ARTICLE OPEN ACCESS

Clinical and molecular characterization ofKCNT1-related severe early-onset epilepsyAmyMcTagueMBChBUmeshNair BScHons SonyMalhotra PhD EstherMeyer PhDNatalie Trump PhD

Elena V Gazina PhD Apostolos Papandreou MBBS Adeline Ngoh MBBS Sally Ackermann MBChB

Gautam Ambegaonkar MBBS MRCPCH Richard Appleton MA(oxon) Archana Desurkar MBBS

Christin Eltze MBBS MPhil Rachel Kneen BMedSci BMBS FRCPCH Ajith V Kumar MD MRCP

Karine Lascelles MBBS MRCPCH Tara Montgomery BMBS BMedSci FRCP Venkateswaran Ramesh MBBS

Rajib Samanta MBBS FRCPCH Richard H Scott PhD Jeen Tan MBChB MRCPCH

William Whitehouse MBBS MRCP Annapurna Poduri MD MPH Ingrid E Scheffer MBBS PhD

WK ldquoKlingrdquo Chong FRCR J Helen Cross MBChB PhD Maya Topf PhD Steven Petrou PhD

and Manju A Kurian MRCPCH PhD

Neurologyreg 201890e55-66 doi101212WNL0000000000004762

Correspondence

Dr McTague

amctagueuclacuk

or Dr Kurian

manjukurianuclacuk

AbstractObjectiveTo characterize the phenotypic spectrum molecular genetic findings and functional con-sequences of pathogenic variants in early-onset KCNT1 epilepsy

MethodsWe identified a cohort of 31 patients with epilepsy of infancy with migrating focal seizures(EIMFS) and screened for variants in KCNT1 using direct Sanger sequencing a multiple-genenext-generation sequencing panel and whole-exome sequencing Additional patients with non-EIMFS early-onset epilepsy in whomwe identifiedKCNT1 variants on local diagnostic multiplegene panel testing were also included When possible we performed homology modeling topredict the putative effects of variants on protein structure and function We undertook elec-trophysiologic assessment of mutant KCNT1 channels in a xenopus oocyte model system

ResultsWe identified pathogenic variants in KCNT1 in 12 patients 4 of which are novel Most variantsoccurred de novo Ten patients had a clinical diagnosis of EIMFS and the other 2 presentedwith early-onset severe nocturnal frontal lobe seizures Three patients had a trial of quinidinewith good clinical response in 1 patient Computational modeling analysis implicates abnormalpore function (F346L) and impaired tetramer formation (F502V) as putative disease mecha-nisms All evaluated KCNT1 variants resulted in marked gain of function with significantlyincreased channel amplitude and variable blockade by quinidine

ConclusionsGain-of-function KCNT1 pathogenic variants cause a spectrum of severe focal epilepsies withonset in early infancy Currently genotype-phenotype correlations are unclear although clinical

These authors contributed equally to this work

From Molecular Neurosciences (AM EM A AN MAK) Developmental Neurosciences UCL Great Ormond Street Institute of Child Health Department of Neurology (AM A AN CE JHC MAK) and Neuroradiology (WKC) Great Ormond Street Hospital for Children London UK Florey Institute of Neuroscience and Mental Health (UN EVG IES SP) Melbourne Australia Department of Biological Sciences (SM MT) Institute of Structural and Molecular Biology Birkbeck College University of London Regional MolecularGenetics Laboratory (NT RHS) North East Thames Regional Genetics Service and Department of Clinical Genetics (AVK RHS) Great Ormond Street Hospital London UKDepartment of Paediatric Neurology (SA) Red Cross War Memorial ChildrenrsquosHospital Cape Town South Africa Department of Paediatric Neurology (GA) AddenbrookersquosHospitalCambridge Roald Dahl EEG Unit (RA) Department of Neurology and Department of Neurology (RK) Alder Hey Childrenrsquos Hospital Liverpool Department of Paediatric Neurology(AD) Sheffield Childrenrsquos Hospital Clinical Neurosciences (CE JHC) Developmental Neurosciences UCL Great Ormond Street Institute of Child Health London Institute ofInfection and Global Health (RK) University of Liverpool Department of Paediatric Neurology (KL) Evelina Childrenrsquos Hospital Guys and St ThomasrsquoNHS Foundation Trust LondonDepartment of Clinical Genetics (TM) Northern Genetics Service Department of Pediatric Neurology (VR) Great North Childrenrsquos Hospital Newcastle Upon Tyne Department ofPaediatric Neurology (RS) University Hospital Leicester Childrenrsquos Hospital Department of Paediatric Neurology (JT) Royal Manchester Childrenrsquos Hospital Department ofPaediatric Neurology (WW) Nottingham University Hospitals NHS Trust UK Epilepsy Genetics Program (A Poduri) Department of Neurology Boston Childrenrsquos Hospital De-partment of Neurology (A Poduri) Harvard Medical School Boston MA University of Melbourne (IES) Austin Health and Royal Childrenrsquos Hospital Australia and Department ofMedicine (SP) Royal Melbourne Hospital University of Melbourne Australia Dr Malhotra is currently at the Department of Biochemistry University of Cambridge UK

Go to NeurologyorgN for full disclosures Funding information and disclosures deemed relevant by the authors if any are provided at the end of the article

The Article Processing Charge was funded by The Wellcome Trust Medical Research Council

This is an open access article distributed under the terms of the Creative Commons Attribution License 40 (CC BY) which permits unrestricted use distribution and reproduction in anymedium provided the original work is properly cited

Copyright copy 2017 The Author(s) Published by Wolters Kluwer Health Inc on behalf of the American Academy of Neurology e55

outcome is poor for the majority of cases Further elucidation of disease mechanisms may facilitatethe development of targeted treatments much needed for this pharmacoresistant genetic epilepsy

GlossaryADNFLE = autosomal dominant nocturnal frontal lobe epilepsy EIMFS = epilepsy of infancy with migrating focal seizuresEOEE = early-onset epileptic encephalopathy NFLE = nocturnal frontal lobe epilepsy RCK = regulator of potassiumconductance SLACK = sequence like a calcium-dependent potassium channel WT = wild-type

Autosomal dominant pathogenic variants in KCNT1 encod-ing the sodium-activated potassium channel are identified ina wide spectrum of epileptic disorders with variable age atonset and cognitive outcome These include severeearly-onset epileptic encephalopathies such as Ohtaharaand West syndromes12 and epilepsy of infancy withmigrating focal seizures (EIMFS)3ndash14 as well as auto-somal dominant and sporadic severe nocturnal frontallobe epilepsies (ADNFLE and NFLE)101516 but thegenotype-phenotype relationship appears to be unclearWe undertook detailed clinical molecular genetic andfunctional characterization of a cohort of patients withKCNT1-related epilepsy

MethodsPatient recruitmentWe recruited patients with EIMFS (n = 31) to a research studyinvestigating the genetic basis of early-onset epileptic en-cephalopathy (EOEE) between 2011 and 2016 followingan earlier national surveillance study4 Inclusion criteria wereepilepsy with onset at lt2 years and unknown etiologyDiagnostic criteria for EIMFS were as described in the pre-vious study4 Patients were recruited at Great Ormond StreetHospital London UK and by referral from other centers inthe United Kingdom and internationally Two patientswho had routine local diagnostic multiple gene panel testingrevealing KCNT1 variants were also included

Standard protocol approvals registrationsand patient consentsWe obtained written informed consent from families inwhom research genetic investigations were undertaken Thestudy was approved by the National Research Ethics Service(London-Bloomsbury Research Ethics Committee reference13LO0168 Integrated Research Application System pro-ject identifier 95005) We collected anonymized data frompatients tested on the diagnostic next-generation sequencingpanel (n = 3) as part of an approved case note review project(Great Ormond Street Hospital Research and DevelopmentDepartment 16NM11)

Genetic testingWe used a variety of different methods (table e-1 httplinkslwwcomWNLA6) including direct Sanger sequencing

multiple gene panel testing with the TruSeq Custom Ampli-con panel and SureSelect panel exome sequencing (e-Methods httplinkslwwcomWNLA8 tables e-1 and e-2httplinkslwwcomWNLA6) and diagnostic chromo-somal microarray

Homology modelingHMMscan17 against Pfam (database of sequence-based do-main families)18 identified 2 domains in the sequenceof human KCNT1 (isoform 1) ion channel (PF07885 atposition 278ndash346) and calcium-activated BK potassiumchannel alpha-subunit family (PF03493 at position495ndash598) (e-Methods)

Electrophysiologic assessment of mutantKCNT1 in xenopus oocyte modelWe introduced variants into a wild-type (WT) humanKCNT1 expression construct19 using QuikChange Light-ning Site-Directed Mutagenesis Kit (Agilent TechnologiesSanta Clara CA) cDNAs were transcribed in vitro(mMessage mMachine Ambion Austin TX) Oocyteswere prepared and 2-electrode voltage clamp recordingwas performed after 14 to 24 hours of expression We alsorecorded currents before and after the application ofQuinidine (e-Methods)

ResultsClinical and molecular genetic features ofKCNT1 mutationndashpositive patientsClinical presentationWe identified pathogenic variants in KCNT1 in 12 patients 5through direct Sanger sequencing 2 from whole-exome se-quencing and 5 from the Great Ormond Street Hospital di-agnostic panel (5 of 800 tested patients with EOEEdevelopmental delay)

Clinical features are summarized in table 1 Median age atseizure onset was 35 weeks (range 1 dayndash6 months) Mostpatients developed seizures consistent with EIMFS Twopatients (patients 3 and 11) presented with severe early-onsetNFLE characterized by asymmetric tonic posturing and laterfencing posture We noted similar frontal seizure semiology inpatients with EIMFS (eg patient 12) All patients developedaxial hypotonia and upper motor neuron signs emerged in 3

e56 Neurology | Volume 90 Number 1 | January 2 2018 NeurologyorgN

Table 1 Clinical and genetic features for 12 patients with KCNT1 mutations

Patient

CDSproteinchange Inheritance

Dxmethods ECS

Ageatonset Initial seizure type

Subsequentseizure types

Autonomicfeatures

MD (age atonset)

Additionalfeatures

Bestdevelopmentalstage attained

Best responseto treatment

Previous functionalvalidation

1a c811GgtTV271F

Unknown(parentalDNA notavailable)

WES SS EIMFS 2 wk HV eye jerking oralautomatisms FM upperlimbs

Asymmetric tonicposturing MyGTCS

Facialflushing

mdash mdash No developmentalmilestonesachieved

None Gain of function428

2 c820CgtAL274I

De novo SS NGSP EIMFS 1 d HV ED and jerking TSupper limbs

FM seizures offace and arm

mdash mdash mdash No developmentalmilestonesachieved

None(includingquinidine)

No

3 c862GgtAG288S

De novo NGSP NFLE 6 mo Predominantly nocturnalasymmetric tonicposturing

ED chokingnoises fencingposture briefgeneralized TSand GTCS

Facialflushing

mdash Right-sidedneglectincreased toneon right sideperipheralhyperreflexia inlower limbs

Grasps objects andstanding briefly at25 y vocalizing andbabbling

None resultedin seizurefreedom

Gain offunction58ndash1013262833

4 c1038CgtGF346L

De novo SS EIMFS 7 wk Exaggerated startle reflexwarm water clonicMyevolved to HV and EDtonic posturing upperlimbs FM all limbs

Rapid alternatingED facialgrimacing leadingto airwayobstruction

Droolingsalivationapneas

HK MDaffectingupper limbs(18 mo)

Coarse facialfeatures gumhypertrophy

Normaldevelopment until10 wk thenregression with lossof social smile andhead control

None No

5 c1504TgtGF502V

Maternalinheritancelikelysomaticmosaicism

SS EIMFS 3 mo Behavioural arreststaring upward eyerolling HV and ED to eitherside asymmetric tonicposturing and elevation oflimbs

Flexor spasmsinvolving thetrunk clonicseizures of limbseyelid twitchinggelastic seizures

HK MDdisorderinvolvinghead and alllimbs (18mo)

Cleft of hardpalate

Early social smileand visualinteraction lostafter onset ofepilepsy

KD withvigabatrineffect later lostQuinidine-markedreduction inseizures

No

6 c2687TgtAM896K

De novo NGSP EIMFS 2 wk Brief FM all limbs(twitching)

HV dystonicposturing upperlimbs ED andjerking

Facialflushingnoisybreathing

HKmovementperioralmusclestonguehand andwrists (2 y)

Systemicproliferativevasculopathy ofpulmonary andmediastinalvessels

No developmentalmilestonesachieved

None(includingquinidine)

No

7 c2849GgtAR950Q

Paternallyinherited

SS EIMFS 5 mo HV TS TS gelasticseizures

mdash HK MD (14mo)

mdash Normal untilseizure onset(smile and headcontrol) regressionat 5 mo with nofurtherdevelopment

None No9

Continued

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Table 1 Clinical and genetic features for 12 patients with KCNT1 mutations (continued)

Patient

CDSproteinchange Inheritance

Dxmethods ECS

Ageatonset Initial seizure type

Subsequentseizure types

Autonomicfeatures

MD (age atonset)

Additionalfeatures

Bestdevelopmentalstage attained

Best responseto treatment

Previous functionalvalidation

8 c2800GgtAA934T

De novo SS EIMFS 4 wk HV ED fisting of hands Asymmetric tonicposturing ED oral9 automatisms

Facialflushing

Limbdystoniaand severescoliosis (18mo)

Peripheralhypertonia

Babbling somedegree of headcontrol

Steroids andKD incombinationat 7ndash12 mo

Gain offunction348920

9a c2800GgtAA934T

De novo WES EIMFS 2 wk HV ED vocalization T10S withadversivecomponent

Facialflushingpupillarydilation

mdash Gastrointestinaldysmotility

Partial head controlsmiling

Nitrazepam at5 mo

10 c2800GgtAA934T

De novo NGSP EIMFS 3 wk HV ED with pupil jerkingFM arm and face oralautomatisms

HV ED droolingTS with adversivecomponent FMupper limbs

Facialflushing

mdash mdash Smiling visualawareness somehead control rolling

Stiripentollevetiracetamandclonazepam incombination

11 c2800GgtAA934T

De novo NGSP NFLE 8 wk Focal motor seizureshands ED and jerking TSupper limb and FMcontralateral lower limbSeizures only in sleepStopped at 4ndash5 mo

From 11 mo TSwith fist clenchingED asymmetrictonic posturingmainly from sleep

mdash mdash Right-sidedweakness withperipheralhyperreflexia

Walking beforeregression at 11mobest subsequentstage sittingindependently

No sustainedresponse

12 c2800GgtAA934T

De novo NGSP EIMFS 3 wk ED with eye flickering HVTS upper limbs

FM upper andlower limbs withlip smackinghand fisting HVand ED fencingposture of arm

Facialflushing

mdash mdash Social smile andreaching for objectsuntil regression andloss of these skills at5 mo

KD andlacosamide incombination

Abbreviations CDS = coding sequence Dx = diagnostic ECS = electroclinical syndrome ED = eye deviation EIMFS = epilepsy of infancy with migrating focal seizures FM = focal motor GTCS = generalized tonic-clonic seizuresHK = hyperkinetic HV = head version KD = ketogenic diet MD =movement disorder My =myoclonic seizures NFLE = nocturnal frontal lobe epilepsy NGSP = next-generation sequencing panel SS = Sanger sequencing TS =tonic seizuresa Previously described by McTague et al4

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patients Four patients had a choreiform movement disorder(onset 14ndash24 months) 1 patient developed generalized dys-tonia at 18 months Onset of hyperkinesia was not related tomedication (including vigabatrin) nor triggered by in-tercurrent illness Initial age at presentation disease courseresponse to medication brain MRI and EEG findings weresimilar for both KCNT1 pathogenic variantndashpositive and ndashnegative patients from the cohort However 5 of 12 patho-genic variantndashpositive patients with EIFMS presented witha severe movement disorder compared to 2 of 19 KCNT1-negative cases Most had extensive uninformative laboratorymetabolic and genetic investigations Abnormal muscle re-spiratory chain enzyme activity for complex I andor II wasdetected in patients 4 and 8 of uncertain significance (table e-3 httplinkslwwcomWNLA6) For patient 4 the musclebiopsy was taken during an intercurrent illness and repeatedafter clinical recovery revealing a more borderline result Inpatient 8 borderline abnormalities in complex I and II ratioswere found Neither patient had other systemic biochemicalor radiologic features of mitochondrial disease or concurrentsodium valproate treatment

In general neurodevelopmental outcome was markedlyimpaired in all patients with EIMFS All patients had a trial ofat least 5 different medications Response to treatment wasin general poor (table 1) Three of 8 patients who receivedthe ketogenic diet in combination with other antiepilepticdrugs responded with asymp75 seizure reduction Threepatients were treated with quinidine Patient 2 received 40mgkgd without adverse events but with no effect on sei-zure burden Patient 5 was treated with quinidine at 40 mgkgd leading to a marked reduction in seizure frequencyPatient 6 showed some initial transient reduction in seizurefrequency at 30 mgkgd For this patient the unexpecteddevelopment of a severe proliferative pulmonary and medi-astinal vasculopathy resulted in life-threatening pulmonaryhemorrhage Investigations failed to identify an underlyingvasculitis and quinidine was subsequently withdrawn Thepatient later died despite initial successful pulmonaryembolization

EEG featuresAll patients with an EIMFS phenotype had a ldquomigratingrdquo ictalfocus with discrete ictal involvement of differing cortical areaswithin the same EEG (table e-4 httplinkslwwcomWNLA6) Although not always evident at initial presentation itdeveloped by 7 months of age in most patients Periods ofEEG suppression or burst suppression were noted in 8 of 12patients 6 of these patients had seizure onset in the first 4weeks of life Further atypical EEG features included a gener-alized electrodecremental response in 5 patients and hypsar-rhythmia in 1 patient

Radiologic featuresNeuroimaging was available for review in 11 of 12 patients Themajority developed predominantly frontal cerebral atrophy by 3years of age (figures e-1A and e-1B httplinkslwwcom

WNLA7 table e-5 httplinkslwwcomWNLA6) Cere-bellar atrophy was also evident in 4 patients (figure e-1C)We noted an open operculum in the first 6 months of life inpatient 4 (figure e-1B) Delayed myelination was evident in9 of 11 patients who had imaging after 3 months of age Insome patients early brain imaging was normal Magneticresonance spectroscopy was abnormal with a relativelyreduced N-acetylcholine peak in 3 of 4 patients

Molecular genetic findingsWe identified 12 patients with pathogenic variants in KCNT1(table 1 and table e-6 httplinkslwwcomWNLA6) 8have been previously reported and 4 are unpublished4591016

Eight of 12 patients had C-terminus variants of whom 5 hadthe commonly reported variant A934T We have identified 4(including 2 unpublished) pathogenic variants causingEIMFS namely V271F L274I G288S and F346L located inor between transmembranes 5 and 6 All are missense variantsthat are predicted to be pathogenic (table e-6) affecting highlyconserved amino acid residues (figure e-2 httplinkslwwcomWNLA7) and are not reported in 1000 Genomes theExAC database or the Exome Variant Server20ndash24 For 9 of 12cases variants occurred de novo Parental DNA was notavailable for patient 1 In patient 5 we found the sameKCNT1variant in an asymptomatic mother and her affected child Wenoted a lower heterozygous peak on Sanger sequencing ofboth salivary and blood-derived maternal genomic DNA(figure e-3) which may reflect somatic mosaicism In patient7 the variant was inherited from the unaffected father with nodifference in peak size on Sanger sequencing (figure e-4) Therecurrent A934T variant was identified in 5 patients 4 with anEIMFSpresentation and 1 (patient 11)with anNFLEphenotype

Protein homology modeling of mutant KCNT1Homology modeling was performed for 2 novel mutationsF346L located in the ion channel domain (residues270ndash353) and F502V located in the gating region (residues373ndash1174 although residues 1045ndash1174 could not bemodeled) F346 is located on the inner helix of the trans-membrane pore (figure 1 A and B) It is part of the hydro-phobic cavity which mediates interactions between the inner-membrane helices of 2 adjacent subunits (figure 1C) and is thusresponsible formaintaining the stability of the open conformationIn the modeled closed-state conformation the helix containingF346 and the inner helix from the other protomer undergoconformational changes (figure e-5 httplinkslwwcomWNLA7) Therefore mutation to leucine (F346L) is likely to de-stabilize the open state by perturbing the hydrophobic inter-actions because the side chain of leucine is smaller (figure 1D)affecting the equilibrium between the closed and open states Inaddition the packing arrangement in the K+ channels involvingthe pore and the inner helix is known to be critical for the stabilityof the tetrameric assembly ion conduction function and cationselectivity Thus F346Lmight be detrimental to these functions25

Within each protomer of the KCNT1 gating region there are2 tandem RCK domains (RCK1 and RCK2) that serve as

NeurologyorgN Neurology | Volume 90 Number 1 | January 2 2018 e59

Figure 1 Modeling the ion channel and gating apparatus of KCNT1

(A) Side view of the homologymodel of the KCNT1 ion channel (residues 278ndash346) as a tetramer F346 is present on the edge of the inner helix (in gold) and interactswith the inner helix of the adjacent subunit in the tetrameric arrangement Membrane position is shown in spheres (B) Top view of the tetramer arrangement of theion channel and location of F346 on the inner helix (C) F346 is part of the hydrophobic cavity (shown as surface) which mediates interactions between the innermembranehelices of the 2 subunits F346 is shown in green the surrounding hydrophobic residues are shown in red (D)Onmutation to leucine (F346L in green) thehydrophobic interactions between the2 subunits are likely tobe reduced (black circle) because the side chain of leucine ismuch shorter thanphenylalanine (E)Modelof a dimer of the gating ring (residues 373ndash1044 residues 1045ndash1174 could not be modeled) which is a tetramer (dimer of the modeled dimer) Each subunitpossesses 2 RCK domains RCK1 (in blue) and RCK2 (in gold) F502 (in green) is present in the RCK1 domain near the intersubunit interface (assembly interface) TheRCK1-RCK2 intrasubunit interface ispurple (residues fromRCK1)andorange (residues fromRCK2) Thedimer interfaces formedbybothRCK-1andRCK-2are indicatedby anarrow (F) F502 (green) and its neighboring hydrophobic residues (red) includingW476withwhich it could potentially formapi-pi interaction Distance betweenthe centroid (spheres) of the 2 rings (F502andW476) is 47 A and theanglebetween the ringplanes is 273deg (G) F502V could abolish the formationof thepotential pi-piinteraction with W476 and is likely to reduce the hydrophobic interactions (black circle) because the side chain of valine is smaller than that of phenylalanine

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regulators of potassium conductance (figure 1E) These formflexible intrasubunit and intersubunit (figure 1E) interfacesthat facilitate functional tetramer formation26 F502 is locatedin RCK1 and predicted to form a pi-pi interaction with W476from αD (figure 1F) F502 is also surrounded by a number ofhydrophobic residues (I472 L473 A475 V500 and A503)which may play a role in stabilizing the gating ring (figure 1F)The amino acid substitution F502V is predicted to result indestabilization of these hydrophobic interactions given thesmaller valine side chain (figure 1G) and abolition of po-tential pi-stacking with resultant disruption of the stable as-sembly interface

Electrophysiologic assessment ofmutant KCNT1We evaluated the 4 previously unpublished variants andV271F which we previously described4 and was recentlystudied in a xenopus oocyte system27 All mutations resulted inan increased current magnitude compared toWT (figure 2A)We noted that for variants V271F and F346L the rate ofactivation was slowed at higher voltages compared toWT andin others (M896K F502V and L274I) the activation rateswere generally faster thanWT (figure 2A) Investigation of thecurrent-voltage relationship showed that mutant channelswere very weakly voltage dependent and in some casesvoltage dependence of steady-state activation was essentiallyabsent (figure 2 B and C) with only a residual Goldman-Hodgkin-Katz rectification Assessment of average peak cur-rents at 10 mV revealed a significant difference between bothindividual mutant channels and summated data compared toWT (figure 2 D and E)

Effect of 300 μmolL quinidine onmutant KCNT1Quinidine 300 μmolL had variable current-blocking effects indifferent mutant channels For F346L peak current wascompletely insensitive to quinidine although it had some ef-fect on activation kinetics (figure 3A) The differential sensi-tivity of KCNT1 mutants to quinidine was clearly shown inthe current-voltage relationship (figure 3B) and percentage ofinhibition at maximum current 80 mV (figure 3C) There issome correlation between the in vitro studies and clinicalresponse in patient 5 (figure 3) M896K had the most markedin vitro blockade by quinidine and patient 6 showed someinitial clinical response F346L showed no quinidine responseat all and the patient harboring this mutation was not treatedwith quinidine

DiscussionWe report a cohort of patients with early-onset epilepsy as-sociated with pathogenic variants in KCNT1 which encodesthe sodium-activated potassium channel KCa41 (sequencelike a calcium-dependent potassium channel [SLACK]Slo22) KCNT1 is widely expressed throughout the brain aswell as in the dorsal root ganglia kidney and heart and isresponsible for slow hyperpolarization after bursts of action

potentials2829 KCNT1 also has direct interactions withFragile X-related protein29 Compared with other potassiumchannels KCNT1 is involved in a highly extensive proteinnetwork suggesting a putative role in cognitive developmentalprocesses382830

To date KCNT1 variants have been reported in a wide rangeof epilepsies (table e-7 httplinkslwwcomWNLA6)1ndash163132 We identified patients with the same variant as-sociated with varying electroclinical phenotypes (table 1)Phenotypic variability has been reported within single familiesin which different individuals may present with eitherADNFLE or EIMFS10 Such intrafamilial variation in pheno-type is also described in SCN1A kindreds Dravet syndromefebrile seizures and a variety of other generalized epilepsiesmay be reported in the same family33 Furthermore whilethe majority of variants in our cohort occurred de novo 2patients inherited variants from an unaffected parent Themechanisms underlying phenotypic variability and trueapparentnonpenetrance are unclear but may be related to somatic mosa-icism variant type other geneticepigenetic factors or differentialexpression of alternative KCNT1 transcripts910293435

The majority of patients with pathogenic KCNT1 variants inour cohort had electroclinical EIFMS although this is likely toreflect ascertainment bias Indeed 2 of the 5 KCNT1-positivepatients identified by the diagnostic panel from a larger cohortof 800 patients with EOEEdevelopmental delay had anNFLE-like presentation Although movement disorders areincreasingly reported in other severe early-onset geneticepilepsies they appear to be rare in KCNT1 epilepsy36 Wedescribe several atypical EEG features Generalized electro-decrement and hypsarrhythmia more classically associatedwith infantile spasms have been previously described inEIMFS2491031 EEG suppression classically seen in Ohtaharasyndrome37 has been only rarely described in EIMFS49 Ex-tensive diagnostic investigations undertaken in patients withKCNT1 mutations were unyielding other than abnormal re-spiratory chain enzyme analysis of muscle tissue in 2 patientsThe relevance of these findings is not clear but secondarymitochondrial effects may be evident in KCNT1 epilepsy asoften reported in other severe drug-resistant epilepsies38

Other genetic and environmental influences onmitochondrialfunction may also play a role

KCNT1 tetramers form a transmembrane sodium-activatedpotassium channel Each subunit consists of 6 transmembranedomains with an extended cytoplasmic carboxy (C-) terminus(figure 4) Themajority of reported pathogenic variants (tablee-7 httplinkslwwcomWNLA6) as seen in this studyare located in the C-terminus with clustering around the RCKand nicotinamide adenine dinucleotidendashbinding domains(figure 4) More recently several variants have been identifiedwithin transmembrane domain 5 and in the pore-formingregions between transmembrane domains 4 and 5458ndash10 (tablee-7) and this study also demonstrates epilepsy-associatedmutations in transmembrane domains

NeurologyorgN Neurology | Volume 90 Number 1 | January 2 2018 e61

To date different model systems have been used to determinethe functional effects ofKCNT1 variants15710131927 Our proteinhomology structural modeling data predict abnormal gating orprotein instability within the pore-forming region as a putativedisease mechanism In silico modeling of G288S has predictedsimilar detrimental effects5 while Y775H is predicted to affectsodium sensitivity of the channel27 KCNT1 variants maytherefore alter structural properties of the protein contributingto altered channel function Consistent with previousreports13131927 (table e-7 httplinkslwwcomWNLA6) ourxenopus oocyte model demonstrated that KCNT1 pathogenicvariants display a gain-of-function effect with increasedcurrent amplitude (figure 2) Previous studies have sought tocorrelate disease severity with the degree of gain of

function119 However in keeping with more recent studies8

such correlation was not evident in our study KCNT1 variantsresult in an increased Po (probability of the channel beingopen) which may be due to increased mutant channel coop-erativity or altered sodium sensitivity827 In a recent study so-dium removal from the pipette solution had a less negativeeffect on G288S channel amplitude than WT suggestingreduced sodium sensitivity in the mutant13 Heterotetramerformation may be of importance in vivo In 1 study mutantKCNT1 homomers revealed a more marked gain of functionthan mutant WT heteromers13 A significant remainingquestion is howKCNT1 gain-of-function variants with predictedeffects on neuronal hyperpolarization result in epilepsy28

Altered voltage sensitivity may result in KCNT1 channels

Figure 2 Functional investigation of KCNT1 mutations in a xenopus oocyte model

(A) Representative current traces obtained from oocytes expressing WT and EIMFS mutants (M896K F502V V271F F346L and L274I) Oocytes were held atminus90 mV and stepped from minus80 to 80 mV for 600 milliseconds every 5 seconds Scale bars apply to all traces (B) Current-voltage relationships for WT (n = 32)M896K (n = 15) F502V (n = 13) V271F (n = 9) F346L (n = 11) and L274I (n = 12) Currents were averaged and then normalized to the value at a test potential of80 mV (Imax) (C) Comparison of current-voltage relationships between WT (solid circles n = 32) and EIMFS mutations (M896K [squares n = 15] F502V[triangles n = 13] V271F [hexagons n = 9] F346L [diamonds n = 11] and L274I [inverted triangles n = 12]) Currents were averaged and then normalized tothe value at a test potential of 80mV (Imax) (D) Average peak currents at 10 mV for WT (n = 44) M896K (n = 19) F502V (n = 16) V271F (n = 10) F346L (n = 11)and L274I (n = 12) channels Peak currents for eachmutant channel at 10mVwere compared to the peak currents for theWT channel at 10mV p lt 0001p lt 00001 (E) Comparison of pooled WT (n = 44) and EIMFS (n = 68) currents at 10 mV p lt 00001 EIMFS = epilepsy of infancy with migrating focalseizures WT = wild-type

e62 Neurology | Volume 90 Number 1 | January 2 2018 NeurologyorgN

opening at more depolarized potentials allowing a persistenthyperpolarizing current with resultant interneuronal dis-inhibition as reported in SCN1A-related epilepsy39 Con-versely increased repolarization permitting more frequentand rapid action potentials may also play a role2734

Recently quinidine has been identified as a novel therapyfor patients with KCNT1-related epilepsy In in vitromodels quinidine has been shown to reduce the abnormalincrease in mutant KCNT1 channel amplitude19 For 1patient with EIMFS with the KCNT1 variant R428Q invitro testing showed quinidine sensitivity and treatmentresulted in a dramatic improvement in seizure control withneurodevelopmental gains37 However in more recentstudies patient response has been variable and not alwaysas predicted by in vitro studies11 Indeed another patient

with the same variant (R428Q) but different epilepsyphenotype (unclassified EOEE) failed to respond to quin-idine albeit at a later stage in the disease course14 Mostrecently a patient withWest syndrome had a good responsebut only with a higher dose of 60 mgkgd2 Clinical re-sponse may possibly be determined by the specific variantother genetic factors epilepsy phenotype and drug timingwithin a therapeutic window In our series we treated 3patients with quinidine and 1 patient showed a clinical responseOne patient developed a severe pulmonary vasculopathy afterwhich quinidine was discontinued Systemic vasculitis has beenreported with quinidine treatment40 While investigations in ourpatient did not reveal overt evidence of vasculitis the observedpulmonary dysfunction may represent an adverse drug-relatedevent The precisemechanism of KCNT1 blockade by quinidineis unclear and it is possible that the disease mechanism for

Figure 3 Effect of quinidine on xenopus oocytes expressing hKCNT1 channels

(A) Representative current traces obtained from oocytes expressing WT and EIMFS mutants (M896K and F346L) with application of vehicle (ND96) and 300μmolL quinidine Oocyteswere held atminus90mVand stepped from minus80 to 80mV for 600milliseconds every 5 seconds Scale bars apply to all traces (B) Current-voltage relationships forWT (n = 32) M896K (n = 15) F502V (n = 13) V271F (n = 9) F346L (n = 11) and L274I (n = 12) hKCNT1 channels in the presence of vehicle(ND96) and 300μmolL quinidine Currentswere averagedand then normalized to the value at a test potential of 80mV (Imax) (C) Average percent inhibition at80 mV of WT (n = 31) and EIMFS (M896K n = 15 F502V n = 13 V271F n = 9 F346L n = 11 and L274I n = 12) hKCNT1 channels by quinidine (300 μmolL)depicting the variable degree of block by 300μmolL quinidine (1-way analysis of variance followed by Bonferroni post hoc analysis) p lt 01 EIMFS = epilepsyof infancy with migrating focal seizures WT = wild-type

NeurologyorgN Neurology | Volume 90 Number 1 | January 2 2018 e63

F346L perhaps involving abnormal channel-opening dynamicsas suggested by themodeling data is notmodifiable by quinidineOur data suggest that quinidine should be considered as a ther-apeutic option for patients with KCNT1 variants but used withcaution Larger studies will provide further guidance about clin-ical utility patient selection optimum age at administration anddose Other KCNT1 modulators including bepridil and clofili-lum have been identified as possible alternative therapies28 Likequinidine bepridil has been shown in vitro to reversibly blockmutant KCNT1 channels at a lower concentration than WTchannels13 However similar to quinidine potential cardiaceffects and lack of specificity may limit use in patients

Pathogenic variants in KCNT1 cause a wide spectrum of se-vere epilepsies typically associated with impaired neurologic

development and significant disease burden As demonstratedin vitromodel systemsmay be useful to validate putative variantsand to confirm pathogenicity although genotype-phenotypecorrelations remain unclear Evaluation of new therapies in-cluding KCNT1-specific blockers remains a research priorityfor this devastating pharmacoresistant group of epilepsies

Note added in proofRecently 3 patients with de novo KCNT1 mutations andmassive systemic to pulmonary collateral artery formationpresenting with pulmonary hemorrhage requiring emboliza-tion were described These patients had not been treated withquinidine However the mechanism remains unclear andfurther investigation of the expression and role of KCNT1 inthe cardiovascular system is required41

Figure 4 Schematic diagram of the location of mutations in KCNT1 in this and previously published studies

KCNT1 encodes sequence like a calcium-dependent potassium channel (SLACK) which forms tetramers (top left) or heteromers with KCNT2 or sequence like anintermediate conductance K channel (SLICK) The structure comprises 6 transmembrane domains with a pore-forming region regulator of potassium conductance(RCK) and nicotinamide adenine dinucleotidendashbinding (NAD-B) domains EIMFS phenotypes are shaded in purple ADNFLE or NFLE in pink others (Ohtaharasyndrome leukoencephalopathy focal epilepsy EOEEWest syndromeunaffected) inorangeMutationsgiving rise togt1phenotypeareshadedwitha combinationofthe corresponding colors Novel mutations identified in this study are outlined in green those identified in previous studies in turquoise ADNFLE = autosomaldominant nocturnal frontal lobe epilepsy EIMFS = epilepsy of infancy with migrating focal seizures EOEE = early-onset epileptic encephalopathy NFLE = nocturnalfrontal lobe epilepsy

e64 Neurology | Volume 90 Number 1 | January 2 2018 NeurologyorgN

Author contributionsAmy McTague Umesh Nair Sony Malhotra and Esther Meyerhave contributed to draftingrevising themanuscript for contentincluding medical writing for content study concept or designacquisition of data analysis or interpretation of data NatalieTrump has contributed to draftingrevising the manuscript forcontent including medical writing for content acquisition ofdata analysis or interpretationof data ElenaVGazina ApostolosPapandreou and Adeline Ngoh have contributed to draftingrevising the manuscript for content acquisition of data analysisor interpretation of data Sally Ackermann and Gautam Ambe-gaonkar have contributed to draftingrevising the manuscript forcontent acquisition of data Richard Appleton has contributed todraftingrevising the manuscript for content including medicalwriting for content acquisition of data analysis or interpretationof data Archana Desurkar has contributed to draftingrevisingthemanuscript for content acquisition of data Christin Eltze andRachel Kneen have contributed to draftingrevising the manu-script for content including medical writing for content acqui-sition of data analysis or interpretation of data Ajith V Kumarand Karine Lascelles have contributed to draftingrevising themanuscript for content acquisition of data Tara Montgomeryhas contributed to draftingrevising the manuscript for contentacquisition of data analysis or interpretation of data Ven-kateswaran Ramesh and Rajib Samanta have contributed todraftingrevising the manuscript for content acquisition of dataRichard H Scott has contributed to draftingrevising the man-uscript for content acquisition of data analysis or interpretationof data Jeen Tan has contributed to draftingrevising the man-uscript for content acquisition of data William Whitehouse andAnnapurna Poduri have contributed to draftingrevising themanuscript for content including medical writing for contentacquisition of data Ingrid E SchefferWK ldquoKlingrdquoChong and JHelen Cross have contributed to draftingrevising the manu-script for content including medical writing for content acqui-sition of data analysis or interpretation of data Maya TopfSteven Petrou and Manju A Kurian have contributed todraftingrevising the manuscript for content including medicalwriting for content study concept or design acquisition of dataanalysis or interpretation of data

AcknowledgmentThe authors thank the patients and their families for theirparticipation in this study They thank all clinicians referringpatients with EIMFS for inclusion in the research study (DrMike Pike Dr Stefan Spinty Dr Ailsa McLellan Dr MaryKing Dr Andrew Curran Dr Hans Randby Dr Linda deMeirleir Dr Jost Richter Dr Elaine King DrMartin PiepkornDr Mary OrsquoRegan Dr Ariane Biebl Dr Gary McCullagh andDr Siobhan West) They thank Erin Heinzen PharmD PhDfor whole-exome sequencing of 5 patients at the Duke Centerfor Genomic Medicine Durham NC

Study fundingThis project was supported by the National Institute forHealth Research Biomedical Research Centre at Great

Ormond Street Hospital for Children NHS Foundation Trustand University College London

DisclosureA McTague was funded by a Medical Research CouncilClinical Research Training Fellowship (MRL0014971) theMedical Research Foundation for travel and conference fees(MRF-007-0003-STD-MCTAG) a Grass Foundation TravelBursary from the American Epilepsy Society and a JuniorInvestigator travel scholarship from the University of SouthCalifornia U Nair and S Malhotra report no disclosuresrelevant to the manuscript E Meyer is funded by SPARKSN Trump reports no disclosures relevant to the manuscriptEV Gazina is funded by the Australian Research Council(APP1106027) A Papandreou is funded by a joint ActionMedical Research and British Paediatric Neurology Associa-tion research training fellowship A Ngoh is funded byGuarantors of Brain Friends of Landau Kleffner Syndromeand Action Medical Research S Ackermann and G Ambe-gaonkar report no disclosures relevant to the manuscript RAppleton served on the scientific committees for and receiveda single honorarium fromNovartis and GWPharma and is theChief Investigator for the EcLiPSE Study funded by an Na-tional Institute for Health Research Health Technology As-sessment grant (12127134) A Desurkar C Eltze RKneen AV Kumar K Lascelles T Montgomery V RameshR Samanta and RH Scott report no disclosures relevant tothe manuscript J Tan received an industry-funded bursary forconference travel (pound300) from UCB Pharma W Whitehouseserves on the editorial board of NeuroEducation A Poduri ison the Scientific Advisory Board of the Dravet SyndromeFoundation serves as an associate editor for Epilepsia andcontributing editor for Epilepsy Currents and is on the editorialboard for Annals of Neurology She receives research supportfrom the National Institute of Neurological Disorders andStroke (NINDS) Citizens United for Research in Epilepsyand the Boston Childrenrsquos Hospital Translational ResearchProgram IE Scheffer serves on the editorial boards of Neu-rology and Epileptic Disorders may accrue future revenue ona pending patent WO61010176 Therapeutic Compoundthat relates to discovery of PCDH19 gene is one of theinventors listed on a patent held by Bionomics Inc on di-agnostic testing of using the SCN1A gene WO2006133508has received speaker honoraria or scientific board consultingfees from Athena Diagnostics UCB GSK Eisai and Trans-genomics has received funding for travel from Athena Diag-nostics UCB Eisai and GSK and has received researchsupport from the National Health and Medical ResearchCouncil of Australia NINDS Health Research Council ofNew Zealand US Department of Defense and March ofDimes WK Chong reports no disclosures relevant to themanuscript JH Cross is on the advisory boards of ShireGSK Eisai Vitaflo UCB and Takeda for which remunerationhas been made to her department has a patent in applicationwith Vitaflo and is the chief investigator for clinical trials withVitaflo GWPharma and Zogenix for which remuneration hasbeen made to the department M Topf reports no disclosures

NeurologyorgN Neurology | Volume 90 Number 1 | January 2 2018 e65

relevant to the manuscript S Petrou is scientific founder ofPraxis Precision Medicine Boston and a Scientific AdvisoryBoard Member of Pairnomix Minneapolis and has receivedshares for both Dr Petroursquos research is supported by the LuluFoundation VitafloNestle Citizens United for Research inEpilepsyMallinckrodt Pharmaceuticals Clarus Ventures DHBFoundation Australian Research Council National Health andMedical Research Council and SCN2A Research FoundationMA Kurian receives researchgrant support from a WellcomeTrust Intermediate Clinical Fellowship (WT098524MA) Goto NeurologyorgN for full disclosures

Received November 7 2016 Accepted in final form September 262017

References1 Martin HC Kim GE Pagnamenta AT et al Clinical whole-genome sequencing in

severe early-onset epilepsy reveals new genes and improves molecular diagnosis HumMol Genet 2014233200ndash3211

2 Fukuoka M Kuki I Kawawaki H et al Quinidine therapy for West syndrome withKCNT1 mutation a case report Brain Dev 20173980ndash83

3 Barcia G Fleming MR Deligniere A et al De novo gain-of-function KCNT1 channelmutations cause malignant migrating partial seizures of infancy Nat Genet 2012441255ndash1259

4 McTague A Appleton R Avula S et al Migrating partial seizures of infancy ex-pansion of the electroclinical radiological and pathological disease spectrum Brain20131361578ndash1591

5 Ishii A Shioda M Okumura A et al A recurrent KCNT1 mutation in two sporadiccases with malignant migrating partial seizures in infancy Gene 2013531467ndash471

6 Vanderver A Simons C Schmidt JL et al Identification of a novel de novo pPhe932Ile KCNT1 mutation in a patient with leukoencephalopathy and severe epi-lepsy Pediatr Neurol 201450112ndash114

7 BeardenD StrongA Ehnot J DiGiovineMDlugosDGoldberg EMTargeted treatmentof migrating partial seizures of infancy with quinidine Ann Neurol 201476457ndash461

8 KimGE Kronengold J Barcia G et al Human slack potassium channel mutations increasepositive cooperativity between individual channels Cell Rep 201491661ndash1672

9 Ohba C Kato M Takahashi N et al De novo KCNT1 mutations in early-onsetepileptic encephalopathy Epilepsia 201556e121ndashe128

10 Moslashller RS Heron SE Larsen LHG et al Mutations in KCNT1 cause a spectrum offocal epilepsies Epilepsia 201556e114ndashe120

11 Mikati MA Jiang Y-H Carboni M et al Quinidine in the treatment of KCNT1positive epilepsies Ann Neurol 201578995ndash999

12 Allen NM Conroy J Shahwan A et al Unexplained early onset epileptic encepha-lopathy exome screening and phenotype expansion Epilepsia 201557e12ndashe17

13 Rizzo F Ambrosino P Guacci A et al Characterization of two de novo KCNT1mutations in children with malignant migrating partial seizures in infancy Mol CellNeurosci 20167254ndash63

14 Chong PF Nakamura R Saitsu H Matsumoto N Kira R Ineffective quinidine therapyin early-onset epileptic encephalopathy with KCNT1 mutation Ann Neurol 201679502ndash503

15 Heron SE Smith KR Bahlo M et al Missense mutations in the sodium-gated po-tassium channel gene KCNT1 cause severe autosomal dominant nocturnal frontallobe epilepsy Nat Genet 2012441188ndash1190

16 Hildebrand MS Myers CT Carvill GL et al A targeted resequencing gene panel forfocal epilepsy Neurology 2016861605ndash1612

17 Eddy SR Accelerated profile HMM searches PLoS Comput Biol 20117e100219518 Finn RD Mistry J Tate J et al The Pfam protein families database Nucleic Acids Res

200938D211ndashD22219 Milligan CJ Li M Gazina EV et al KCNT1 gain of function in 2 epilepsy phenotypes

is reversed by quinidine Ann Neurol 201475581ndash59020 1000 Genomes Available at httpbrowser1000genomesorgindexhtml Accessed

June 14 201621 ExAC Browser (beta)|Exome Aggregation Consortium Available at httpexac

broadinstituteorg Accessed June 14 201622 NHLBI Exome Sequencing Project (ESP) Available at httpevsgswashington

eduEVS Accessed June 14 201623 PolyPhen-2 Available at httpgeneticsbwhharvardedupph2 Accessed June 14

201624 Available at httpproveanjcviorggenome_submit_2php Accessed June 14

201625 Doyle DA Morais Cabral J Pfuetzner RA et al The structure of the potassium

channel molecular basis of K+ conduction and selectivity Science 199828069ndash77

26 Yuan P Leonetti MD Pico AR Hsiung Y MacKinnon R Structure of the humanBK channel Ca2+-activation apparatus at 30 A resolution Science 2010329182ndash186

27 Tang Q-Y Zhang F-F Xu J et al Epilepsy-related slack channel mutants lead tochannel over-activity by two different mechanisms Cell Rep 201614129ndash139

28 Kaczmarek LK Slack slick and sodium-activated potassium channels ISRNNeurosci20132013354262

29 Brown MR Kronengold J Gazula V-R et al Amino-termini isoforms of the slack K+channel regulated by alternative promoters differentially modulate rhythmic firingand adaptation J Physiol 20085865161ndash5179

30 Kim GE Kaczmarek LK Emerging role of the KCNT1 slack channel in intellectualdisability Front Cell Neurosci 20148209

31 Allen AS Berkovic SF Cossette P et al De novo mutations in epileptic encephalo-pathies Nature 2013501 217ndash221

32 Arai-Ichinoi N Uematsu M Sato R et al Genetic heterogeneity in 26 infants witha hypomyelinating leukodystrophy Hum Genet 201513589ndash98

33 Scheffer IE Zhang Y-HH Jansen FE Dibbens L Dravet syndrome or genetic(generalized) epilepsy with febrile seizures plus Brain Dev 200931394ndash400

34 Lim CX Ricos MG Dibbens LM Heron SE KCNT1 mutations in seizuredisorders the phenotypic spectrum and functional effects J Med Genet 201653217ndash225

35 Chen H Kronengold J Yan Y et al The N-terminal domain of slack determines theformation and trafficking of slickslack heteromeric sodium-activated potassiumchannels J Neurosci 2009295654ndash5665

36 Howell KB McMahon JM Carvill GL et al SCN2A encephalopathy a major cause ofepilepsy of infancy with migrating focal seizures Neurology 201585958ndash966

37 Ohtahara S A study on the age dependent epileptic encephalopathy No To Hattatsu197792ndash21

38 Folbergrova J Kunz WS Mitochondrial dysfunction in epilepsy Mitochondrion20121235ndash40

39 Yu FH Mantegazza M Westenbroek RE et al Reduced sodium current inGABAergic interneurons in a mouse model of severe myoclonic epilepsy in infancyNat Neurosci 200691142ndash1149

40 Lipsker DWalther S Schulz R Nave S Cribier B Life-threatening vasculitis related toquinidine occurring in a healthy volunteer during a clinical trial Eur J Clin Pharmacol199854815

41 Kawasaki Y Kuki I Ehara E et al Three cases of KCNT1 mutations malignantmigrating partial seizures in infancy with massive systemic to pulmonary collateralarteries J Pediatr Epub 2017 October 5

e66 Neurology | Volume 90 Number 1 | January 2 2018 NeurologyorgN

SOURCE ARTICLE NPuborg22ubfy

Clinical and molecular characterization ofKCNT1-related severe early-onset epilepsyAmy McTague MBChB Umesh Nair BSc Hons Sony Malhotra PhD Esther Meyer PhD Natalie Trump PhD

Elena V Gazina PhD Apostolos Papandreou MBBS Adeline Ngoh MBBS Sally Ackermann MBChB

Gautam Ambegaonkar MBBS MRCPCH Richard Appleton MA(Oxon) Archana Desurkar MBBS

Christin Eltze MBBS MPhil Rachel Kneen BMedSci BMBS FRCPCH Ajith V Kumar MD MRCP

Karine Lascelles MBBS MRCPCH TaraMontgomery BM BS BMedSci FRCP Venkateswaran RameshMBBS

Rajib SamantaMBBS FRCPCH RichardH Scott PhD Jeen TanMBChBMRCPCHWilliamWhitehouseMBBS

MRCP Annapurna Poduri MD MPH Ingrid E Scheffer MBBS PhD W K ldquoKlingrdquo Chong FRCR

J Helen Cross MBChB PhD Maya Topf PhD Steven Petrou PhD and Manju A Kurian MRCPCH PhD

Neurologyreg 20189024 doi101212WNL0000000000004762

Correspondence

Dr McTague

amctagueuclacuk or

Dr Kurian

manjukurianuclacuk

Study questionWhat are the phenotypic molecular genetic and functional char-acteristics of KCNT1 variants that cause early-onset epilepsy

Summary answerGain-of-function KCNT1 mutations can cause diverse severefocal epilepsies with onset in early infancy but genotype-phenotype relationships remain unclear

What is known and what this paper addsAutosomal dominant pathogenic variants of the sodium-activatedpotassium channel gene KCNT1 are associated with a broadspectrum of epileptic disorders The study explores the clinicalmolecular genetic and functional properties of a cohort ofpatients with KCNT1-related epilepsy

Participants and settingThirty-one patients with epilepsy of infancy with migrating focalseizures (EIMFS) were recruited from 2011 to 2016 Twopatientswhohad undergone routine genetic testing that includedKCNT1 analysis were also included The patients wererecruited at Londonrsquos Great Ormond Street Hospital and byreferrals from UK and international centers

Design size and durationThe patientsrsquo KCNT1 variants were identified via multiplemethods and homologymodeling was performed ThemutantKCNT1 variants were electrophysiologically assessed ina Xenopus oocyte model

Main results and the role of chancePathogenic KCNT1 mutations were detected in 12 patientsThese patients exhibited diverse symptoms The ages at onsetranged from 1 day to 6 months and all had neurodevelopmentalimpairments Treatment outcomes were generally poor ThepathogenicKCNT1 variants included 8 previously reported and4 unreported variants Homology modeling for 2 unreportedvariants indicated that they would induce abnormal gating orprotein instability Electrophysiologic assessments of 5

variants including the 4 unreported ones revealed abnormalchannel functions including increased current magnitudesrelative to the wild-type (WT) channel and variable blockadeby quinidine in keeping with the clinical response

Bias confounding and other reasonsfor cautionFocusing on patients with EIMFS may have introduced as-certainment bias

Generalizability to other populationsPathogenic KCNT1 mutations have been detected in patientswith epileptic conditions other than EIMFS including noc-turnal frontal lobe epilepsy Such mutations may have differenteffects in other conditions

Study fundingpotential competing interestsThis study was funded by the UK National Health Service andUniversity College London Some authors report receiving fund-ing personal compensation and advisory committee appoint-ments from various companies journals scholarly societies andgovernment agencies Go to NeurologyorgN for fulldisclosures

A draft of the short-form article was written byM Dalefield a writer with Editage a division of Cactus Communications The authors of the full-length article and the journal editors edited and approved the final version

24 Copyright copy 2017 American Academy of Neurology

SHORT-FORM ARTICLE

DOI 101212WNL0000000000004762201890e55-e66 Published Online before print December 1 2017Neurology

Amy McTague Umesh Nair Sony Malhotra et al -related severe early-onset epilepsyKCNT1Clinical and molecular characterization of

This information is current as of December 1 2017

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ISSN 0028-3878 Online ISSN 1526-632XWolters Kluwer Health Inc on behalf of the American Academy of Neurology All rights reserved Print1951 it is now a weekly with 48 issues per year Copyright Copyright copy 2017 The Author(s) Published by

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