Association between SCN1A gene polymorphisms and drug resistant epilepsy in pediatric patients

Association

between

SCN1A

gene

polymorphisms

and

drug

resistant

epilepsy

in

pediatric

patients

Lucia

Margari

a,

*

,1

,

Anna

R.

Legrottaglie

a,1

,

Alessandra

Vincenti

b,1

,

Giangennaro

Coppola

c

,

Francesca

F.

Operto

a

,

Maura

Buttiglione

a

,

Amalia

Cassano

b

,

Nicola

Bartolomeo

b

,

Maria

A.

Mariggiò

b,1

a

ChildNeuropsychiatryUnit,DepartmentofBasicMedicalSciences,NeuroscienceandSenseOrgans,UniversityofBari“AldoMoro”,Italy

b

DepartmentofBiomedicalSciencesandHumanOncology,UniversityofBari“AldoMoro”,Italy

c_{ChildNeuropsychiatryUnit,DepartmentofMedicine,SurgeryandDentistry,UniversityofSalerno,Italy}

ARTICLE INFO

Articlehistory:

Received12October2017

Receivedinrevisedform29December2017 Accepted2January2018 Availableonlinexxx Keywords: Drug-resistance Epilepsy Intronicpolymorphisms SCN1A ABSTRACT

Purpose:“SingleNucleotidePolymorphisms(SNPs)”couldbeanimportantexplanationofdrugresistance inepilepsy.Theaimofthisstudywastoinvestigateifgeneticpolymorphisms(SNPs)oftheSCN1Agene couldinfluencetheresponsetoanti–epilepticdrugs(AED)andiftheycouldpredisposetoadrug resistantepilepsyinpediatricpatients.

Methods:WeinvestigatedSNPsinexonandintronicregionsoftheSCN1Ageneinasampleof120 pediatric patients, in both drug-resistant and drug-responsive patients. Association between polymorphismsand refractoryepilepsywereinvestigatedbycomparingSNPs inexonandintronic regionsbetweenthetwogroups.Thegenotypesofeachintronicpolymorphisminthedrug-resistant groupwasanalyzed.Oddsratiosandconfidenceintervalswerecalculated.

Results:NoneoftheSNPsidentifiedinexonsoftheSCN1Agenewereassociatedwithdrug-resistance.In the intronic regions, a statistically significant difference was found in the prevalence of three polymorphismswasfoundbetweenthetwopatientgroups(rs6730344A/C,rs6732655A/T,rs10167228A/ T).Theanalysisofthegenotypesofeachintronicpolymorphisminthedrug-resistantgrouprevealedthat theAAandATgenotypesforthers1962842polymorphismareassociatedwithanincreasedriskof developingdrugresistancecomparedtoTTgenotype.

Conclusion:Theintronicrs6730344,rs6732655andrs10167228polymorphismsoftheSCN1Agenearea potentialriskfactorsfordrugresistance.AAeATgenotypeofthers1962842intronicpolymorphismalso emergedasariskfactorinthedrugresistantgroup.Therefore,polymorphismsoftheSCN1Agenecould playaroleintheresponsetoAEDinpatientswithdrug-resistantepilepsy,withimportantimplications forclinicalpractice.

©2018BritishEpilepsyAssociation.PublishedbyElsevierLtd.Allrightsreserved.

1.Introduction

Epilepsyisoneofthemostcommonneurologicaldisordersin children,withanincidencerangingfrom50to120/100.000/year [1].AlthoughseveralAEDhavebeendevelopedinthecourseof time,itisestimatedthatabout20%ofpatientsfailtorespondto standard therapies and continue to experience debilitating refractory seizures [1,2]. These patients are classified as

“drug-resistantepilepsy”(DRE),aclinicalconditioncharacterized by poor prognostic implications that include prematuredeath, physicalinjury,psychosocialdysfunctionand reducedqualityof life. DRE represents a major handicap for the patient, with important repercussions on social and health costs [3]. The InternationalLeagueagainstEpilepsytaskforcedefinedDREasthe failureof adequatetrials oftwotolerated,appropriatelychosen and used AED schedules (whether as monotherapies or in combination) to achieve sustained seizure freedom [3,4]. The mechanisms underlying the development of drug-resistance in epilepsyarecomplexandnotfullyunderstood[5]:thetwo well-knownhypothesesfor understandingthebiologicalmechanism underlying multidrug resistance are thetarget and transporter hypotheses[6,7].Thereisanindividualvariationontheoptimal dose,theeffectivenessandtheoccurrenceofadverseeventsofan

*Corresponding authorat:ChildNeuropsychiatryUnit,DepartmentofBasic MedicalSciences,NeuroscienceandSenseOrgans,UniversityofBari“AldoMoro”; PiazzaGiulioCesaren.11,Italy.

E-mailaddress:lucia.margari@uniba.it(L.Margari).

1

Theseauthorscontributedequallytothiswork.

https://doi.org/10.1016/j.seizure.2018.01.002

1059-1311/©2018BritishEpilepsyAssociation.PublishedbyElsevierLtd.Allrightsreserved.

ContentslistsavailableatScienceDirect

Seizure

AED.The responseof anindividualpatient toa specificAEDis generallyunpredictable[8,9]. Itis influencedby manydifferent parameterssuchas:thepathophysiologyoftheepilepsyitself;the interaction of an AED with its target(s) (pharmacodynamic effects);thepharmacokineticsoftheAED,involvingmechanisms of absorption, distribution, metabolism and elimination [9]. Although manyfactors may contributeto variabilityof clinical outcome in individual patients, unpredictability may partially result fromgenetic variation [10]. Recentdevelopments in the pharmacogeneticsofAEDprovidenewprospectsforpredictingthe efficacyoftreatmentandpotentialside-effects.Thetreatmentof epilepsy appropriately offers a model for the application of pharmacogenetics in clinical practice, in view of the high prevalenceofthisdisorder,thewidevarietyofindividualresponse todrugtreatmentandthepossibilityofquantifyseizurecontrol [11].Therefore,understandingtheDREpharmacogeneticcausesis criticalinordertopredictdrugresponsehenceprovidingabasis forpersonalizedmedications.

SNPs,variationsinthesingle-siteDNA,arethemostfrequent formsofsequencevariationsinthehumangenome,whichmay affectefficacy,tolerability,safety,anddurationofactionofadrug andtheyareemergingaspotentialcandidatesoftheDRE[10].The effectofthegeneticpolymorphismsonthedrugmetabolismis significant,sowewillconsiderhowgeneticvariationswithinthese processesmayaffecttheeffectivenessofAED.Followingthecourse ofanAEDfromitsabsorptioninthegastrointestinaltracttodrug distributiontothebrain,drugactionatbraintargetsandfinallyits metabolismandelimination,eachprocesscanbeinfluencedbythe presence of genetic variations that can affect transporters and targetproteins,resultinginalterationoftheeffectivenessofthe treatment [10]. The role of voltage-gated ion channels in epileptogenesisofboth geneticand acquiredepilepsies,aswell astargetsinthedevelopmentofnewAED,isveryimportant[12]. BecausemanyAEDactprimarilyassodiumchannelsblockers,the SCN1A gene coding for voltage-gated sodium ion channels represents an attractive candidate for investigating the link between genetic polymorphisms and clinical response. So far, severalSNPsinthesodiumchannelgeneshavebeendescribed,but only a few have been found to have a significant role in the differentneurologicaldisorders[13].Inviewofthesocialimpact thatmayhaveapoorresponsetotreatmentwithAED,intermsof seizurecontrolandadverseevents,weconsideredthecorrelation between the patient's phenotype (e.g., drug response) and genotype(genepolymorphisms).Therefore,theobjectiveofthis research was to study the genetic polymorphisms that could influencetheresponsetoAEDandthepredispositiontodevelop drugresistanceinpatientsundergoingoneormoreAED.Thegenes codingfortargetproteinsofAEDand,specifically,forvoltage-gated sodiumion channels werethe polimorphismsselected for this study.

2.Methods 2.1.Participants

Thestudyincluded120epilepticpediatricpatientsborninItaly, attendingtheNeuropsychiatricsServicesattheUniversityofBari andSalernoduringtheperiodfromMarch2013andMarch2014. Sixtypatientswerediagnosedasepilepticdrug-resistantand60as drug-responsive.AccordingtothecriteriaILAE2010,drug-resistant epilepsy was defined as “failure to achieve sustained seizure freedom,despiteadequatetrialsoftwotoleratedandappropriately chosenandusedAEDscheduleswhetherasmonotherapiesorin combination”;drug-responsiveepilepsywasdefinedas“epilepsyin whichthepatientsreceivingthecurrentAEDtreatmentregimen hasbeenseizurefreefora minimumof threetimesthelongest

pre-interventioninterseizureintervalor12months,whicheveris longer”[4].

Theprotocolforthis studywasapprovedbythelocalEthics Committee and a written consent was obtained frompatients’ parentsorlegalguardians.

Foreachpatientenrolled,investigatorscollectedmedicaldata includinghistoryofepilepsyandAEDtreatmentsreceivedatthe timeofthediagnosis,reasonsforanychanges,doseregimensand compliance.

Inclusioncriteriawere:adiagnosisofepilepsy(idiopathicor cryptogenic/symptomatic),accordingtotheInternationalLeague AgainstEpilepsyclassi_fication [14];treatmentwithatleastone AED,longenoughtoachievetheoptimaldose;drug-resistanceand drug-responsiveness,accordingtothecriteriaILAE2010;written consentobtainedfromaparentorlegalguardian;agebetweenone andseventeenyears.

Exclusioncriteriawere:epilepticpatientsinclinicalremission, with gradual withdrawal of therapy; epileptic patients with therapy titration phase; patients with previous history of encephalitisorbraintumor.

Inbothdrug-resistantanddrug-responsivegroups,westudied polymorphisms in the SCN1A gene whose mutations may be involved in mechanisms of epileptogenesis. The criteria that guidedtheselectionofthestudiedSNPsarethefollowing:weused dbSNPdatabaseofNCBI(http://www.ncbi.nlm.nih.gov/snp/)that providedthepolymorphismsintheSCN1Agenewiththerelative allelic frequencyand wechose allelicvariants whose expected frequencywasdeemedinformative.Onthebasisofthiscriteria,we investigated in exon regions SNPs rs146733308, rs35735053, rs3749029, rs112767060, rs10613159, rs12617205, rs112157737; in non-coding regions SNPs rs6730344, rs6432858, rs6732655, rs1962842,rs10167228,rs10194956andrs11690962.

2.2.Leukocytesisolationfromwholeblood

Leukocytes isolation was conducted using Emagel (Piramal Healthcare,NorthumberlandUK)withheparin(5U.I/mlEmagel). Ten cc of whole peripheral blood from each patient were collectedintubeswithEDTA.EqualvolumeofEmagelwithheparin was added toblood and suspension was mixed on a rotor for 10min.Themixturewasallowedtoresttopelletredbloodcells, aftersupernatantwascollectedandcentrifugedat1600rpmfor 10min.Leukocyteswerewashedin5ml1XPBS,centrifugedat 1600rpmfor10minandvortexedin1ml0,2%NaClfor1minto remove red blood cells, then 1ml 1.6% NaCl was immediately added.Suspensionwascentrifugedat1200rpmfor10min,then pelletwasmixedin2mlNaCl0.9%.Onehundredandeighty

m

lof this solutionwere collected and leukocytes were stained with 20

m

l Trypan Blue solution to cell counting using Burker’s chambers.

2.3.DNAextraction

DNAzol1 Reagent (Life Technologies, Carlsbad CA) was employedtoconductDNAextraction.Briefly10
106_leukocytes

weremixedin1mlDNAzol1Reagentandincubatedfor2–3minat room temperature. 500

m

l 100% EtOH were added to cellular suspensionandtubewasreversedaslongasDNAwasvisible.The mixture was centrifuged at 10000g for a few seconds and supernatantwas removed.Thepelletwas washedtwicein1ml 75%EtOhand atfinallyresuspendedin100

m

l8mMNaOHand 10

m

l0,1MHEPES.

The DNAconcentrationwas measuredat spectrophotometer and the solution was diluted with H2O RNasi and DNasi free

2.4.Polymerasechainreaction(PCR)

Eachpolymorphicregionwasamplifiedusing100ngDNA:5

m

l 10XPCRBuffer,3

m

l25mMMgCl2,2

m

l10mMdNTPsmix,0.5

m

l

AmpliTaqGold5U/

m

l(LifeTechnologies,CarlsbadCA)and1

m

lof specificprimer(IDTInc.,Coralville,IA).Primer3(v.0.4.0)software

(http://bioinfo.ut.ee/primer3-0.4.0/primer3/) and polymorphic

sequencesubmittedindbSNPdatabase(http://www.ncbi.nlm.

nih.gov/snp/)wereusedtodrawspecificprimers.Table1shows

primersandannealingtemperatureutilizedtoamplify polymor-phismin SCN1A genes.Amplificateswere analyzedat Gel Doc (Biorad) after electrophoresis on 2% agarose gel stained with etidiumbromide.

2.5.Restrictionfragmentlengthpolymorphisms(RFLP)

Ten

m

lofPCR productswereutilizedtocarry outrestriction fragmentlengthpolymorphism.

Endonucleases able to discriminate between wild type and polymorphic sequence were identified by NEBcutter1

(http://tools.neb.com/NEBcutter2/).

The REBsites (http://tools.neb.com/REBsites/index.php) programwasusedinsteadtopredictrestrictionfragmentslength. EnzymeswerepurchasedbyNewEnglandBiolabs(Ipswich,MA) andThermoScientific(CarloErbaReagents,CornaderoIT)anda digestion mixture was prepared as manufactures recommend. MslI,PsiI,SphI,ApoI,BseNI,BbsI,MspA1I,MspI,TaqI,HaeIII,RsaI wereemployedasrestrictionenzymes.

2.6.Statisticalanalysis

Correlation between polymorphism and refractory epilepsy wasinvestigatedcomparinggroupofdrug-responsiveand drug-resistantpatientsandcalculatingPvaluebychisquaretest.Within theresistantgroupitwasanalyzedwhetherthereisagenotype that increases the risk for drug resistance. Odds ratio were calculatedwhenp<0.05.

3.Results

Atotalof120epilepsypediatricpatientswereenrolledinthis study,64(53,3%)maleand56(46,7%) female,60(50%)patients werediagnoseddrug-resistantand60(50%)haddrug-responsive epilepsy.AccordingtotheclassificationILAE1989ofepilepsy[14], inthegroupofdrugresistantpatients,39(65%)had localization-relatedepilepsy,onewithidiopathicepilepsy,27with symptom-atic, 11 with cryptogenic; 21 (35%) patients had generalized epilepsy,8symptomaticand13cryptogenic.Inthegroupofdrug responderpatients,45(75%)hadlocalization-relatedepilepsy,35 withidiopathicepilepsy,8symptomatic,2cryptogenic;15(25%) patientshad generalizedepilepsy,11withidiopathicepilepsy,3 with symptomatic and 1 cryptogenic. The average age of the patientswas 11.4years,withnosignificantdifferencesbetween thedrug-resistant(11.2years3.88DS)versusthedrug respon-sivepatients(11.7years3.87DS).Theaverageageattheonsetof thefirstseizurewas6.9years(4.1DS)fortherespondersand4.8 years(3.4DS)forthenon-responders.NoneofthestudiedSNPs oftheexonicregionoftheSCN1Agenewereassociatedwith drug-resistance.Intheintronicregionsofthesamegene,astatistically significantdifferencewasrevealedforthreeofthepolymorphisms studied (rs6730344A/C, rs6732655A/T, rs10167228A/T), in the genotypedistributionofthetwogroups(p<0.05).Foreachofthe three SNP with p<0.05 theodds ratio was also calculated, to evaluatethepossibleassociationbetweena particulargenotype anddrugresistance.Theresultswereasfollows:

-rs6730344A/C: theAC genotypeemerged as a risk factorfor drug-resistance,withO.R.=1.58(Fig.1);

-rs6732655A/T: theAA genotype emerged as a riskfactor for drug-resistance,withO.R.=2.25(Fig.2);

-rs10167228A/T: theAA genotypeemergedasa riskfactorfor drug-resistance,withO.R=1.89(Fig.3).

DNA electrophoretic gels show patients genotyping for individualintronicpolymorphisms(Fig.4).

Theanalysisofthegenotypesofeachintronicpolymorphismin thedrug-resistantgroupfoundthatrs1962842polymorphismAA genotype(OR:3,33)andATgenotype(OR:2695)haveabiggerrisk ofdevelopingdrugresistancethanTT(OR:0,348).

a)rs6730344A*/CdigestedwiththePsiIenzyme.Cuttingwiththis enzymeproducestwobandsof180and109bpinpolimorphic sequence(A*).

b)

Table1

SCN1Apolymorphisms,primersandannealingtemperature.Thefirst7areexonic SNPsandthelast7areintronicSNPs.

SNPs Primers Ta

rs146733308G/T FOR:50_{-CATCTTTGCCTTCTTGCTCA-3}0 ₅₄_C

REV:50_{-CAAAATCAAAGGTGGGGCTA-3}0

rs35735053C/T FOR:50_{-GTGCTTACAGACGCCACCTT-3}0 ₅₂_C

REV:50_{-TGCAGTGGACATGGTCAGAT-3}0

rs3749029G/A FOR:50_{-ttcatggcttccaatccttc-3}0 ₅₄_C

REV:50-ttccacaattggctttgtca-30

rs112767060T/A FOR:50-TCACATTTTTCCATCGAGCA-30 54C REV:50_{-GTGGTTGTGAATGCCCTTTT-3}0

rs10613159-AAC FOR:50_{-tgtgccatgctggtgTATTT-3}0 ₅₂_C

REV:50_{-GCTGTGTGGGGAGTGGATAG-3}0

rs12617205G/A FOR:50_{-TGGGAGTTGACAATCACTGG-3}0 ₅₄_C

REV:50-CATTTTCCAGCTGCGAGTTT-30

rs112157737T/C FOR:50-CATCTTTCTCTTCCCCACCA-30 54C REV:50_{-TGCAAATACTTCAGCCCTTTC-3}0

rs6730344A/C FOR:50_{-GGGGATTATACAAGGGCAAG-3}0 ₅₄_C

REV:50_{-CAAAATTTGTGGAAGAAGACCA-3}0

rs6432858C/T FOR:50_{-CAGGTGGCAAAAGCTTCATT-3}0 ₅₄_C

REV:50_{-AATGTGGTGCTTCTCAAATTGT-3}0

rs6732655A/T FOR:50-TCCCAAGTAGCTGGGACTACA-30 52C REV:50-GCCTTAGAACCACCTTTATAAAA-30

rs1962842G/A FOR:50_{-CATGGGCAAAATGTTGTCAG-3}0 ₅₄_C

REV:50_{-CACGCCCGCCTAATTTTT-3}0

rs10167228A/T FOR:50_{-CCAAATGGTGACACAGTGAA-3}0 ₅₄_C

REV:50_{-GCCTTGATCACTTGTAGGACTTTT-3}0

rs10194956A/G FOR:50_{-CATGGGCATGGAATAAAACA-3}0 ₅₂_C

REV:50-CCAATCTTACACAGATTGACCA-30

rs11690962G/T FOR:50-ATCCTTGGCATCACTCTGCT-30 52C

REV:50_{-TGATACCTTCAGTTGGGCTTTT-3}0 Fig1.PolymorphicalleleAisshowedas*.TheACheterozygousgenotypeismore prevalentinthedrugresistantgroupthaninthatofthedrugresponders.

rs6732655A*/TgenotypedwithTaqIenzyme.Therecognition andtherespectivecuttingleadstoformationoftwobandsof95 and155bpinwildetypesequence(T).

c)rs10167228A*/T.Theenzymeusedforthispolymorphism,RsaI, produces two restriction fragments of 97 and 107bp in polimorphicsequence(A*).

4.Discussion

Despite the widespread availability of AEDs with different mechanismsofaction,morethanathirdofpatientswithepilepsy isresistanttothedrugs.Twentypercentofpatientsinpediatricage exhibit a resistance to many AED and they are deemed to be “pharmacologicallyintractable_”.Uncontrolledcrisesandexposure tohighdosesofnumerousandineffectiveAEDleadtosignificant neuropsychiatric (depression, anxiety disorder) and social im-pairment,lowerqualityoflife, greatercomorbidity (intellectual disability,attentionandlearningproblems),higherriskofdeath [1]. Sudden unexpecteddeath in epilepsy(SUDEP) is themost commontype ofdeathinpatientswithdrug-resistant epilepsy. Othercausesofdeathinpatientswithepilepsymaybedirectly relatedtoseizures(accidentaltrauma,drowning,burns)ortothe underlyingcauseofepilepsy[10].ThecausesofaDREarecomplex and multifactorial and this can make it difficult to obtain a statisticallysignificantassociationwithaSNP[15].Becausemany AEDactprimarilyasblockersofsodiumchannels,thegenecoding for the SCN1Avoltage-gated sodiumchannels is an interesting candidate to study the relationship between genetic polymor-phismsindrugtargetsandclinicalresponse[11,13,16].

The SCN1A gene, coding for the

a

subunit of the sodium channelvoltage-dependent,isthemostfrequentlymutatedgene indifferentformsofepilepsyofchildhoodandindifferenttypesof infantile epileptic encephalopathies [17]. For the high rate of mutation in the SCN1A gene, polymorphisms were selected primarilytobefoundwithinthecodingregions,toidentifysome causesofdrugresistanceinthesepolymorphisms.

In ourstudy, thedetectionof polymorphisms(rs146733308, rs35735053, rs3749029, rs112767060, rs10613159, rs12617205, rs112157737)oftheexonicregionsofSCN1Agene,coveringthe whole study sample, did not produce statistically significant results. As for the small sample size, this result is not to be considered definitive, butrather to beconfirmed enlarging the numberofpatientsinthestudy.

Indeed,mostofthepolymorphismspresentinthedatabases (95%)arepresentinthenon-coding(intron,intergenicpromoteror zones)whichconstitutethemajorityofthegenome.Clinicaltrials have suggested that intronic polymorphism rs3812718G>A of SCN1AgeneisinvolvedinclinicalresponsetotreatmentwithAED, buttheliteraturedatainthisregardarecontradictory.

Tateetal.[18]reportedanassociationbetweenthefunctional polymorphism rs3812718 (c.603-91G>A) of SCN1A gene and pharmacological response, in terms of effective dosage of CBZ andPHT.Thereafter,astudyinvolving71Chinesepatientsfailedto replicatetheseresults,althoughaminimalimpactoftheSCN1A polymorphism was not eliminated [19]. Two other replication studies_–the_firstcarriedouton228Japaneseepilepticpatients [20]and thesecondon369Australian epilepticpatients[21] – havefailedtoidentifyanassociationbetweenthepolymorphism anddosesofsodiumblockersdrugs,althoughtheJapanesestudy described an association between genotype AA of c.603-91 polymorphism and unresponsiveness to CBZ [13,20]. These differentdatacouldbeexplainedbymanyfactors,includingsize and heterogeneity of the sample, selection bias and other confoundingfactors.Furthermore,wemustconsiderthelimited supportofasingleSNP:infact,asthegeneticcomponentofthe drugresponseisusuallypolygenic[10],theimpactofasinglegene couldbeinfluencedbyothergenesandbyenvironmentalfactors [13].

Fig.2.PolymorphicalleleAisshowedas*.TheAAhomoozygousgenotypeismore prevalentinthedrugresistantgroupthaninthatofthedrugresponders.

Fig.3.PolymorphicalleleTisshowedas*.TheAAhomozygousgenotypeismore prevalentinthedrugresistantgroupthaninthatofthedrugresponders.

Fig. 4.DNA electrophoretic gels showing patients genotyping for individual polymorphisms.

Recently,Yipetal.[16]havestudiednineSNPswithinSCN1Ain 519Caucasianepilepticpatientswithknownresponsestatusfor sodium channel blocking AED: firstly, the only association observed was for rs10188577; after correction for potential confounding factors, the association for rs10188577 was only marginallysignificant(P=0.049).Therefore,itseemsunlikelythat rs10188577couldbeamajordeterminantofresponsetoAED[16]. We investigated polymorphisms rs6730344, rs6432858, rs6732655,rs1962842,rs10167228,rs10194956 and rs11690962 within the intronic regions of the SCN1A gene, in both drug-responsiveanddrug-resistantgroups.Asthesepolymorphismsare locatedinintronicregions,theirpresencecouldinterferewiththe mechanisms of alternative splicing and therefore alter the conformation of the functional domains of the Na+ _channel;

moreover,theycouldresult in unstableprimary transcript and thereforein decreased synthesisof the Na+ _{channel.Our study}

revealed a statistically significant difference (P<0.05) on the distributionof genotypesbetween the two groups of epileptic patients(drug resistant and drug sensitive) for three of these polymorphisms(rs6730344,rs6732655andrs10167228),allowing to assign to these polymorphisms a role in the response to antiepilepticdrugs.Inparticular,assessingapossibleassociation betweena specificgenotypeand phenotype drugresistance by oddsratio,wefoundthatthegenotypers6730344A/C(O.R.=1.58), rs6732655A/T (O.R.=2.25) and rs10167228A/T (O.R.=1.89) appearto berelated tothedrugresistance phenotype, as they have a prevalent genotype distribution in the group of drug-resistantpatients.Wealsoinvestigatedwhetherwithinthegroup ofpatientswith drug-resistantepilepsy therewas a significant genotypefordrugresistancerisk. Rs1962842polymorphism AA genotype(OR:3,33)andATgenotype(OR:2695)haveanincreased riskofdevelopingdrugresistancethanTT(OR:0,348)amongthe sevenanalyzedintronic polymorphisms(rs6730344,rs6432858, rs6732655,rs1962842,rs10167228,rs10194956andrs11690962). Howcouldpolymorphismslocatedinnon-codingregionsaffect theresponsetodrugs?Thepresenceofsignificantpolymorphisms located in non-coding regions has stimulated us to further investigate the significance of this result. We used Conserved domain software (http://www.ncbi.nlm.nih.gov/Structure/cdd/

wrpsb.cg)todeterminethedomainspresentinthesequenceof

thegeneSCN1A.Then,eachpolymorphisminourstudywasputin relationtothefunctionaldomainsoftheprotein.Rs10167228is positionedbetweentwoexonsthatencodefora domainwhose functionhasnotbeencharacterized,DUF34511.Thisdomainhas been found only in eukaryotes and it is associated to other functional domains as pfam06512 (Na_trans_assoc) and pfam00520(Ion_trans).

rs6730344 (p=0.0462 rs6730344 AC o.r=1.58) is located betweenaregionthatencodeforpfam06512.Thisdomain,also knownasNa_trans_assoc,includesaregionfoundexclusivelyin eukaryoticsodiumchannelsortheirsubunits,manyofwhichare voltage-gated.Membersveryoftenalsocontainbetweenoneand fourcopiesofpfam00520(Ion_trans)and,lessoften,onecopyof pfam00612(IQcalmodulin-bindingmotif).

rs6732655(p=0.017rs6732655AAo.r.=2.25)issurroundedby exonscodingforthedomainpfam00520(Ion_trans).Thisfamilyis characterizedby6transmembranehelicesinwhichthelasttwo helicesflankaloopwhichdeterminesionselectivityandinsodium channelsthedomainisrepeatedfourtimes.However,rs6732655 inisoforms1,2and3(respectivelyNM_001165963,NM_006920 andNM_001165964)islocatedbetweentwoexonsthatinaddition tocodeforpfam00520alsoencodeforcl21557domain(Yip1).The Yip1 integral membrane domain contains four transmembrane alphahelicescharacterizedbythemotifsDLYGPandGY.

It hasbeenwidelyacknowledgedthatintronsareanintegral partoftheregulationofgeneexpression.Non-codingRNAswithin

intronsarecommonlyproducedthroughthepost-splicingprocess and arespecificsignals ofgene transcription events, impacting manyothergenesandmodulatingtheirexpression.Anassociation betweenhumanintronsandncRNAshasapronouncedsynergistic effectwithimportantimplicationsforfine-tuninggeneexpression patternsacrosstheentiregenome[22].

Therefore, although introns are non-coding regions, the presence of polymorphisms in these sequences may result in alterationsintheencodedprotein,becauseintronicpolymorphic sequences flanking exons can result in destruction of existing splicingsitesorinthecreationofnewones[23]. Moreover,the presence of intronic polymorphisms could interfere with the formationofthespliceosome[24,25].

5.Conclusion

Consideringthepsychosocialimpactofdrug-resistantepilepsy, the knowledgeof themechanisms of drug resistancebecomes important in the development of AED and clinical practice. Pharmacogenomics constitute an emerging field with high potentialtoinfluenceourdecisionsforthetreatmentofepilepsy inthefuture.Literaturedatareportassociationsbetweenspecific genetic polymorphisms and drug resistance; although these results may not always be replicated in subsequent studies, however, thestudyof polymorphisms ofgenes involvedin the metabolismofAEDisaveryattractiveresearchline.

Finally, ourstudy showed a significant association between rs6730344, rs6732655 and rs10167228 polymorphisms in the intronicregionsoftheSCN1Ageneandrefractoryepilepsy,thus emerging as risk factors for drug resistance. SCN1A gene polymorphismsmayplayaroleintheresponsetoAEDinpatients with drug-resistant epilepsy, with important implications for clinicalpractice,whichhasthehighestgoalofatailoredtherapy. Thesepharmacogeneticdatacouldassistcliniciansinpredicting patientdrugresponsephenotypeandallowformolecular-based designofgenotypespecifictherapeuticswithgreatertolerability and efficacy.Thiscould bethebasis ofa personalized epilepsy treatment aiming at improving patient quality of life. Further studies are needed to confirm these results and complete our journey through the pharmacogenetic causes of drug resistant epilepsy.

Multicenter studieswill benecessary toreachthegoalof a broaderunderstandingofpharmacogenomicmechanismsinthe epilepsies.Moreover,identificationofpolymorphismsofcandidate genes,whichmayinfluenceAEDresponse andresistance,could leadtonewknowledgeofthemolecularmechanismsunderlying drug-resistantepilepsyand the developmentofnew and more effectivedrugs.

Conflictsofinterest None.

Funding

Thisresearchdidnotreceiveanyspecificgrantfromfunding agenciesinthepublic,commercial,ornot-for-profitsectors. References

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