Genes and molecular mechanisms involved in the epileptogenesis of idiopathic absence epilepsies

Review

Genes

and

molecular

mechanisms

involved

in

the

epileptogenesis

of

idiopathic

absence

epilepsies

O

¨ zlem

Yalc¸ın

*

DepartmentofMolecularBiologyandGenetics,T.C.IstanbulArelUniversity,Tepekent-Buyukcekmece,Istanbul,Turkey

1. Introduction

Idiopathic absence epilepsies (IAE) are types of generalized epilepsies (IGE) that are not preceded or occasioned by other disorders,butareinstead,causedbycomplexgeneticfactors.1_The

mostcommonformsofIAEsarechildhoodabsenceepilepsy(CAE) andjuvenileabsenceepilepsy(JAE).However,absenceseizurescan be associated with other subtypes of IGEs, such as juvenile myoclonicepilepsy(JME).The clinicalfeatures ofCAEare non-convulsiveepilepticseizurescharacterized bythebrief(5–20s) andsuddenlossofconsciousnesswithageneralizedsynchronous, bilateral,2.5–4Hzspikeandslowwavedischarge(SWD)in the electroencephalogram(EEG).2,3_{Theonsetoftheabsenceseizures}

havinga frequency of up to200 per day (pyknoleptic),begins between the ages of 3 and 8, with seizures disappearing spontaneously at adolescence. JAE, on the other hand, is characterized by non-pyknoleptic absence seizures, primarily occurring after 10 years of age. Unlike CAE,these seizuresare associated frequently with other epileptic symptoms, such as generalizedtonic-clonicseizures(GTCS)andmyoclonicjerks.

Experimentsonanimalmodelsrevealedthatthethalamusand thecortexarebothinvolvedinthegenerationofSWDs.4,5_Thereare

three main components of a thalamocortical network: (1) Thalamocorticalrelayneuronswhichtransferinputsfromalarge number ofsourcestopyramidalneuronsintheIII–IVandV–VI layersofthecortexthroughexcitatorysynapticconnections.(2) LayerVIpyramidalcellsofthecortexwhichsendbackexcitatory inputstothethalamus.(3)InhibitoryGABAergicinterneuronsin the thalamic reticular nucleus (nRT), which receive excitatory inputsfromaxoncollateralsofthereciprocalthalamocorticaland corticothalamic pathways. When activated, these GABAergic neuronssendinhibitoryinputstothethalamusandalsotoeach other,butnottothecortex.6

When a thalamocortical circuit works properly, a burst of synchronizedoscillationswithafrequencyof10Hzwilloccur.In this circuit, thalamocortical neurons induce excitatory post-synapticpotentials(EPSPs)inGABAergicnRTneuronsviaNMDA andnon-NMDAreceptors.Low-thresholdcalciumchannelsinnRT neurons are activated leading to the opening of Na channels, initiating action potentials. The activation of these inhibitory neurons induces inhibitory postsynaptic potentials (IPSPs) in thalamocortical neurons via GABAA receptors. This inhibitory

phaseabolishestheburstfiringinthecircuitforatime.Duringthis hyperpolarized state, low-threshold calcium channelsin thala-mocortical neurons recover from inactivation. The calcium channels open, depolarizing the membrane, making the cell available for the next burst of action potentials. However, abnormal activity in this circuit would disrupt the alternating

ARTICLE INFO Articlehistory: Received10May2011

Receivedinrevisedform2December2011 Accepted4December2011

Keywords:

Childhoodabsenceepilepsy Juvenileabsenceepilepsy GABAchannels Calciumchannels Spikewavedischarges

ABSTRACT

Idiopathic absence epilepsies (IAE), that have high prevalence particularly among children and adolescents,arecomplexdisordersmainlycausedbygeneticfactors.Childhoodabsenceepilepsyand juvenileabsenceepilepsyareamongthemostcommonsubtypesofIAEs.Whiletheroleofionchannels hasbeentheprimaryfocusofepilepsyresearch,theanalysisofmutationandassociationinbothpatients withabsenceepilepsiesandanimalmodelsrevealedtheinvolvementofGABAreceptorsandcalcium channels,butalsoofnovelnon-ionchannelproteinsininducingspikewavedischarges(SWD).Functional studiesonamutatedvariantoftheseproteinsalsosupporttheirroleintheepileptogenesisofabsence seizures.StudiesinanimalmodelspointtoboththethalamusandcortexastheoriginofSWDs:the abnormalitiesinthecomponentsofthesecircuitsleadingtoseizureactivity.Thisreviewexaminesthe currentresearchonmutationsand susceptibilityallelesdeterminedinthegenesthatcodeforthe subunitsofGABAreceptors(GABRG2,GABRA1,GABRB3,GABRA5,GABA(B1)andGABA(B2)),calciumchannels (CACNA1A,CACNA1G,CACNA1H,CACNA1I,CACNAB4,CACNAG2andCACNG3),andnovelnon-ionchannel proteins,takingintoaccounttheresultsoffunctionalstudiesonthesevariants.

ß2011BritishEpilepsyAssociation.PublishedbyElsevierLtd.Allrightsreserved.

*Correspondingauthor.Tel.:+902128600480;fax:+902128600314. E-mailaddress:ozlemyalcin@arel.edu.tr.

ContentslistsavailableatSciVerseScienceDirect

Seizure

j o urn a lhom e pa g e :ww w . e l se v i e r. c om / l oca t e / y se i z

1059-1311/$–seefrontmatterß2011BritishEpilepsyAssociation.PublishedbyElsevierLtd.Allrightsreserved. doi:10.1016/j.seizure.2011.12.002

cyclesofexcitatoryandinhibitoryactivity,leadingto3–4Hzspike wavedischarges.Forexample,increasedexcitatoryactivityinthe cortexorthalamocorticalregionwouldleadtoalongerburstof nRT neurons firing, causing longer IPSPs mediated by GABAB

receptorsviaG-proteincoupledKchannelsinthethalamocortical region.During this long hyperpolarizedstate,calciumchannels open,initiatingseveralactionpotentialsineverycycle,resultingin paroxysmalspikesandslowwavedischarges.

In addition to possible abnormal activity in the cortex or thalamocorticalregions, there areother suggestedmechanisms explainingthegenerationofabnormalspikewavedischarges.For example,thelossofGABAAreceptormediatedinhibitionbetween

thalamicreticularcellscausesthesecellstoproducelongerIPSPs on thalamocortical cells mediated by GABAB receptors and

enhancementoflowthresholdCa2+.7

Thisreviewsummarizesnotonlythemutationsand suscepti-bility alleles determinedin patients and animal modelsin the candidateionchannelandthenovelnon-ionchannelgenesbut alsoexaminesthefunctionalresultsofthesevariationsthroughthe mechanismofSWDsinthethalamocorticalnetwork.

2. Geneticetiologyofidiopathicabsenceepilepsies

Becausethecellularmechanismofabsenceseizuresindicates theinvolvementofionchannelsinthepathogenesisofabsence epilepsies; gene analysis carried out both on patients and on animalmodels,revealedbothmutationsandsusceptibilityalleles ingenesthatcodeforthesubunitsofGABAreceptorsand Ca2+

channels (as shown in Table 1). Unfortunately, most of these variationsarerare andrestricted toafew a patients;thus, the components of the complex genetic factors involved in the triggeringofabsenceseizuresarestillunclear.However,inrecent years,researchintothesefactorshascontinued,andthefocuson using novel non-ion channel genes and approaches, such as studyingthecopynumbervariations(CNV),epigenetic modifica-tions,and exome sequencing,hasdrawnmoreattentionto the importanceofunderstandingtheplaceofgeneticcomponentsin absenceseizures.

2.1. GABAreceptors

GABAis themain inhibitoryneurotransmitter inthecentral nervoussystem.Itinteractswithtwomajorsubtypesofreceptors, GABAAandGABAB,whichareinvolvedinthegenerationofspike

wavedischargesduringabsenceseizures.

2.1.1. GABAAreceptors

IonotropicGABAAreceptorsmediatefastsynapticinhibitionin

thecentralnervoussystem.BindingofGABAtoGABAAreceptors

allowsaninfluxofCI_{ionswhichcauseshyperpolarizationofthe}

membraneandinhibitionofactionpotentials.8_GABA

Areceptors

arepentamericstructures,consistingoffiveoutofatleast18 sub-units(

a

1-6,

b

1-3,

g

1-3,

d

,

e

,

u

,

r

1-3).9 _{Each subunit has four}

transmembranedomainswiththesecondtransmembraneofeach subunitformingthechannelpore.10_{Thegenesthatcodeforthese}

subunitsarelocalizedasclustersondifferentchromosomes.The mostprevalentGABAAreceptorthroughoutthebrainisformedby

the

a

1,

b

2,andthe

g

2subunits,encodedbyGABRA1,GABRB2and GABRG2geneslocalizedon5q34–35.AnotherGABAgenecluster (GABRA5,GABRB3andGABRG3)resideson15q11–12,codingforthe

a

5,

b

3and

g

3 subunits. These receptorscanbemodulated by steroids, barbiturates and benzodiazepines. The role of GABAA

receptors in epileptic activity has been studied extensively; researchersfoundthatGABAAreceptoragonists,suchas

barbitu-rates and benzodiazepines, suppress seizures while theGABAA

blockers, such as bicuculline, penicillin and picrotoxin induce epilepticactivityinmodelanimals.

InanalysisofGABAAreceptorsinabsenceseizures,theresearch

hasforthemostpartfocusedonthe

g

2subunit,whichwasthefirst mutationidentifiedintheGABRG2geneinafamilywithCAEand febrileseizures.11_The

_g

_{2subunitisknowntoberesponsiblefor}

modulationofbenzodiazepineandreceptortargeting.8_Mutation

intheCAEfamily,argininetoglutaminesubstitutionataminoacid 43(Arg43Glu),ledtothelossofcurrentthroughGABAreceptors duetoanimpairmentintrafficfromthereceptortothemembrane. Functionalstudiesinthisareapointedoutthatthesubunitwith themutation is stuckinthe endoplasmicreticulum,leading to decreasedsurfaceexpression.12_{Arg43Glumutationwasassessed}

inamousemodelwhichwasconstructedbytheinsertionofan Arg43Glu mutation in the heterozygous state.13 The mouse showedasimilarphenotypeindicatingchildhoodabsenceepilepsy andconfirmingthecausativeroleofthe

g

2subunit.Areductionof inhibitioninthecortexofthemousewasdetected,markingthe cortexasthestartregionofSWDs.Further,thalamicburstingis subjecttoinputsfromthecorticalregion,andthus,inhibitionin thecortexwouldaltertheseinputs,triggeringSWDs.

AsecondmutationinGABRG2wasalsofoundinafamilywith CAEandfebrileseizures.14_{TheIVS6+2T!Gmutationdisrupteda}

putativesplicesite,whichprobablycaused atruncatedprotein. Despite these findings, contradictory results in Japanese and Chinese populations limited the potentialimpact of this study because mutation analysis and association studies revealed negativelinkageofCAEtoGABRG2inthesepopulations.15,16

The first study to understand the role of GABRA1 in the pathogenesis of epilepsyincluded 61 JME, 38 JAE,and 29 CAE patients,revealednolinkage.17Furtherstudyidentifiedmutations inaJMEfamily18_{andaCAEpatient.}19_{IntheCAEpatient,thesingle}

basepairdeletion(975delC)hadcausedaprematurestopcodon. Functionalstudiesshowedthatthetruncatedreceptorcouldnot integrateintothemembraneandthuscausednocurrent.Dueto haploinsufficiency there were losses in the function of the inhibitory action of GABA channels in the thalamic neurons. However, in the Japanese population, attempts to find any causative mutation in typical and atypical absence patients revealednegativeresults.15

The GABRB3 gene codes for the

b

3-subunit of the GABAA

receptor.20_{Associationanalysiscarriedoutwithdifferentethnic}

groups for GABRB3gene displayed possibleassociations in CAE patients.21–23InUrak’sstudy,acommonpromoterhaplotypewas foundtobeatahigherfrequencyinCAEpatients.Areportergene assay was carried out to illustrate the possible effects of this haplotypeontranscription.Theresultshowedthatthisparticular haplotype reduced the transcriptionallevel of GABRB3 gene by interferingwiththebindingsiteoftheneuron-specific transcrip-tionalactivatorN-Oct-3.However,reducedlevelsofthe

b

3subunit wouldhavealsodecreasedthelevelofinhibitoryGABAAreceptors.

ThisdiseasesusceptiblehaplotypewhenassessedintheGerman IGEsamplesdidnotconfirmthatthishaplotypewascommonin absencepatientsofdifferentethnicgroups.24

Differences based on ethnicity were further illustrated by mutations found in GABRB3 in 4 families of 48 CAE Mexican familiesofAmericanIndianorSpanishEuropeandescent.Three mutations resided in the exon1a in signal peptide, while the Gly32Arg mutation in exon 2 affected protein maturation, topology, assembly, and subcellular localization of a GABA receptor,resultinginhyperglycosylation.25

GABRB3deficientmiceshowedabnormalEEGactivityincluding generalizedtonicclonicseizures,clonicandmyoclonicseizures,as wellasbehavioralarrestduringtheabnormalEEGactivity,similar to that found in absence seizures.26 _{However, there was no}

evidencethatthemicehad experiencedpureepilepticseizures;

O¨.Yalc¸ın/Seizure21(2012)79–86 80

Association and mutation analysis of the candidate genes for idiopathic absence epilepsies.

Gene Protein DNA Mutation/association analysis Protein mutation Clinical features and origin of Patient(s)/reference Functional study

GABRG2 g2 subunit of GABAAreceptor c.245G > A R43Q CAE patients with febrile seizures in a large family11 Reduced GABAAreceptor currents12

IVS6 + 2T > G – CAE patient with febrile seizures14

Probably truncated protein

GABRA1 a1subunit of GABAAreceptor 975delC Premature

stop codon

CAE patient19 _{No GABA}

Areceptor current

GABRB3 b3 subunit of the GABAAreceptor Significant association

of a common haplotype

– 45 Austrian CAE patients21

Reduced transcriptional level of GABRB3 gene

c.31C > T P11S CAE Mexican families with American Indian

and Spanish European ancestry25

Altered protein maturation, topology, assembly and subcellular localization of GABA receptors

c.44C > T S15F

c.G962 > A G32R

CACNA1A a1A subunit of P/Q channels 5733C > T R1820stop A patient with severe phenotype of absence

seizures and ataxia39

Reduced Ca2+

current

439G > A E147K In a family with absence seizures and ataxia40 _{Reduced Ca}2+_{channel function}

CACNA1G a1G subunit of LVA channels c.1709C > T A570V A Japanese patient with sporadic case of JME

and with a history of early CAE46

Increased Ca2+

current and faster inactivation decay rates

c.3295G > A A1099T Japanese CAE patients46

No alteration in Ca+

currents

c.3728G > A R1243Q No alteration in Ca+

currents

CACNA1H a1H subunit of LVA channels 562C > A F161L Chinese CAE patients52

Increased T-type channel activity53–55

923G > A E282K 1445T > A C456S 1574G > A G499S 2022C > T P648L 2310G > A R744Q 2322C > T A748V

47152C > T P314S Chinese CAE patient56

Predicted to change potential phosphorylation site

48684C > T P492S Predicted to change the charge status

of the channel and phosphorylation site

47247C > T N345N Predicted to alter potential splicing site

49016C > G L602L Predicted to affect transcription

factor binding site

49067A > G S619S Predicted to affect transcription factor

binding site or splicing Significant association of SNP

(rs2745150) in intron 11

– 100 Chinese CAE patients56 _{Predicted to change potential splicing site}

Significant association of SNP (rs9934839) in exon 9 and haplotype

– 218 Chinese CAE patients57

Predicted to change the transcription binding site

CACNG3 g3 subunit of neuronal

voltage-gated calcium channel

Strong association of 3 common SNPs (rs4787924, rs965830,

rs2214437) and a haplotype

– 217 CAE trios and the 65 nuclear

pedigrees with Caucasian origin66

–

ME2 Malic enzyme 2 Association of a nine-SNP haplotype – 68 JAE patients with GTCS67 _–

Glud1 glutamate dehydrogenase 833C > T R221C 3 children in the same family with photosensitive

myoclonic absence epilepsy and HI/HA68

Decreased level of GABA in patients compared to the controls

JRK/JH8 JRK jerky homolog (mouse) c.1367C > T T456M CAE patient evolving to JME70

–

LGI4 leucine-rich, glioma inactivated 4 Association of a polymorphism

(c.1914GC > AT) – 42 CAE patients72 – O ¨ . Yalc ¸ın / Seizure 21 (2012) 79–86 81

insteadtheyshowedfeaturesoftheAngelmansyndrome,whichis known tohavedeletionson15q11–13.PatientswithAngelman syndrome, like theGABRB3 deficient mice,experience different typesofepilepticseizuressuchas,atypical absence,myoclonic, atonic,tonic,andtonic-clonicseizures.Themiceweretreatedwith carbamazepine,awell-knownantiepilepticdrug(AED),whichis used for thetreatment of focal epilepsies. After treatment, the absenceseizureswereaggravated,asisthecaseinhumanswith absence seizures, indicating an involvement of absence-like pathophysiologyinthesemice.27,28

The

a

5-subunitofGABAAreceptors(GABRA5)of50CAEpatients

werealsosubjectedtomutationanalysis;however,nocausative variationwasidentified.23

2.1.2. GABABreceptors

Metabotropic GABABreceptors mediate their activity via

G-coupledproteinsbyactivatingK+_andCa2+_{ionchannels,second}

messenger systems, phospholipase C, and adenylate cyclase.10 These proteins are seven transmembrane receptors, and the functional GABAB receptor is formed from heterodimers of

GABA(B1) and GABA(B2) subunits. These receptorsproduce both

slow and prolonged inhibitory signals, mainly located in the presynapticterminals,whichhaveanessentialrolein neurotrans-mitterrelease.

Themicemodels,withknockeddownGABA(B1)andGABA(B2)

subunits,displayedspontaneousSWDs,whichindicatedapossible role for GABAB receptors in absence epilepsy.29,30 Also, in the

neocortexoftheWag/rijrat(oneoftheestablishedanimalmodels for human absence seizures), researchers found a reduced expressionandfunctionofGABABreceptor.31Althoughtheanimal

models emphasized the possible role of GABAB receptors in

absenceseizures,mutationandassociationanalysisperformedon ChineseCAEpatientsagainrevealednegativeresults.32

2.2. Calciumchannels

Calcium channels are voltage dependent channels whose conductancedependsonchangesintransmembranepotential.In excitablecells,theyconduct Ca2+_{ionswhichfunctioninmuscle}

contraction,aswellasreleasinghormonesandneurotransmitters throughadiversecalciuminvolvedprocesses.33Calciumchannels arecomposedofonemain

a

1subunit,i.e.anintegralmembrane proteinanditssmallerauxiliarysubunits(

b

,

a

2,

d

and

g

).34The

biologicalandphysiologicalpropertiesofcalciumchannelsdepend on the

a

1 subunits, consisting of four repeats of the six transmembranedomains.Asinallothervoltagegatedchannels, thesedomainsincludeoneS4segmentwhichfunctionsasvoltage sensor,aselectiveP-loop,andS6segmentthatformstheinnerpart ofthechannel.TheC-terminusofthe

a

1proteinisalsoessentialfor interactionwiththeauxiliarysubunits,Ca2+_{-calmodulin-mediated}

inactivationandforG-proteinregulation.35_{Thereareatleasttwo}

distinct classes of Ca2+ _{channels depending on the voltage}

requirementforactivation.Low-voltage-activated(LVA)channels activateafterasmalldepolarizationofthemembranewhile high-voltage-activated(HVA)channelsfunctioninthecaseofa larger depolarization.Calciumchannelsarepossibletargetsforinduction ofSWDsbecauseoftheirexcitatoryfunctioninthethalamocortical region. Ethosuximide, which is an essential AED used to treat absenceseizures,isalsoknowntosuppressT-channelcurrents.36

Attempts to locate epilepsy genes in both animal models and absencepatientsrevealedthatboththeLVAandHVAchannelsplay rolesinthepathogenesisofabsenceseizures(showninTable1). 2.2.1. HVAcalciumchannels

HVA calcium channels include L-, P/Q-, N- and R-subtypes dependingontheirdifferentelectrophysiologicaland

pharmaco-Table 1 (Continued ) Gene Protein DNA Mutation/association analysis Protein mutation Clinical features and origin of Patient(s)/reference Functional study SLC2A1 Glut1 glucose transporter c.668G > C R223P Early onset absence epilepsy 74 Decreased level of glucose transport c.971C > T S324L c.376C > T R126C c.680-11G > A 227–228ins PPV c.971C > T S324L Aglo-saxon absence families with different syndromes 75 – c.668G > C R223P INHA inhibin alpha subunit Significant association of SNP (rs7588807) in intron 1 – 7 2 Turkish JAE patients 76 – n.370C > T R124C Turkish JAE patients – n.525C > G H175Q n.747G > A L249L

O¨.Yalc¸ın/Seizure21(2012)79–86 82

logical properties, along with amino acid identity.37 _{P/Q type}

calciumchannels,mainlyfoundinpresynapticterminal distribu-tion, are known to have an essential role in modulating neurotransmitterrelease.38_{Therefore,dysfunctionofthese}

chan-nelswouldimpairthebalancebetweenneuronalinhibitionand excitationleadingtoburstfiring.

In a patientwith severephenotype ofabsence seizuresand ataxia,forthefirsttime,anonsensemutationhasbeenidentifiedin the

a

1AsubunitofP/Qchannels(CACNA1A).39_{Thegeneresidedon}

19p13,withthemutationlocatedintheC-terminusoftheprotein, whichcausedaprematurestopcodon.Functionalstudiesshowed thatthis mutationhad adominantnegativeeffect,leadingtoa reducedCa2+_{current.Asecondmutationwasalsoidentifiedinthe}

CACNA1Aofafamilywiththreegenerationsofabsenceandataxia intheirmedicalhistory.40The439G!Anucleotidetransitionin exon3inheterozygousstatecausedE147Kaminoacid substitu-tionin thesecondtransmembrane segment of domainIof the channel.Functional studies forthis mutation revealeda partial reductionincalciumchannelfunctionduetoimpairmentintraffic tothemembrane.

Intwoanimalmodels,thetottering andleanermouse, which showedboththeabsenceandataxiaphenotypes,mutationswere foundinCACNA1A(Cav2.1).Inthetotteringmousethemutationwas located in the S4–S5 linker region of the third transmembrane domainnearthepore-formingregionofthechannel.41_Thismutation

reduced the whole-cell current density and voltage dependent inactivation during a prolonged depolarization in dissociated Purkinjesomas.42Intheleanermouse,themutationinCACNA1A hadmoresevereeffects,itslocationintheC-terminus,reducedboth currentdensityandopenprobabilityofsingleP/Qtypechannels.43

While Cav2.1 channels appear to be good candidates for absenceseizures,bothpatientsandmousemodelsfailedtoexhibit not only pure absence seizures, but also other neurological disorders like ataxia and dystonia. Therefore, mutations in CACNA1Acouldhavebeenthecauseoftheotherdiseases,with anotherlocus responsible for theepileptic phenotype. Another possibilitycouldbethatmalfunctionsinotherproteins,secondary tothemutationsinCACNA1A,weretheactualcauseoftheabsence seizures.28_{Recentstudiesindicatethatthis secondpossibilityis}

morelikely:specificallyastudyonmousemodelswithCACNA1A mutations,whereanincreaseinthecurrentofLVAchannelswas measuredinthethalamocorticalregionofthebrain.44_Inmutant

micewithnullmutationofCACNA1A,LVAcurrentwaselevatedin thethalamocorticalregionandthemicewerefoundtobemore likelytohavespikewavedischarges.45

2.2.2. LVAcalciumchannels

Therearethreegenesthatcodeforthe

a

1subunits,whichhave low-voltage activation, namely CACNA1G (Cav3.1), CACNA1H

(Cav3.2) and CACNA1I (Cav3.3). CACNA1G is located on the

17q21.33,andthis channelismainlyexpressedin the thalamo-cortical neurons where the spike wave discharges occur. As a potentialcandidategene,CACNA1Gwasscreenedin73Japanese and 50 non-Japanese patients. Of this group, 13 variants were identifiedand5ofthosecausedaminoacidsubstitutions.46_Oneof

thesevariantswasfoundinapatientwithasporadiccaseofJME andahistoryofearlychildhoodabsenceepilepsy.Themutation caused alanine tovaline amino acid exchangeat position 570, locatedintheintracellularportionoftheproteinwithinI-IIloop. Themutationwasnot foundin360control samplesandwhen expressedinHEKcells,themutatedchannelscausedalargerCa2+

current compared to the wild type, however the result was statistically insignificant. However, other variants may have affectedthealternativesplicingofthegene:atleastfivedifferent isoformswithdifferentkineticsandsteady-statepropertieshave beenidentifiedfortheCav3.1channel.47

Mutationanalysiswasalsoperformedon48Chinesepatients who were shown to have a similar genomic structure to the JapaneseintheHapMapproject.Unfortunately,theresearchdid not reveal any pathological change, but six newvariants were detected.Anassociationanalysiswascarriedouttodetermineif there was a significant difference between controls, but it exhibitednegativeassociation.48

ToassessthepossibleroleoftheCav3.1channelsininducing

spike wave discharges, CACNA1G was deleted in mice models. Interestingly,animalsbecameresistanttospikewavedischarges, duetotheLVAT-typechannelbeingabolished.49_{Inanotherstudy,}

CACNA1G was overexpressed in a mouse with low and high transgene copy numbers. This led to an elevation in

a

1G expression, and consequently, in thefunctionalT-type currents inthalamicneurons.50BothtransgeniclinesshowedSWDbutdid not indicate muchof a difference inthe frequency ofseizures. Further, the mouse did not exhibit any other neurological disorders; providing direct evidencethat an increase in Ca

v

3.1 didleadtopureabsenceseizures.

TheCACNA1Hgene(Ca

v

3.2)islocatedonchromosome16p13.3 andexpressedinthethalamicreticularnucleus.49_{Itisextensively}

alternatively spliced and generates a family of variant tran-scripts.51_{Thedifferentvariantsshiftvoltage-domainforgating,the}

kineticsofactivation,inactivationandrecoveryfrominactivation, andthemagnitudesandvoltagemidpointsforfunctionalwindow currents.Thus,changesthataffecttheESEregulatorysitesinexons orsplicinginintronicregionscouldpredisposeseizures.Thefirst mutationanalysisinCACNA1Hwascarriedoutwith118Chinese CAEpatientsandrevealed12missensemutationsin14patients who were in a heterozygous state.52 _{These mutations were}

introducedintohumanCav3.2acDNAandtransfectedinto

HEK-293 cells for whole-cell patch-clamp recordings.53–55 _T-type

channelactivitywasfoundtohaveincreasedinallmutanttypes causing SWD in absence seizures due to a shift in activation potentials.Thechannelswereactivatedinresponsetoasmaller voltagechange,orachangeintherateofrecoveryofchannelsfrom theinactivatedstate(deinactivation),oranincreaseinthesurface expressionofthechannels.

ThepossibleroleofCACNA1Hinabsenceepilepsywas further confirmed by a study performed on another Chinesepopulation wherefiveexonicvariations(P314S,N345N,P492S,L602LandS619S) and nine rare intronic variations were identified. While it was predictedthatexonicvariationswouldchangeeitherthe transcrip-tion binding, or splicing sites,or the secondary structure of the channel,acommonvariation(rs2745150)inintron11wasfoundto be highly associated with CAE, and was suggested to alter the potentialsplicingsite.56LateronLiangetal.carriedoutanassociation analysison218ChineseCAEpatientsandbothacommonvariation (rs9934839)inexon9ofCACNA1Handacommonhaplotypecovering thegenewerefoundtobesignificantlyassociatedbybotha case-controlstudyandatransmissiondisequilibriumtest.57

The CACNA1H gene was also screened in 192 Chinese IGE patientswhiletheresearcherslookedforacommonsusceptibility allele.58_{Thefour variantsfoundin patients werealsofoundin}

some of the patient’s unaffected family members, indicating a polygenicinheritanceofIGEs.Thesevariantswerealsoassessedfor functionalstudiesand showntohaveincreasedT-typecurrents with one exception; the A480T variant which did not lead to differentcurrentwhencomparedtothewildtype.59_Considering

the alternative splice variants, this variant may affect the regulationof transcription,causing theexpressionof a channel which is more prone to excitation. However, evaluation of CACNA1HinpopulationsofCaucasianoriginrevealednolinkage ormutationinthisgene.60_{Thesecontradictoryresultsemphasize}

the presence of population specific susceptibility alleles in complexdisorders.

In a polygenic rat model of absence epilepsy (GAERS), a mutation was found in exon 24 causing Arg158Pro.61 Further studies showed that CACNA1H had two splice variants in the thalamus,onewithexon25andonewithout.Themutationcaused significantlyquickerrecoveryfromchannelinactivationandlarger Ca2+_{influxesduringhigh-frequencybursts,butonlywhenitwas}

on thevariantwithexon 25. Therefore,themutations and the splicedvariantshouldbeconsideredtogetherwhenstudyingthe functionof thechannel.These splicevariants couldprovidean explanationofthemechanismofepilepticseizuresinrelationto thefollowing questions:whyare theytemporal,have celltype specificeffects,andwhyareseizurespresentincertainagegroups, butnotbeforeorafterthatperiod?

The CACNA1I gene is located on the 22q13.1 and mainly expressedinthethalamic reticularnucleus.49However, whena mutational analysis was done with Chinese CAE patients, no mutationsweredetected.62

Non-poreformingmodulatorysubunitsofCa2+channelsisalso a possiblecandidate for burst firingas it can regulate channel function,assembly,andlocalization.The

b

4subunitcaninteract withboth

a

1A(P/Qtype)and

a

1B(N-type)subunits.Amutation identifiedinthe

b

4subunitgene(CACNAB4)ofanepilepsyanimal model (the lethargic mouse) displayed epileptic seizures and ataxia.63 _{Thismutation caused a truncatedcytoplasmicprotein}

and possibly caused other

b

subunits to coassemble with

a

subunits compensating for the mutation at the hippocampal synapses.44_{However,thethalamus}

_b

_{4subunitisexpressedhighly,}

whereasthe

b

1–

b

3subunitsarenot;suggestingthattheremay not be a compensatory mechanism in the thalamus for the inhibitoryfunctionandthus,andthemutationmayleadtoSWD. Mutations were also identified in the

g

2 subunit gene (CACNAG2)ofanotherepilepsyanimalmodel(stargazermouse).64

ThissubunithasthepotentialtointeractwithbothHVAandLVA channel types. The

g

2 subunit plays a role in elevating the inactivationofcalciumchannels.65_{Inbothmutantmicetherewas}

anelevationofLVAcurrent,astherewasinthecaseofthetottering andtheleanermutantmicewhichhavemutationsinCACNA1A.44

In humanstudies,the

g

3 subunitgene (CACNG3)locatedon chromosome16p13.1-p12,wasfoundtobeassociatedwithCAEin the European population: confirming the distinct roles of regulatory subunits in channel function, and consequently, in epilepticseizures.66

2.3. Non-ionchannelgenes

Epilepsy hasfor themostpart beenconsidered a channelo-pathy.However,mutationsandsusceptibilityvariationsinnovel non-ionchannelgeneshavealsobeenidentifiedasthepossible causesof thedisease(see Table1). TheMalicenzyme2 (ME2), locatedonchromosome18,codesforthemitochondrialenzyme thatconvertsmalatetopyruvateandisalsoinvolvedinneuronal synthesisoftheneurotransmitterGABA.Inapatientgroupof88 JMEand68JAEwithEGTCS,ME2wasfoundtobeassociatedwith allsubtypesinarecessivemodel.67_{35%ofcaseswerehomozygous}

forthenineSNPsthatcovertheME2geneanditspromoter,while only8%ofcontrolswerehomozygous.

In a familywithhyperinsulinism/hyperammonemia (HI/HA), three children had both photosensitive myoclonic absence epilepsyandmutationsintheglutamatedehydrogenase(Glud1) gene(10q23.3).ThepatientsalsohadlowlevelsofGABAcompared tothecontrolgroup.68

In a mouse model of absence epilepsy, named ‘‘jerky’’, mutationswereidentifiedintheJRKgene.Thehomologof this gene(JRK/JH8)residesinhumanson8q24,whichisacandidate regionbasedon linkagestudies.69_{Further, araremutation was}

identifiedinthecaseofaCAEpatientwhoevolvedtoJME.70_The

proteinofthegenehassimilaritiestoseveralnuclearregulatory proteins, suggesting that it might function as a DNA-binding protein.71

Apolymorphismintheleucine-rich,gliomainactivated4(LGI4) genewasfoundtobeassociatedwithchildhoodabsenceepilepsy; however,thepathologicaleffectofthisvariantwasnotclear.72_This

generesideson19q13.11andhasarecessivemodelofinheritance inabsenceseizures.

Inrecentyears,anovelgene,SLC2A1,hascometobeviewedas oneofthemostprevalentcausativegenesinearly-onsetabsence seizures(thoseoccurringbefore4yearsofage).Thediseaseisnot common and patients may have additional medical conditions suchasmovementdisordersandintellectualimpairment.73_The

generesideson1p34.2,codingfortheGLUT-1glucosetransporter, which carriesglucose acrosstheblood-brainbarrier. Mutations havebeenidentifiedin10%ofexaminedpatientswithearlyonset absenceseizuresintheSulsetal.study,andin12%ofpatientsin theMullenetal.study.74,75Functionalstudiesonthesemutations pointedoutthereducedtransportcapacityofmutantproteins.

Anassociationstudywith205TurkishIAEpatientsrevealeda strongassociationofa commonSNP(rs7588807)intheinhibin alphaprecursorgene (INHA)in 2q36toJAE and/ortoIAEwith GTCS.76 _{The INHA gene has also been screened through DNA}

analysis and three potentially damaging mutations have been identified. Inhibin protein, commonly known as a gonadal glycoprotein,inhibitsthesecretionoffollicle-stimulatinghormone which in turn induces the production of progesterone and estradiol.Thishasalsobeenshowntohaveexpressionindifferent partsofthebrainwithunknownfunction.Themutationsandthe associatedSNPininhibinproteinarepredictedtohaveeitheran indirecteffectonabsenceseizures,asprogesteronewasshownto have enhanced SWD through allopregnanolone, a positive modulator of GABAA receptors,77 or to have a direct effect by

increasingtheexcitabilityofthebrain. 3. Conclusion

Thesetypesofmutationsingenesthatcodeforthesubunitsof GABAandCa2+_{channels,identifiedinbothpatients andanimal}

models,explainthecellularmechanismofabsenceseizuresand thegenerationofSWD.Lossoffunctionmutationsfoundinthe subunitsofGABAAandGABABreceptorsarethereasonfortheloss

of inhibition in the thalamacortical network, while thegain of function mutationsinthe subunitsof LVACa2+channels cause excitatoryactivity,leadingtoasusceptibilitytoseizures. Muta-tionsinCACNA1A,CACNAB4,andCACNAG2alsoreducethefunction oftheHVAchannels,butthisreductionresultsinanincreaseinthe LVAcurrentinthethalamocorticalregionofthebrain. Unfortu-nately,themutationsandsusceptibilityallelesinionchannelsare rareandrestrictedtoafewpatientsorpopulations(seeTable1). However,thisdoessupportthepossibilitythattheonsetofSWDs may vary among individuals with similar or different absence syndromes,showingthecomplexityofthisdisease.Thus,negative resultsinonegene,inonepopulation,shouldnotmarkthegeneas ‘‘notacandidate’’forabsenceseizures,ashasbeenthecasewith many of the causativemutationsidentified in CACNA1H in the Chinese population which appeared tocontradict the negative resultsofstudiesinvolvingCaucasianpatients.

A recent study examining the exonic variations of 237 ion channel subunitgenesinboth healthy individualsandpatients withidiopathicepilepsies(IE)confirmsthehighgenetic heteroge-neityandcomplexpathogeneityinIE.78_{Theresultsofthisstudy}

suggestthata singlevariant,evenifitseffectwasshowntobe pathogenicbyfunctionalstudies,maynotberesponsibleforthe disease, becausemissense mutationsin ionchannelshavebeen identifiedinbothstudygroups.Instead,eachindividualmayhave

O¨.Yalc¸ın/Seizure21(2012)79–86 84

a unique channotype (ion channel sequence variation profile) variantpatternwhichdeterminesthenetwork’sexcitabilityand thepossibilityofpossessing theepilepticphenotype.Therefore, mutationanalysisincomplexdiseaseslikeepilepsyshouldnotbe restricted to single ion channel studies; instead, using next generation sequencing methods, such as exome sequencing, wouldbeamoreefficientwaytorevealanindividual’schannotype andthepossibilityofriskforthedisease.

Inlargefamiliesofepilepsywherethereisamajorgene,the genotype-phenotyperelationsareclearerasthemutationsinthe disease-related gene in other independent large families of epilepsyconfirmtheinvolvementofthegeneinthepathogenesis ofthedisease.However,inthecomplexsubtypesofepilepsy,like absence epilepsies, it is difficult to establish the link between genotype and phenotype, because the susceptibility variant identified by genetic studies may have little effect in the pathogenesisofthedisorder.Thus,functionalstudiesareessential tovalidatethemutationsinthestudyofcomplexdisorders.79For example,intheDNAanalysisofCACNA1GintheSinghetal.study,a possible pathological mutation (A570V) was identified. The locationofthemutationin thechanneland theabsenceof the mutationin360controlsamplesstronglysupporttheargumentfor thepathologicalroleofthegene.However,functionalanalysisof themutated proteinrevealed an insignificant difference in the channelfunctionwhencomparedtothewild-typeprotein.

Besides the known function of the ion channel genes in membrane excitability, in recent years novel non-ion channel mutationshavebeguntoemergeinabsenceepilepsies.Somecould beexplained through themechanism of SWD by low levelsof GABAinpatients;however,someofthemarestillinneedoffurther functionalstudiesinordertoverifytheirrolesinabsenceseizures. InothersubtypesofIGEs,novelapproachessuchasdeletion/ duplication analysis and next generation sequencing methods havealreadyrevealednovelcausativevariations.80_{Inthefollowing}

years,inordertoclarifythecomplexpathogenesisoftheabsence seizures,thesearchforthemutationsandsusceptibilityalleleswill bereinforcedbystudiesinCNV,CGHanalysis,exomesequencing, andepigeneticmodificationstudies.

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