhumans. In this hospital-based study, we enrolled 372 patients with epilepsy and 199control subjects. We selected rs3773364 A>G polymorphism in SYN2 gene andanalyzed its distribution in north Indian patients with epilepsy and control subjects.Genotyping was carried out by polymerase chain reaction-restriction fragment lengthpolymorphism (PCR-RFLP) method. According to the results obtained, SYN2 ‘‘AG’’ ge-notype frequency was significantly higher in patients with epilepsy versus control sub-jects in north Indian population (P 5 0.02, OR 5 1.55, 95% CI 5 1.06–2.26). After sub-classification, we observed higher frequency of AG genotype in idiopathic patients ascompared to control subjects (P 5 0.01, OR 5 1.67, 95% CI 5 1.08–2.56). There wereno significant differences in genotypic (AG: OR 5 0.80, P 5 0.377; GG: P 5 0.628, OR5 1.17) or allelic (P 5 0.86, OR 5 1.03) frequency distributions in patients with multi-ple drug resistance versus patients with drug-responsive epilepsy. Results from ourstudy indicate the involvement of SYN2 gene polymorphism in conferring risk to epi-lepsy; however, the genetic variant does not seem to modulate drug-response in epi-lepsy pharmacotherapy. Synapse 64:403–408, 2010. VVC 2009 Wiley-Liss, Inc.

INTRODUCTION

Epilepsy is a common multifactorial neurologicaldisorder, with higher prevalence in developing coun-tries like India (Strzelczyk et al., 2008). Overall prev-alence rate of epilepsy in India is 5.59 per 1000 popu-lation, with no statistically different rates betweenmen and women, urban or rural (Sridharan and Mur-thy, 1999). As compared to Western nations, in India,higher proportion of febrile seizure cases develop epi-lepsy; this may also be a cause of higher prevalencerates of active epilepsy in Indian population (Banerjeeet al., 2009). Also, certain forms of epilepsies havehigh incidence/prevalence in certain geographicalareas or races such as hot water epilepsy in SouthIndia (Gururaj and Satishchandra, 1992). The princi-ples and success of treatment also differ considerably

in developing and developed countries. Epilepsy beinga major neurological problem, it is worthwhile toinvestigate the environmental as well as genetic fac-tors in the disease predisposition and pharmacother-apy in countries like India.

Since the discovery of the first human epilepsyassociated ion channel gene, mutations in at least 25different genes have been described (http://www.ncbi.nlm.nih.gov/sites/entrez?db 5 OMIM). How-

Contract grant sponsor: Department of Biotechnology (DBT).

*Correspondence to: Balraj Mittal, Department of Genetics, Sanjay GandhiPostgraduate Institute of Medical Sciences, Lucknow, Uttar Pradesh 226014,India. E-mail: balraj@sgpgi.ac.in or bml_pgi@yahoo.com

Received 29 June 2009; Accepted 11 August 2009

DOI 10.1002/syn.20740

Published online in Wiley InterScience (www.interscience.wiley.com).

VVC 2009 WILEY-LISS, INC.

SYNAPSE 64:403–408 (2010)

ever, the strength of the evidence for these genes hav-ing a pathogenic role in epilepsy varies. Monogenicidiopathic epilepsies account for only 1–2%; however,most of them are believed to be polygenic (Weber andLerche, 2008). According to Epilepsy Genetic Associa-tion Study database (http://www.epilepsygenes.org/page/show/EGAD), more than 50 association studieshave been performed and genetic variants of severalgenes such as neuronal sodium channels, potassiumchannels, and GABA receptors have been found to beassociated in a variety of epilepsy phenotypes (Helbiget al., 2008; Reid et al., 2009). These genes could alsoaffect drug response behavior to antiepileptic drugs(AEDs) by modulating pharmacokinetics and pharma-codynamics. Recently, Cavalleri et al. (2007) exam-ined common variations across 279 prime candidategenes in samples from four independent research cen-ters (in UK, Ireland, Finland, and Australia), insearch for genetic susceptibility loci in sporadicepilepsy syndrome and seizure disorder. Besidesmany genetic variants in other genes, it was foundthat rs3773364 A>G single-nucleotide polymorphismof Synapsin-2 (SYN2) gene was associated inCaucasian epilepsy patients with history of febrileseizures.

The synapsins are a family of neuronal phosphopro-teins, specifically associated with cytoplasmic surfaceof synaptic vesicle membranes (Geinisman et al.,1990), which account for 9% of total vesicle proteins.These proteins selectively bind to small synapticvesicles in the presynaptic nerve terminal involved insynaptogenesis and are believed to regulate therelease of neurotransmitter (Heierhorst et al., 1999).Synapsins are also associated with the regulation ofneuronal development and maintenance of maturesynapses (Ferreira and Rapoport, 2002). Studies havedemonstrated that the concentration of synapsins isreduced in the brains of human schizophrenics(Browning et al., 1993; Harrison and Eastwood,1998). One of the synapsin family genes, SYN2, whichspans more than 140 kb regions on human chromo-some 3, is composed of 14 exons. Previously, geneticvariants of SYN2 have been reported to be involvedin susceptibility to schizophrenia (Chen et al.,2004a,b; Saviouk et al., 2007) and febrile seizures(Cavalleri et al., 2007).

Epilepsy is also a disorder of disturbances in neuro-transmitter release and neurotransmission. Becauseof population-specific nature of association studies,there is a universal need to replicate the studies indifferent populations. On the basis of functionalknowledge of the gene, we looked for the possibleassociation of rs3773364 A>G intronic polymorphismof synapsin-2 (SYN2) gene in north Indian epilepsypatients. In addition, we also examined the role ofthis polymorphism in influencing drug resistance phe-notype in epilepsy.

MATERIALS AND METHODSPatients and controls

This study comprised 372 patients with epilepsyand 199 healthy control subjects. All patients withepilepsy were recruited from the Department of Neu-rology, Sanjay Gandhi Post Graduate Institute ofMedical Sciences (SGPGIMS), Lucknow, India. Thepatients were classified and investigated by experi-enced neurologists. Exclusion criteria included severeadverse drug reactions, poor compliance with AEDs,unreliable record of seizure frequency, history ofpseudo seizures, alcohol or drug abuse, or any othermalignant diseases such as brain tumor, secondarymetastasis, hepatic failure, or renal failure. Aninformed consent was signed by each participant orresponsible adult, and they were personally inter-viewed for information on ethnicity, seizure frequencyand duration of seizure, compliance, and other habits.The study was approved by local ethics committee.

The control subjects were unrelated persons to thepatients. Both controls and patients with epilepsybelong to the same ethnicity and geographical area.The control subjects were not reported to have a his-tory of epileptic seizures or any other neuropsychiat-ric disorder.

Definition of drug resistance andresponsiveness

The main criterion for drug resistance was theoccurrence of at least four seizures over a period of 1year with three appropriate AEDs at maximum toler-ated doses (Lakhan et al., 2009; Siddiqui et al., 2003).Patients who had undergone surgeries for seizurecontrol were considered refractory, irrespective oftheir outcome after surgery. The patients with epi-lepsy who had complete freedom from seizures for atleast 1 year from last follow-up visit were considereddrug responsive.

Laboratory protocols: sample collection andgenotyping

Blood samples (5 ml) were taken in EDTA vialsfrom the patients, and DNA was isolated using salt-ing out method with slight modifications (Milleret al., 1988). The plasma was separated and stored at2208C for drug level assay. We genotyped a total of372 patients with epilepsy and 199 control subjects.

TABLE I. Primers used in the study

SNP Primer sequence

Restrictionenzyme/products

SYN2 A>G(rs3773364)

50-TCTGCTAGTTCATGGGAGTGG-30 DdeI,146; 81,6550-AGAGCCTTTTCCAACCCAATC-30

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PCR primers and RFLP method was designed usingthe software ‘‘Lasergene-8’’ (Burland, 2000). Single-nucleotide polymorphism in intron-5 rs3773364 A>Gchange created a restriction site in target nucleotidesequence. The PCR yielded a 146 base pair product.After restriction digestion with DdeI, the G allele pro-duced two fragments of 81 and 65 base pairs, respec-tively (Table 1). Restriction endonuclease digestedproducts were separated using 15% polyacryl amidegel electrophoresis, and genotyping patterns wererecorded (Fig. 1). For quality control, 20% of the sam-ples were regenotyped by different laboratory person-nel, and the results found were 100% concordant tothose previously observed.

Statistical methods

The relationship between various genotypes andresponsiveness was examined using binary logisticregression. Association was expressed as odds ratios(OR) or risk estimates with 95% confidence intervals(CI), and it was considered significant at P < 0.05. Allanalyses were performed using SPSS statistical anal-ysis software, Version 15.0 (SPSS, Chicago, IL). Sam-ple size was calculated using the QUANTO 1.1 pro-gram (http://hydra.usc.edu/gxe). The desired power ofstudy was set at 80% using additive model. The rela-tive risk or power calculation was set at 2.

RESULTS

The mean age 6 SD of patients with epilepsy was24.45 6 11.85. Among them, 72.6% (270/372) were

male and 27.4% (102/372) were female. Mean age atthe first seizure was 16.41 6 10.93 years in patientswith epilepsy. Epilepsy was classified as symptomaticand idiopathic. Seizures were classified as generalizedor partial. Among the patients, 58.1% (216/372) of thepatients had generalized seizures, and 41.9% (156/372) of the patients had partial seizures. Patientswere also categorized on the basis of their responsestatus to multiple AEDs. 31.72% (118/372) patientswere diagnosed as drug resistant and 67.28% (254/372) were diagnosed as drug responsive. To confirmcompliance, AED levels in plasma were also measuredusing HPLC (Perkin Elmer) in 20% of patients.Mean carbamazepine, phenytoin, and valproate levelswere 8.26 6 5.25, 11.27 6 8.12, and 68.0 6 36.22 lg/ml, respectively, which were in the therapeutic range.We were not able to correlate drug levels in patientswith different genotypes because sample size wasvery small for the given drugs and particulargenotypes.

Association of SYN2 polymorphism andsusceptibility to epilepsy

We determined the genotypic and allelic frequenciesin 372 patients with sporadic epilepsy and 199 controlsubjects by PCR-RFLP assay (Table II). In our studyof control population, the observed genotype distribu-tion of SYN2 polymorphisms was consistent withHardy–Weinberg equilibrium (P 5 0.73). Frequencyof AG genotype of SYN2 rs3773364 A>G polymor-phism was significantly higher in patients with epi-lepsy versus control subjects (Table II; P 5 0.02, OR5 1.55, 95% CI 5 1.06–2.26). However, we did notobserve significant differences in GG genotype or Gallele between patients with epilepsy and control sub-jects (Table II). We found that distribution of AG ge-notype (P 5 0.019, OR 5 1.67, 95% CI 5 1.08–2.56)was significantly differed in patients with idiopathicepilepsy versus control subjects (Table III), while stat-istically significant differences were not observed inpatients with symptomatic epilepsy versus patientswith idiopathic epilepsy (data not shown).

SYN2 polymorphism in drug resistance epilepsy

Genotype or allelic frequencies of SYN2 polymor-phism did not differ significantly in patients with

TABLE II. Distribution of SYN2 A[G gene (rs3773364) polymorphism; genotype and allele frequenciesin patients with epilepsy versus control subjects

Genotypes/allelesaAll patients withepilepsy (N 5 372)

Controls subjects(N 5 199) P-value Odds ratio (95% CI)

AA 119 (32.0%) 83 (41.7%) – ReferenceAG 194 (52.2%) 87 (43.7%) 0.022 1.55 (1.06–2.26)GG 59 (15.8%) 29 (14.6%) 0.192 1.41 (0.83–2.40)Aa 432 (58.1%) 253 (63.6%) – ReferenceGa 312 (41.9%) 145 (36.4%) 0.071 1.26 (0.98–1.61)

aAlleles.

Fig. 1. Representative gel picture for SYN2 A>G (rs3773364)genotyping. Lane 1, homozygous AA genotype; lane 2, homozygousGG; lane 3, heterozygous AG genotype; L, 50 bp DNA ladder.

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drug resistance versus patients with drug responsiveepilepsy for AG (P 5 0.37, OR 5 0.80, 95% CI 50.49–1.63) and GG genotypes (P 5 0.62, OR 5 1.17,Table IV). The variant G carrier allele frequency alsodid not differ in both the groups indicating noinvolvement of studied genetic polymorphism in ther-apeutic response to AEDs.

DISCUSSION

In this study, we observed SYN2 rs3773364 ‘G’ al-lele to be associated with overall as well as idio-pathic epilepsy susceptibility in a north Indian popu-lation. However, the SYN2 gene polymorphism didnot seem to modulate drug response in epilepsypharmacotherapy. In accordance with the HapmapProject Third Phase Data, there is a wide variationin distribution of frequency of the major allele ‘A’ranging from 35.5% (Chinese in metropolitan Den-ver) to 95.7% (African ancestry in South West). Thefrequencies of different genotypes (AA 5 41.7%, AG5 43.7%, and GG 5 14.6%) in north Indian controlsubjects were comparable to that reported for Gujar-ati (people from a Western state in India) Indians(AA 5 30.1%, AG 5 44.6%, GG 5 25.3%) livingin Houston, TX (http://www.hapmap.org/cgi-perl/gbrowse/hapmap3_B36/).

In this study, SYN2 rs3773364 ‘G’ polymorphismwas found to be associated with overall epilepsy sus-ceptibility. The association was observed in patientswith heterozygous AG genotype. After segregation ofpatients with symptomatic and idiopathic epilepsies,significant association was observed in idiopathic epi-lepsy that included patients with grand-mal, petit-mal, and myoclonic epilepsy, etc., and many of these

forms have defined or definitive genetic loci. Thispolymorphism also conferred certain risk in sympto-matic patients (OR 5 1.42, P 5 0.12) for AG geno-type, but it did not attain statistical significance. Itmay be added that most of the symptomatic patientshad neurocysticercosis (NCC) as CT finding andmany patients in this group also had triggering/pre-cipitating factors such as stroke, migraine and headinjury, etc. Some of these forms also have defined ordefinitive genetic loci associated, while in otherssome environmental factors and modifier gene maybe playing major roles in seizure susceptibility. Simi-larly, in schizophrenia, case–control and family-basedassociation studies have shown significant involve-ment of SYN2 genetic variant with its susceptibility(Chen et al., 2004a; Lee et al., 2005; Stober et al.,2000).

Cavalleri et al. (2007) analyzed SYN2 SNP infour Caucasian populations, but they found it to beassociated with febrile convulsions only and notwith other seizure syndromes. In our study popula-tion, we studied it in all patients with epilepsy asnumbers of patients with history of febrile seizureswere limited, and therefore we could not study itseparately. Given the fact that, in India, morepatients with febrile seizure develop active epilepsycompared to Western countries (Banerjee et al.,2009), it may also be a cause of differential associa-tion of SYN2 rs3773364 A>G polymorphism in spo-radic seizure disorder. It is also possible that thereare population-specific differences in predispositionto epilepsy in different parts of the world (Macet al., 2007).

The synaptic vesicle-associated proteins are regu-lated by phosphorylation (Greengard et al., 1993)

TABLE III. Distribution of SYN2 genotypes in idiopathic epilepsy versus control subjects

GenotypeIdiopathic

epilepsy (N 5 207)Controls

subjects (N 5 199) P-value OR (95%CI)

AA 64 (30.9%) 83 (41.7%) _ ReferenceAG 112 (54.1%) 87 (43.7%) 0.019 1.67 (1.08–2.56)GG 31 (15.0%) 29 (14.6%) 0.288 1.38 (0.75–2.53)Aa 240 (58.0%) 253 (63.6%) _ ReferenceGa 174 (42.0%) 145 (36.4%) 0.103 1.26 (0.95–1.67)

aAlleles.

TABLE IV. Distribution of SYN2 A>G gene (rs3773364) polymorphism in drug resistant andresponsive patients with epilepsy

Genotypes/allelesaDrug resistant

epilepsy (N 5 118)Drug responsive

epilepsy (N 5 254) P-value Odds ratio (95% CI)

AA 40 (33.9%) 79 (31.1%) – ReferenceAG 56 (47.5%) 138 (54.3%) 0.377 0.80 (0.49–1.63)GG 22 (18.6%) 37 (14.6%) 0.628 1.17 (0.61–2.25)Aa 136 (57.6%) 296 (58.3%) – ReferenceGa 100 (42.4%) 212 (41.7%) 0.86 1.03 (0.75–1.40)

aAlleles.

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that might provide a mechanism for pharmacologicalmanipulation; so, we also checked distribution ofthis polymorphism in patients with epilepsy on thebasis of drug response status but we found no appa-rent genotypic and allelic differences in drug respon-sive and drug refractory epilepsy patients. There aremultiple factors that contribute to drug resistance inepilepsy, e.g., drug-metabolizing enzymes (Depondt,2006) that include mainly cytochrome P450 for AEDmetabolism, multidrug transporter such as ABCB1(Bournissen et al., 2008; Lakhan et al., 2009) anddrug targets that include sodium channels (Lakhanet al., 2009), calcium channels, potassium channels,and GABA receptors. So, the overall genetic effect ofthese genes may have greater role in drug respon-siveness. Apart from genetic variations, other factorssuch as type of epilepsy, duration of seizure, andnumber of seizures prior to initiation of drug ther-apy (Schiller and Najjar, 2008) may also beresponsible for differences in drug responsiveness.

Calverri et al. (2007) attributed the rs3773364 poly-morphism to be present in promoter region. However,based on our search results, it was found to be pres-ent in intronic region of the gene (http://May2009.archive.ensembl.org/q 5 rs3773364). Thefunction of this variation is still not known. This SNPis located in intron-5, which lies in between thesequence that code for actin-binding domain in thesynapsin-2 protein (UniProtKB/Swiss-Prot Q92777).It is possible that this SNP affects splicing of theseexons that code for active domain in the protein, thusaltering the neurotransmitter release, which may inturn provide susceptibility for epilepsy. It has alsobeen observed that two other polymorphisms in thenearby region of gene are in linkage disequilibriumwith the SYN2 polymorphism (Cavalleri et al., 2007).So, there is a possibility that other loci in close prox-imity or in linkage disequilibrium to this site mayinfluence susceptibility to epilepsy.

In summary, our results support that SYN2 is animportant gene in predisposition to epilepsy but theSNP under study does not modulate therapeuticresponse in north Indian patients with epilepsy. Asthis polymorphism is present in the intronic region,it would be necessary to establish functional role ofthis polymorphism in epilepsy phenotypes and sei-zure susceptibility. In addition, it is warranted to per-form and replicate such association studies in specificclinical phenotypes of epilepsy such as febrile seizureor juvenile myoclonic epilepsy (JME) with largersample size to draw definitive conclusions.

ACKNOWLEDGMENTS

The study was supported by fellowships providedby CSIR and DST New Delhi, Government of India.

We acknowledge support from Dr. N.J. Gogtay, KEMHospital, Mumbai, for drug level assays.

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