Severe neonatal seizures: From molecular diagnosis to precision therapy? §
M. Milh, P. Cacciagli, C. Ravix, C. Badens, A. Le´pine, N. Villeneuve, L. Villard
Aix-MarseilleUniversite´,Inserm,Servicedeneurologiepe´diatrique,GMGFUMR_S910,264,rueSaint-Pierre, 13385Marseille,France
1. Introduction
Early epileptic encephalopathies (EEE)are aheterogeneous groupofdiseasescharacterizedbytheassociationofsevere epilepsyandpermanentabnormalneurologicalexamination, as part of a developmental disorder. These are serious diseases, with severe prognosis in terms of neurological disability.Giventheearlyandimmediatelyseverenatureof epilepsy, it is very difficult to determine whether the developmental disorder is secondary to seizure activity (seizuresand interictalactivities)ortogenetic abnormality byitself(mutationinanionchannel,forexample). So,the termofepilepticencephalopathyisprobablyusedbyexcess, atleastinsomecases[1].Currently,thevastmajorityofEEEis incurable.TheAEDhasagreaterorlessereffectonseizure frequency, but do not change drastically the long-term
prognosis,apartfromrareexceptions.ThecausesoftheEEE areheterogeneous,but mostofthem areprobablygenetic, withanMRInotpresentinganystructuralabnormalitiesthat canexplainit.Currently,severaldozenofgeneswereinvolved intheEEE[2].Herewewilldescribethephenotypeassociated tothemostfrequentlymutatedgenesfoundintheEEE,andwe will question the usefulness of finding a mutation in the therapeuticstrategy.
2. Relative frequency of different EEE genes:
results of a prospective study of 63 patients with a severe epilepsy beginning before 3 months followed in Marseille
InordertoassesstherelativefrequencyofmajorgenesofEEE, wehaveprospectivelystudiedatotalof70patientswithEEE, abstract
Earlyonsetepilepticencephalopathies(EOEE)areheterogeneousgroupofsevereepilepsies thatstillneedtobebetterdefinedandcharacterized.Onageneticpointofview,several dozenofgeneshavebeenassociatedwithEOEE,andtodate,itisdifficulttofindacommon mechanismto explainEOEE.In thisshort review,we showthattwomainsgenes are involvedinEOEE:STXBP1andKCNQ2.FocusingonKCNQ2relatedEOEE,weshowthata relativelysimilarphenotypecanberelatedtovariousconsequencesofmutationsonasingle gene.ThiswillprobablychallengethetreatmentofEOEEpatients.
E-mailaddress:mathieu.milh@ap-hm.fr.
andtestedseveralgenes(Fig.1).Bydefinition,epilepsybegan before3monthsandMRIdidnotfindobviousstructuralcause of this epilepsy. Patients were tested for STXBP1, KCNQ2, KCNQ3,SCN2A,SCN8A,KCNT1,ARX,SLC13A5.Theyallhad CGHarray.
No mutation of these genes has been found in 37/
63patients.ThemostfrequentlymutatedgenewasKCNQ2 (5/70,8%),followedbySCN8A(4/70,6%),STXBP1(3/70,5%), KCNT1(3/70,5%),TBC1D24(3/70,3%).Wefoundonemutation ofARX,SLC13A5,SCN2AandKCNQ3(1/70).In2patients,an abnormalityinCGHallowedtoexplaintheEEE.Fourpatients werecarryingamutation inothergenes.Overall, themost frequentlymutatedgenesfoundinourcohortwereKCNQ2, STXBP1,SCN8AandKCNT1.
ThemajorityofpatientsEEEdidnot,however,carryany abnormalityinthegenesthatwetested.
This study put the light on two major genes of EEE:
STXBP1andKCNQ2(Fig.2).ThekeyfeaturesofSTXBP1and KCNQ2relatedepilepsieswere:
forKCNQ2:very earlyonset(firstweek);out ofhandvery frequent tonic asymmetric seizures with blockpnea and cyanosis,interictalEEGeitherdiscontinuousorshowinga
suppression-burst pattern,no or poorefficacy of Pheno- barbital[3];
forSTXBP1:onsetwithinthefirstthreemonthsoflife,mostly duringthefirstmonth,motorseizuresbutlessfrequentand bettertolerated,oftenfocal.InterictalEEGshowingeithera discontinuity (50 %) or focal spikes [4]. In a specific retrospectivecohortofpatientswithanOhatharasyndrome, therateofmutationofthesetwogeneswashigh(seeabove).
3. Targeted therapy trials
Therecentadvancesingenediscoveryleadtothegenerationof variousdedicatedanimalmodelsaimedtofindpathophysio- logicalhypothesisthatcouldexplaintheepilepsyand/orthe developmentalimpairment.Functionalstudiesmadeatdiffe- rentlevels(cellular,neuronal,network,entireanimalmodel) could also lead to the discovery of potential targets and treatmentsthatcouldcorrectabiologicaleffectofaspecific mutation.Here,wewilldealonlywiththerapiesthathavebeen proposed/developedafterthediscoveryofthegeneinvolvedin epilepsy,aspartofatranslationalresearchprogram.Numerous targeted therapiesfrom clinicalresearchsuchstiripentol in Dravet syndrome, or vitamin B6 in the mutation of the Antiquitinareexcludedfromthispresentation.
Wewilldiscussthreerecentexamplesoftargetedtherapy, data from research on animals models: RETIGABINE and KCNQ2mutations;QUINIDINEandmutationsofKCNT1and ARXandCDKL5.
3.1. RetigabineandKCNQ2mutations
Retigabine acts as anactivator ofneuron-expressed KCNQ- channels,therebyreducingneuronalexcitability.Patientswith a KCNQ2 mediatedepileptic encephalopathy may especially benefitfromthistargetedtherapy,asrapidcontrolofseizures could potentially improve developmental outcome. Specific clinicalstudiesarestillneededtotestthishypothesis.Electro- physiological analysis on xenopus oocytesor on CHO cells showed that the majority of KCNQ2 mutations lead to a measurableeffectonpotassiumcurrent,atthesubthreshold levelatwhichthesechannelsareknowntocriticallyinfluence neuronalfiring,eitherbygloballyreducingcurrentamplitudes orbyadepolarizingshiftoftheactivationcurve.Inthesecases, Applicationofretigabinepartiallyreversedtheseeffectsforthe majorityofanalyzedmutations[5].However,somemutationsof KCNQ2donotaltersignificantlythechannelpropertiesinterm ofcurrentintensity,buteithertheexpressionofthechannel,or evenitslocalizationwithintheneuronalmembrane[6].These alterationsareobviouslynotcorrectedbyretigabine.Moreover, some gain of function mutations have been described in patientswithasimilarphenotype[7].Patientscarrying such mutationscouldevenbeaggravatedbyretigabine.
3.2. EstradiolandARXmutations
ARXisatranscriptionfactorselectivelyexpressedinneuronal precursorsandadultinhibitoryinterneurons.Severalstudies have shown that somemutations impaired cell migration.
ARXrelatedepilepsyisoftensevereandofearlyonset.The
KCNQ2 STXBP1 SCN8A KCNT1 SCN2A TBC1D24 ARX SLC13A5 KCNQ3 aCGH Other causes Unknown
Fig.1–Geneticanalysisof70patientswithEEE.
KCNQ2 STXBP1 Other
Fig.2–RelativeproportionofKCNQ2and
STXBP1mutationsinacohortof60patientswithan Ohtaharasyndrome.SixtypatientswithanOhathara syndromeweretestedforKCNQ2andSTXBP1.Thecohort wasbuiltwithpatients’DNA,comingfromseveralcenters ofFrance,withadiagnosisofOhtaharaSyndrome.
most common seizure type related to ARX mutation is Epilepticspasms.Usingananimalmodel(micemutatedfor ARX), Olivetti et al. showed that a brief estradiol (E2) administrationduringearlypostnataldevelopmentprevented spasms in infancy and seizures in adult mutants. E2 was ineffectivewhendeliveredafterpubertyor30daysafterbirth.
They showed that early E2 treatment restored the pool of GABAergicinterneurons.Thus,thispreclinicalstudyshowed forthefirsttimethatanearlytherapeuticalinterventionmay correct or improve a developmental disorder due to an interneuropathy[8].
3.3. QuinidineandKCNT1mutations
Mutations in KCNT1 have been implicated in autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) and epilepsy of infancy with migrating focal seizures (EIMFS).
Using non-neuronal cells with transfection of mutated channels,theyshowedthatKCNT1mutationsimplicatedin epilepsy cause a marked increase in function and that exposure to quinidine significantly reduces this gain of functionforallmutationsstudied[9,10].
4. Limits of the precision therapy approach
Thefirstlimitationisthatallthepathophysiologicalconse- quencesofthemutationofaparticulargenearenotknown.
For example, the most severe KCNQ2 mutations alter the functionofthechannel,butalsoitslocationintheneuron[5].
Thelatteralterationisobviouslynotcorrectedbyretigabine.
Moreover,todate,dozensofgeneshavebeenimplicatedin theEEP.Mostfrequentsarefoundmutatedinlessthan20%of cases, certain genes are described only a few cases. The developmentofpersonalizedmedicinewouldrequireforeach gene, creating a reliable animal model, validation of pre- clinicalstudiesandclinicaltrials,whichisnotrealistic.The discoveryofcommonphysiopathologicallanesinEEPthrough fundamentalstudieson themechanismsofepilepsyon an immaturebrain,wouldbeofgreathelp.
Finally,theeffectivenessofaparticularmoleculeistested onitsabilitytodecreaseseizurefrequencyinanimalmodels.
However,theseverityofEEEiscertainlylinkedtoseizures,but notonly.Itisnotimpossiblethatthemechanismsinvolvedin seizureandinthedevelopmentaldisorderarenotthesame.In thiscase,thetargetchosentomeasuretheeffectivenessofa drug(seizurefrequency)wouldnotbegood[11].
Disclosure of interest
Theauthorsdeclarethattheyhavenocompetinginterest.
Acknowledgements
Wewouldliketothankallthepatientsandfamilies.Wealso wanttothankallthecliniciansandgeneticiststhatparticipate tothecollaborativeprojectonEOEE.
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