clinical features and outcome of 6 new patients carrying de ...renzo guerrini, md, frcp...

Carla Marini MD PhDMichele Romoli MDElena Parrini PhDCinzia Costa MD PhDDavide Mei MScFrancesco Mari

MD PhDLucio Parmeggiani MDElena Procopio MDTiziana Metitieri

Psyd PhDElena Cellini PhDSimona Virdograve MScDalila De Vita BScMattia Gentile MD PhDPaolo Prontera MD PhDPaolo Calabresi MDRenzo Guerrini

MD FRCP

Correspondence toDr Guerrinirenzoguerrinimeyerit

Clinical features and outcome of 6 newpatients carrying de novo KCNB1 genemutations

ABSTRACT

Objective To describe electroclinical features and outcome of 6 patients harboring KCNB1mutations

Methods Clinical EEG neuropsychological and brain MRI data analysis Targeted next-generation sequencing of a 95 epilepsy gene panel

Results The mean age at seizure onset was 11 months The mean follow-up of 113 years docu-mented that 4 patients following an infantile phase of frequent seizures became seizure freethe mean age at seizure offset was 425 years Epilepsy phenotypes comprisedWest syndromein 2 patients infantile-onset unspecified generalized epilepsy myoclonic and photosensitiveeyelid myoclonia epilepsy resembling Jeavons syndrome Lennox-Gastaut syndrome and focalepilepsy with prolonged occipital or clonic seizures in each and every one Five patients haddevelopmental delay prior to seizure onset evolving into severe intellectual disability withabsent speech and autistic traits in one and stereotypic hand movements with impulse controldisorder in another The patient with Jeavons syndrome evolved into moderate intellectual dis-ability Mutations were de novo 4 missense and 2 nonsense 5 were novel and 1 resulted fromsomatic mosaicism

Conclusions KCNB1-related manifestations include a spectrum of infantile-onset generalized orfocal seizures whose combination leads to early infantile epileptic encephalopathy including WestLennox-Gastaut and Jeavons syndromes Long-term follow-up highlights that following a stormyphase seizures subside or cease and treatment may be eased or withdrawn Cognitive and motorfunctions are almost always delayed prior to seizure onset and evolve into severe persistent impair-ment Thus KCNB1 mutations are associated with diffuse brain dysfunction combining seizuresmotor and cognitive impairment Neurol Genet 20173e206 doi 101212NXG0000000000000206

GLOSSARYACMG 5 American College of Medical Genetics and Genomics AED 5 antiepileptic drug ASD 5 autism spectrum disorderCSWS 5 continuous spikes and waves during slow-wave sleep EIEE 5 early infantile epileptic encephalopathy ESP 5Exome Sequencing Project ExAC 5 Exome Aggregation Consortium FS 5 febrile seizures ID 5 intellectual disabilityNGS 5 next-generation sequencing TCS 5 tonic-clonic seizures VPA 5 valproic acid

The KCNB1 gene encodes the pore-forming and voltage-sensing b subunit of the voltage-gated potassium (K1) channel subfamily 2 (Kv21) that is expressed throughout the brain1ndash3

and plays essential roles in regulating neuronal excitability contributing to action potentialrepolarization4 and dynamic modulation of neuronal activity5ndash7 De novo heterozygousmissense (number 5 13) and nonsense (number 5 1) mutations of KCNB1 have beenreported in 14 patients with neurodevelopmental disorders including epilepsy of variableseverity in 13 of them8ndash15 Probands carrying KCNB1 mutations resulting in severe epilepsy

From the Pediatric Neurology Unit (CM EP DM FM TM EC SV DDV RG) Neurogenetics and Neurobiology LaboratoriesNeuroscience Department A Meyer Pediatric Hospital University of Florence Neurology Unit (MR CC PC) Department of MedicineUniversity of Perugia Ospedale S Maria della Misericordia Child Neurology Service (LP) Hospital of Bolzano Metabolic Unit (EP) A MeyerPediatric Hospital Florence Medical Genetics Unit (MG) Azienda Sanitaria Locale Bari Neonatology Unit and Prenatal Diagnosis (PP)Medical Genetic Unit Ospedale S Maria della Misericordia Perugia Department of Experimental Neurosciences (PC) ldquoIstituto di Ricovero eCura a Carattere Scientificordquo IRCCS Santa Lucia Foundation Rome and IRCCS Stella Maris Foundation (RG) Calambrone Pisa Italy

Funding information and disclosures are provided at the end of the article Go to Neurologyorgng for full disclosure forms The Article ProcessingCharge was funded by the University of Florence

This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License 40 (CCBY-NC-ND) which permits downloading and sharing the work provided it is properly cited The work cannot be changed in any way or usedcommercially without permission from the journal

Neurologyorgng Copyright copy 2017 The Author(s) Published by Wolters Kluwer Health Inc on behalf of the American Academy of Neurology 1

phenotypes have been classified as epilepticencephalopathyndashearly infantile 26 (OMIMEIEE26 600397) Table 1 summarizes theclinical and genetic features of previouslyreported patients

Here we describe the electroclinical fea-tures and long-term follow-up of 6 novel pa-tients carrying de novo missense andnonsense KCNB1 mutations and provide anoverall view of the spectrum of epilepsy phe-notypes associated with KCNB1 mutationsranging from Lennox-Gastaut syndrome toremitting infantile spasms and to mild gener-alized epilepsy with eyelid myoclonia Oursmall cohort of patients also suggests thatthe KCNB1 EIEE26 manifests infantile-onset seizures with a tendency to attenuateor resolve completely over time in some pa-tients Concomitant impairment of cognitiveand motor functions constant and oftensevere persists over time and is at times com-bined with absent speech autism spectrumdisorder (ASD) and psychiatric problemsMutations in this gene which are not rareconfirm that genes coding for potassiumchannels are important genetic contributorsto epilepsies and more broadly to neurodeve-lopmental disorders

METHODS From a highly heterogenous cohort of 873 pa-

tients with pediatric epilepsies either refractory or benign with

a supposed genetic etiology recruited at the Meyer Childrenrsquos

Hospital or referred from other national or international epilepsy

centers for genetic testing we identified and studied 6 patients

carrying de novo variants of the KCNB1 gene (GenBank Acces-

sion Number NM_0049752) Three patients were examined at

the Neurology unit and 3 were referred for genetic testing to our

Neurogenetic Laboratory Collection and analysis of retrospective

clinical EEG neuropsychological neuroimaging data were per-

formed using a specific format filled by the treating specialist

aiming to obtain accurate and homogenous information We

classified seizure types and epilepsysyndromes according to the

International League Against Epilepsy guidelines1617

Molecular genetic testing Genomic DNA was extracted from

peripheral blood leukocytes using a QIAsymphony SP robot

(Qiagen Hilden Germany) according to the manufacturerrsquos pro-

tocol High-quality DNA was quantified using a QuantiFluor

Fluorometer (Promega Madison WI) Targeted resequencing of

a panel including 95 genes associated with epilepsy was per-

formed in all 6 patients Target enrichment and library prepara-

tion were performed using a custom-designed Nextera Rapid

Capture assay (Illumina San Diego CA) and sequencing was

performed on an Illumina MiSeq (Illumina) with a 2 3 150-bp

paired-end protocol as previously described18 Variants were

annotated and filtered using the ANNOVAR tool19 Variants

localized in intronic regions outside the 10-bp exon-flanking

boundaries and in the 59- and 39-UTR regions were excluded

Variants reported in the Exome Aggregation Consortium (ExAC)

database (exacbroadinstituteorg) andor in the 1000 Genomes

Project (in the National Heart Lung and Blood Institute Exome

Sequencing Project [ESP] ESP6500 database evsgswashington

eduEVS) in the Genome Aggregation Database (gnomAD)

(gnomadbroadinstituteorg) with a minor allele frequency 0

01 (1) were dropped out In silico prediction of mutationsrsquo

pathogenicity was obtained using the dbNSFP database (v33)

which provides functional prediction scores on more than 20

different algorithms (sitesgooglecomsitejpopgendbNSFP)

Putative causative variants were analyzed by Sanger sequencing to

confirm the next-generation sequencing (NGS) results in pro-

bands and investigated in the parents to check the inheritance

status We classified variants according to the international

Table 1 Clinical and genetic summary of previously published patients

Patient Ethnicity Phenotype Genotypes Genetic analysis Inheritance Reference

1 Chinese 2 y TC sz spasm drug resistantEEG GSW 3 y regression

pGly379Val WES De novo 15

3 Caucasian EOEE pPhe416Leu pArg312HispTyr533

NGS gene panel De novo 14

1 Caucasian ID severe speech impairment no epilepsy pVal378Leudeg WES De novo 13

2 Caucasian EOEE pGly381Arg pPhe416Leu WES De novo 12

1 Caucasian EE pVal378Aladeg WES De novo 11

2 Asian DD and severe infantile generalized sz pArg306Cys pGly401Arg WES De novo 10

1 Caucasian EE pGly379Arg^ WES De novo 9

3 Caucasian Sz onset 475 y EE hypotonia DDintermittent agitation

pS347Arg WES De novo 8

Sz onset 8 mo drug-resistant sz DDabsent speech stereotyped handwringing movements

pGly379Arg^ WES De novo

Sz onset first year of life TC atypical absence atonicinfantile spasms and focal dyscognitive

pThr374Ile WES De novo

Abbreviations ^ deg 5 recurrent mutations 5 nonsense mutation DD 5 developmental delay EE 5 epileptic encephalopathy EOEE 5 early-onset epilepticencephalopathy GSW 5 generalized spikes and waves ID 5 intellectual disability mo 5 months NGS 5 next-generation sequencing TC 5 tonic-clonicseizures sz 5 seizures WES 5 whole-exome sequencing y 5 years

2 Neurology Genetics

guidelines of the American College of Medical Genetics and

Genomics (ACMG) Laboratory Practice Committee Working

Group20

Standard protocol approvals registrations and patientconsents Written informed consent to disclose clinical informa-

tion was obtained from all parentsguardians of the participants

Written informed consent for genetic testing was obtained ac-

cording to the Paediatric Ethic Committee of the Tuscany Region

(approval no 2014-0000559) in the context of the DESIRE pro-

ject (grant agreement no 602531)

RESULTS The 6 patients (4 females and 2 males) har-boring KCNB1 mutations had a mean age at the timeof the study of 12 years (range 7ndash22 years) Theyrepresented the 075 (6 of 873) of a highly

heterogeneous cohort of patients with potentiallygenetic epilepsies studied with our target resequencinggene panel18 Table 2 summarizes the main clinicalfeatures of each KCNB1 mutationndashpositive patient

Epilepsy The mean age at seizure onset was 11months (median 9 range 5ndash23 months) Four ofthe 6 patients (666) following an initial phase ofactive epilepsy with frequent seizures became seizurefree at a median age of 425 years the mean durationof seizure remission at the last follow-up was 6 years(range 8 monthsndash16 years)

Seizure types (table 2) included infantile spasms(patients 1 and 2) myoclonic seizures with eyelidmyoclonia (table 2 patient 3) febrile seizures (FS)

Table 2 Clinical and genetic summary of the 6 patients reported harboring KCNB1 mutations

ID 1 2 3 4 5 6

Age ysex 7M 8F 22F 12F 8F 17M

Sz onsetoffset

9 mo18 mo 5 mo8 mo 6 mo10 mo 7 y16 y 18 mo FS 23 mo7 y 12 moongoingyearly sz from 8 y

11 mo FS ongoingdaily sz

Sz type Spasms Spasms Myoclonic eyelid myoclonia andself-induced Ab-eyelid myocloniasleep-related TCS

FS focal occipitalprolonged lateralizedmotor monthlyfrequency

TCS atypical Ab Focal Abnonconvulsive statusepilepticus TC tonic

Epilepsy typeor syndrome

West syndrome West syndrome Myoclonic photosensitivegeneralized (Jeavons syndrome)

Focal symptomatic Infantile onsetgeneralized

Lennox-Gastautsyndrome

EEG

Onset Hypsarrhythmia Hypsarrhythmia GSWmyoclonic jerks Focal F-T spikeincreased in sleep

Bilateral SW R L NA

Current Backgr delayedfocal L spikes

Normal Normal backgr GSW and PSWeyelid myoclonia generalizedphotosensitivity

Multifocaldiffuseabnormalities in sleep attimes resembling CSWS

Slow backgrFC dischargesL R

Slow backgrprolongeddischarges ofbilateral SW attimes subcontinuous

Treatment

Previous ACTH ACTH CBZ VPA TPM LEV LTG ESM CLN VPA LEV pyridoxineSTP BDZ PHT

VPA VPA RFN BDZACTH LEV LTGprimidone CBZTPM ESM

Current mdash mdash VPA 1 ESM 1 CLN LTG VPA 1 TPM VPA 1 RFN 1perampanellorazepam 1neuroleptics

Development Developmentaldelay severe ID

Developmentaldelay severe ID

Borderline development 7 y mild IDwith learning disability motor andverbal dyspraxia 11 y moderate ID16 y moderate ID major impairmentof oculomanual coordination andslowness of execution

Developmental delaysevere ID



Other Hypotonialanguage singlewords

No speech R leghyperchromicskin spotbilateralclinodactyly

Mild hypotonia motor and verbaldyspraxia clumsiness enuresis

Significant sleepproblem absent speechASD

Hypotonianonverbalstereotypic handmovementshyperlaxity ataxiatrichotillomaniapavor nocturnus

Hypotonia languagesingle wordsbehavioral disorder

Mutation c1109GA pTrp370

c1747CT pArg583

c916CT pArg306Cys c586AT pIle196Phe c629CT pThr210Met

c1045GT pVal349Phe (mosaic)

Inheritance De novo De novo De novo De novo De novo De novo

Abbreviations Ab 5 absence seizures ACTH 5 adrenocorticotropic hormone ASD 5 autism spectrum disorder backgr 5 background BDZ 5 benzodi-azepine CBZ 5 carbamazepine CLN 5 clonazepam CSWS continuous spikes and waves during slow-wave sleep ESM 5 ethosuximide F 5 frontal FC 5

frontocentral FS 5 febrile seizures GSW 5 generalized spikes and waves ID 5 intellectual disability L 5 left LEV 5 levetiracetam LTG 5 lamotrigine NA5 not available PHT 5 phenytoin PSW 5 polyspike-wave discharges R 5 right RFN 5 rufinamide STP 5 stiripentol SW 5 spike wave sz 5 seizures T 5

temporal TCS 5 tonic-clonic seizures TPM 5 topiramate VPA 5 valproic acid

Neurology Genetics 3

followed by focal seizures (patients 4 and 5) absencesand nonconvulsive status epilepticus (patient 6) andafebrile tonic-clonic seizures (TCS) followed by othergeneralized seizure types (patients 5 and 6) For eachpatient we considered age of symptom appearanceand age at the time of the study and calculated a meanfollow-up of 113 years when last seen (median 85years range 6ndash21 years)

Two patients with infantile spasms (patients 1and 2) classified as West syndrome due to their hyp-sarrhythmic EEG and co-occurring developmentaldelay exhibited an identical phenotype and outcomeBoth patients were immediately treated with adreno-corticotropic hormone (ACTH) after the onset ofspasms with a prompt remission of spasms andEEG improvement After 6ndash7 years of follow-upboth patients with ldquoremitting infantile spasmsrdquoremain seizure free and off antiepileptic drugs(AEDs) Yet their development was delayed and cur-rently sets on severe impairment of cognitive andmotor functions including absent speech

The remaining 4 patients manifested different andheterogeneous phenotypes Patient 3 (table 2) showedan interesting and peculiar phenotype consisting of

myoclonic seizures and absences with eyelid myoclo-nia with persistent generalized photosensitivity Herepilepsy started at 6 months with bilateral myoclonicjerks and was very active for 3 months when treat-ment with valproic acid (VPA) prompted a prolongedyet temporary resolution of symptoms Indeed at age7 years during primary school age myoclonicseizures and absences with eyelid myoclonia withphotosensitivity were observed (figure 1A) Episodesof absences with eyelid myoclonia occurred fre-quently on eye closure or were autoinduced like seenin Jeavons syndrome21 Two generalized TCS duringsleep were also reported around age 15 years Thepatient was trialed with several AEDs but only a com-bination of VPA ethosuximide and clonazepam waseffective at age 16 years At the time of the study thispatient was 22 years old and is still seizure free onAEDs Patients 4 and 5 (table 2) exhibited infantile-onset multifocal and generalized seizures preceded byglobal developmental delay Then patient 4 mani-fested monthly seizures from age 2 to 5 years whenseizures ceased she is currently 12 still on AEDs andseizure free Despite 5 years of seizure freedom hercognitive and motor functions are yet severely

Figure 1 Representative EEG findings in 3 patients

(A) Ictal videopolygraphic EEG recording of patient 3 (table 2) showing discharges of diffuse polyspike waves concomitant with myoclonic jerks evident onthe leads recording from both deltoid muscles (B) Ictal videopolygraphic EEG recording of patient 3 (table 2) showing generalized paroxysmal activityevoked by eye closure with concomitant eyelid myoclonia (video available as supplementary material) (C) Interictal EEG recording of patient 5 showingbifrontal single complexes of spikes and waves with left predominance (D) Interictal sleep EEG recording of patient 4 showing very frequent paroxysmalactivity over the fronto-centro-temporal regions (continuous spikes and waves during the slow-wave sleep pattern)


impaired with absent speech and autistic traitsPatient 5 has ongoing yearly seizures and severecognitive impairment The last patient (patient 6)exhibited the most refractory epilepsy with infantile-onset FS followed by focal seizures frequent absencesnonconvulsive status tonic and TCS configuringa phenotype resembling Lennox-Gastaut syndromeHe is currently 17 and has daily atypical absences andsleep-related TCS Concomitant with such refractoryepilepsy he manifested early developmental delayevolving into moderate to severe intellectual disability(ID) with a behavioral disorder requiring antipsy-chotic drugs

EEG recordings All patients underwent repeatedEEG recordings from seizure onset to current age Ac-cording to their epilepsy phenotype at onset 2 pa-tients presented a severely abnormal EEG withhypsarrhythmia that overtime evolved into a normalEEG in 1 (patient 2) and slow background activitywith rare left temporal spikes in the second patient(patient 1) Video-EEG polygraphic recordings ofthe patient with Jeavons syndrome (patient 3)showed at onset and during follow-up generalizedspike- and polyspike-wave discharges with a prom-inent generalized photoparoxysmal response (figure1B) Several episodes of myoclonia and absences witheyelid myoclonia were recorded (figure 1 A and B)Patient 4 (table 2) with infantile-onset multifocalepilepsy consistently showed slow background withmultifocal or diffuse paroxysmal activity in sleep andsemiperiodic posterior fast activity runs on awaken-ing Some EEG recordings in this patient showed inearly childhood a relevant increase in paroxysmalactivity during sleep resembling the continuous spikeand wave during the slow-wave sleep (CSWS) EEGpattern (figure 1C) The EEG recordings in patient 5(table 2) exhibited slow background activity andintermittent bilateral frontocentral spike and wavedischarges with left predominance (figure 1B) EEGrecordings of patient 6 showed slow background andfrequent at times almost continuous multifocal anddiffuse spike and wave discharges

Cognition and behavior Five of the 6 patients(table 2 patients 1 2 and 4ndash6) had clear-cut devel-opmental delay prior to seizure onset involving bothmotor and cognitive functions Their cognitive out-come evolved into severe impairment with absentspeech and autistic traits in 1 (table 2 patient 4) Inaddition one of them (table 2 patient 5) manifestedstereotypic hand movements and impulse controldisorder consisting of trichotillomania and patient 6(table 2) developed a psychiatric disorder requiringtreatment with neuroleptics

The girl with Jeavons syndrome instead had a de-layed early development (sitting 8 months ambula-tion 15 months and language 2 years) evolving in

childhood into mild cognitive impairment withmotor and verbal dyspraxia and poor coordinationDespite such a cognitive profile she attended primaryschool unassisted While during secondary school sherequired a special needs teacher and further neuro-psychological testing documented moderate ID(Wechsler Intelligence Scale for ChildrenmdashRevisedfull-scale IQ 5 45) Yet she was able to reachsecondary school graduation

Five of the 6 patients acquired independent walk-ing with a variable degree of hypotonia hyperlaxityand mild ataxia in 3 One patient was using a wheel-chair (patient 1)

MRI All patients underwent normal brain MRI

Genetic Heterozygous KCNB1 mutations occurredde novo (table 2 figure 2) and were classified as path-ogenic according to the ACMG guidelines Bothpatients 1 and 2 carried a novel de novo nonsensemutation including the c1109GA [p(Trp370)]variant that is located in the pore domain of the pro-tein and the c1747CT [p(Arg583)] variantwhich falls in the C-terminal domain Patient 3 har-bored the c916CT [p(Arg306Cys)] missense var-iant which is located in the S4 segment of thevoltage-sensor domain of the protein This varianthad previously been reported in a patient withdevelopmental delay and infantile-onset seizures10

Patient 4 carried the novel c586AT [p(Ile196-Phe)] missense variant located in the S1 segment ofthe protein and patient 5 carried the novelc629CT [p(Thr210Met)] missense variantlocated in the first extracellular domain between S1and S2 Patient 6 carried the novel c1045GT[p(Val349Phe)] missense variant located in the S5segment of the pore both NGS and Sangersequencing data showed evidence of somatic mosai-cism (figure e-1 httplinkslwwcomNXGA1)None of the KCNB1 variants were reported in thepublic available allele frequency databases (table e-1httplinkslwwcomNXGA0) and the toolsincluded in the dbNSFP comprising the MetaSVMand MetaLR ensemble scores predicted the 4 mis-sense variants to be damaging (table e-1 httplinkslwwcomNXGA0)

DISCUSSION We describe the electroclinical fea-tures and outcome of 6 patients harboring de novoheterozygous KCNB1 mutations confirming thatmutations in this gene are not rare Indeed in ourhighly heterogenous cohort of pediatric epilepsieswith a supposed genetic etiology nearly 1 of pa-tients carried mutations in this gene

Genetic studies from the ldquooldrdquo linkage analysesstudies to the modern era of whole-exome sequenc-ingwhole-genome sequencing techniques have dem-onstrated that heterozygous mutations in several


genes coding for different K1 channels includingKNCQ2 KCNQ3 KCNA2 KCNA1 KCNC1KCNH1 KCNMA1 HCN2 and KCNT1 areinvolved in epileptic disorders whose severity variesfrom benign forms to early infantile epileptic enceph-alopathy (EIEE)22ndash32 The list of K1 channel genesimplicated in epilepsy includes KCNB1 that was in2014 linked to the EIEE268 At present there are 14patients on record carrying mutations in this geneand 13 of them exhibit epilepsy8ndash15

All patients including the 6 reported here presentwith early motor and cognitive delay and thence-forth with different types of infantile-onset seizuresReview of previously published patients shows thatboth focal and generalized seizures including tonic-clonic tonic atonic absences myoclonic seizuresand infantile spasms can be equally observed (figure3A) Epilepsy often classified as early-onset epilepticencephalopathy has been reported as severe andrefractory to AEDs in most patients In our smallseries 2 of the 6 patients were diagnosed as Westsyndrome (figure 3B) Of the remaining 4 2 hadgeneralized epilepsy with myoclonic seizures and eye-lid myoclonia with photosensitivity resembling Jea-vons syndrome in 1 (figure 3B) Focal epilepsy withprolonged occipital seizures or focal clonic possiblyfrontal seizures was documented only in 1 and thelast sixth patientrsquos had a phenotype consistent withLennox-Gastaut syndrome (figure 3B)

Combining the analysis of the seizure types ofall known patients harboring KCNB1 mutations

(including our 6 probands and the 14 previouslypublished for a total of 20 of whom 19 with seiz-ures) we can observe that (1) TCS are the common-est seizure type observed in 578 (1119) ofpatients (2) focal seizures are the second most com-mon type reported in 47 (919) (3) infantilespasms are also a relatively common manifestationoccurring in 315 (619) (4) approximately 15(3ndash419) of patients have either absences atonic ortonic seizures (5) single patients might have peculiarand unusual phenotypes consisting of CSWS non-convulsive status epilepticus (NCSE) or myoclonicseizures or eyelid myoclonia and photosensitive seiz-ures Of course most patients manifest more than 1seizure type that combined the result in unspecifiedEE or more rarely to known epilepsy syndromesincluding West Lennox-Gastaut and Jeavonssyndromes

Likewise previously published patients in oursmall series seizures started in infancy around 1 yearof age and were also drug resistant at onset Yet overtime in 4 of our 6 patients (666) seizure frequencyattenuated and disappeared still being under remis-sion after a seizure-free period of 6 years Two pa-tients are also at present off AEDs The reason whythere is such a wide spectrum of epilepsy severity inthis and in other EIEE remains a true mystery at pres-ent Thus further speculative discussion is betterpostponed until we improve our understanding aboutthe underlying pathophysiologic mechanism and howindividual genetic backgrounds influence outcomes

Figure 2 Schematic representation of the Kv21 protein with mutation distribution

Structure of the human Kv21 channel including previously published mutations and those reported here (specified under each mutation) The protein topol-ogy was performed using the Protter online tool (29) with the Uniprot accession Q14721 (KCNB1_HUMAN)


Long-term follow-up of our 6 patients showedthat following delay of milestone acquisition anddespite seizure freedom they evolved into moderateto severe impairment of cognitive functions includ-ing psychiatric symptoms and autistic traits Over-all these findings indicate that Kv21 impairedfunction causes diffuse brain dysfunction express-ing in humans as epilepsy and developmental orbehavioral disorders Thus it might be more appro-priate to define patients with KCNB1 mutations ashaving developmental encephalopathy with epi-lepsy An intermingled pathophysiologic relation-ship between the triad of seizuresASDID andpotassium channel genes is further supported bythe identification of mutations in KCNJ10 a genecoding for the inwardly rectifying potassium chan-nel Kir41 in several probands exhibiting seizuresASD and ID33

All mutations reported here were de novo andincluded 4 missense and 2 nonsense variants Noneof the mutations appeared in allele frequency data-bases (table e-1 httplinkslwwcomNXGA0)and all were predicted to be damaging by several pre-diction tools and ensemble scores (table e-1 httplinkslwwcomNXGA0) and novel with the excep-tion of the pArg306Cys falling in the S4 of thevoltage-sensor domain and demonstrated to be

damaging by Saitsu et al10 Patients carrying thepArg306Cys mutation appear to have different phe-notypes The patient we are reporting in this studyhas generalized epilepsy with myoclonic seizures andeyelid myoclonia while the patient described by Sait-su et al10 had a more severe phenotype with earlierseizure onset and heterogeneous drug-resistantseizures including infantile spasms myoclonictonic-clonic and focal seizures The latter patientmanifested also severe ID and macrocephaly unlikethe one we are reporting who had a normal head sizeand moderate ID

While assessing genotype-phenotype correlationswe noted that both pTrp370 and pArg583 non-sense mutations occurred in the 2 patients with remit-ting infantile-onset spasms and severe cognitiveimpairment At present there is a single previouslyreported nonsense mutation14 in a patient with EEyet detailed clinical features are not available for fur-ther correlations

The remaining 2 mutations pIle196Phe and thepThr210Met both predicted to be damaging aremissense variants (table 1) located in the S1 and inthe S1ndashS2 extracellular loop The S1ndashS4 segmentsserve as the voltage-sensing module and the voltagesensitivity of the channel is conferred by a series ofhighly conserved basic side chains in the S4 segment

Figure 3 Graphic representation of seizure type distribution in patients with KCNB1 mutations

(A) Previously published patients (data from 10 of the 13 patients of previously published KCNB1 mutations and epilepsy)and (B) in our series of 6 patients AB 5 absences CSWS 5 continuous spikes and waves during slow-wave sleep FS 5

febrile seizures MY 5 myoclonia NCSE 5 nonconvulsive status epilepticus TCS 5 tonic-clonic seizures


These side chains are stabilized in a transmembraneconfiguration by the formation of fully solvated saltbridges with a set of highly conserved acidic sidechains in S1ndashS334 The pIle196Phe and pThr210Met mutations are located in the S1 con-served acidic side chains and are therefore likely toimpair the structural signature of voltage-sensingmotion between S1ndashS3 and S4 segments35

NGS and Sanger sequencing showed that the pVal349Phe variant was present as mosaicism althoughassociated with the most refractory epilepsy type inour cohort This might correlate with the locationof this mutation in the pore-forming region of thechannel thus supposedly causing a severe alterationof its function A missense mutation located veryclosely at position 347 causing severe impairmentof the channel function and associated with refractoryepilepsy strikingly resembling our patientrsquos pheno-type had previously been reported8

Patch-clamp studies have demonstrated that someKCNB1 variants cause significant alterations of Kv21channel function and increased excitability of corticalneurons Functional studies suggested differentmechanisms including abolished voltage-activatedgating loss of ion selectivity11 gain of depolarizinginward cation8 defects in expression and subcellularlocalization8 disruption of sensitivity and cooperativ-ity of the sensor10 and abolished endogenous Kv21currents10 The role of K1 channels in the neurobiol-ogy of both epilepsy and cognitive impairment hasbeen investigated also using animal models Micelacking Kv21 are strikingly hyperactive defective inspatial learning hypersensitive to convulsants andexhibit accelerated seizure progression36 Moreoverthe Kcnb122 mice model showed neuronal hyperex-citability and confirmed the critical role of Kv21in hippocampal neuronal network homeostaticregulation36

Thus KCNB1-related manifestations comprisea wide spectrum of infantile onset generalized orfocal seizures whose combination leads to EIEEincluding West Lennox-Gastaut and Jeavons syn-dromes Long-term follow-up highlights that follow-ing a stormy phase seizures cease and AEDs mightbe withdrawn at least in some patients Instead cog-nitive and motor functions are almost always delayedprior to seizure onset and evolve into severe persis-tent impairment Altogether these findings suggestthat KCNB1 mutations are associated with diffusebrain dysfunction and that the KCNB1-related clin-ical picture might also be defined as a developmentalldquoencephalopathyrdquo with seizures Further under-standing of the underlying pathophysiology byexperimental studies including animal models isrequired to improve treatment and prognosis ofthese disorders

AUTHOR CONTRIBUTIONSCarla Marini Michele Romoli Elena Parrini and Paolo Prontera study

design data collection and analysis and manuscript preparation Frances-

co Mari Cinzia Costa Lucio Parmeggiani Tiziana Metitieri and Mattia

Gentile data collection and analysis and manuscript revision Davide

Mei Elena Cellini Simona Virdograve and Dalila De Vita genetic study

data collection and analysis and manuscript revision Paolo Calabresi and

Renzo Guerrini study design data analysis and manuscript revision

STUDY FUNDINGThis work was supported by a grant from the EU Seventh Framework

Programme FP7 under the project DESIRE (grant agreement no

602531) (to RG) and by the Italian Ministry of Health (grant agree-

ment no RF-2013-02355240) (to RG)

DISCLOSURECarla Marini has received travel funding andor speaker honoraria at VII

CONGRESSO NAZIONALE SIMMESNndashFirenze December 16ndash18

2015 from SOBI Swedish Orphan Biovitrum has served on the editorial

boards of Epilepsia and Epileptic Disorders and has received research support

from the Italian Ministry of Health and Cariplo Foundation Michele

Romoli Elena Parrini and Cinzia Costa report no disclosures Davide Mei

has received funding for travel or speaker honoraria from BioMarin Phar-

maceutical and serves on the editorial board of Epilepsia Francesco Mari

Lucio Parmeggiani Elena Procopio Tiziana Metitieri Elena Cellini Si-

mona Virdograve Dalila De Vita Mattia Gentile Paolo Prontera and Paolo

Calabresi report no disclosures Renzo Guerrini has served on scientific

advisory boards of Eisai Inc Novartis Zogenix and BioMarin has received

funding for travel andor speaker honoraria from EISAI Novartis UCB

and BioMarin has served on the editorial boards of Epilepsia Progress in

Epileptic Disorders Neuropediatrics Journal of Child Neurology Seizure BMC

Medical Genetics Topics in Epilepsy Journal of Pediatric Epilepsy Epileptic

Disorders European Neurological Journal Neurology and Journal of Embry-

ology amp Developmental Biology receiveshas received royalties from the

publication of Epilepsy and Movements disorders (Cambridge University

Press 2002) Aicardirsquos Epilepsy in Children (Lippincott Williams amp Wil-

kins 2004) Progress in Epileptic Spasms and West Syndrome (John

Libbey Eurotext 2007) Epilepsy and Migraine (John Libbey Eurotext

2009) The Causes of Epilepsy (Cambridge University Press 2011) Dravet

syndrome (John Libbey Eurotext 2011) and Epilepsy and epileptic seizures

(Oxford University Press 2012) and has received research support from

Tuscany Region Research Department EU and EC Programs the Italian

Ministry of Health and the PISA Foundation Go to Neurologyorgng for

full disclosure forms

Received June 17 2017 Accepted in final form September 7 2017

REFERENCES1 Hwang PM Fotuhi M Bredt DS Cunningham AM

Snyder SH Contrasting immunohistochemical localiza-

tions in rat brain of two novel K1 channels of the Shab

subfamily J Neurosci 1993131569ndash1576

2 Maletic-Savatic M Lenn NJ Trimmer JS Differential

spatiotemporal expression of K1 channel polypeptides in

rat hippocampal neurons developing in situ and in vitro

J Neurosci 1995153840ndash3851

3 Du J Tao-Cheng JH Zerfas P et al The K1 channel

Kv21 is apposed to astrocytic processes and is associated

with inhibitory postsynaptic membranes in hippocampal

and cortical principal neurons and inhibitory interneurons

Neuroscience 19988437ndash48

4 Liu PW Bean BP Kv2 channel regulation of action

potential repolarization and firing patterns in superior cer-

vical ganglion neurons and hippocampal CA1 pyramidal

neurons J Neurosci 2014344991ndash5002

5 Misonou H Mohapatra DP Park EW et al Regulation of

ion channel localization and phosphorylation by neuronal

activity Nat Neurosci 20047711ndash718


6 Misonou H Thompson SM Cai X Dynamic regulation

of the Kv21 voltage-gated potassium channel during brain

ischemia through neuroglial interaction J Neurosci 2008

288529ndash8538

7 Fox PD Loftus RJ Tamkun MM Regulation of Kv21 K

(1) conductance by cell surface channel density


8 Torkamani A Bersell K Jorge BS et al De novo KCNB1mutations in epileptic encephalopathy Ann Neurol 2014

76529ndash540

9 Srivastava S Cohen JS Vernon H et al Clinical whole

exome sequencing in child neurology practice Ann Neurol

201476473ndash483

10 Saitsu H Akita T Tohyama J et al De novo KCNB1mutations in infantile epilepsy inhibit repetitive neuronal

firing Sci Rep 2015515199

11 Thiffault I Speca DJ Austin DC et al A novel epileptic

encephalopathy mutation in KCNB1 disrupts Kv21 ion

selectivity expression and localization J Gen Physiol

2015146399ndash410

12 Allen NM Conroy J Shahwan A et al Unexplained early

onset epileptic encephalopathy exome screening and phe-

notype expansion Epilepsia 201657e12ndashe17

13 Latypova X Matsumoto N Vinceslas-Muller C et al

Novel KCNB1 mutation associated with non-syndromic

intellectual disability J Hum Genet 201762569ndash573

14 de Kovel CGF Brilstra EH van Kempen MJA et al

Targeted sequencing of 351 candidate genes for epileptic

encephalopathy in a large cohort of patients Mol Genet

Genomic Med 20164568ndash580

15 Miao P Peng J Chen C et al A novel mutation in KCNB1gene in a child with neuropsychiatric comorbidities with both

intellectual disability and epilepsy and review of literature

Zhonghua Er Ke Za Zhi 201755115ndash119

16 Commission on Classification and Terminology of the

International League Against Epilepsy Proposal for revised

classification of epilepsies and epileptic syndromes Epilep-

sia 198930389ndash399

17 Berg AT Berkovic SF Brodie MJ et al Revised terminol-

ogy and concepts for organization of seizures and epilep-

sies report of the ILAE Commission on Classification and

Terminology 2005ndash2009 Epilepsia 201051676ndash685

18 Parrini E Marini C Mei D et al Diagnostic targeted

resequencing in 349 patients with drug-resistant pediatric

epilepsies identifies causative mutations in 30 different

genes Hum Mutat 201738216ndash225

19 Yang H Wang K Genomic variant annotation and prior-

itization with ANNOVAR and wANNOVAR Nat Protoc

2015101556ndash1566

20 Richards S Aziz N Bale S et al Standards and guidelines

for the interpretation of sequence variants a joint consen-

sus recommendation of the American College of Medical

Genetics and Genomics and the Association for Molecular

Pathology Genet Med 201517405ndash424

21 Caraballo RH Fontana E Darra F et al A study of 63

cases with eyelid myoclonia with or without absences type

of seizure or an epileptic syndrome Seizure 200918440ndash

445

22 Biervert C Schroeder BC Kubisch C et al A potassium

channel mutation in neonatal human epilepsy Science

1998279403ndash406

23 Charlier C Singh NA Ryan SG et al A pore mutation in

a novel KQT-like potassium channel gene in an idiopathic

epilepsy family Nat Genet 19981853ndash55

24 Singh NA Charlier C Stauffer D et al A novel potassium

channel gene KCNQ2 is mutated in an inherited epilepsy

of newborns Nat Genet 19981825ndash29

25 Benarroch EE Potassium channels brief overview and

implications in epilepsy Neurology 200972664ndash669

26 Muona M Berkovic SF Dibbens LM et al A recurrent

de novo mutation in KCNC1 causes progressive myoclo-

nus epilepsy Nat Genet 20154739ndash46

27 Simons C Rash LD Crawford J et al Mutations in the

voltage-gated potassium channel gene KCNH1 cause

Temple-Baraitser syndrome and epilepsy Nat Genet

20154773ndash77

28 Du W Bautista JF Yang H et al Calcium-sensitive potas-

sium channelopathy in human epilepsy and paroxysmal

movement disorder Nat Genet 200537733ndash778

29 Nava C Dalle C Rastetter A et al De novo mutations in

HCN1 cause early infantile epileptic encephalopathy Nat

Genet 201446640ndash645

30 Barcia G Fleming MR Deligniere A et al De novo gain-

of-function KCNT1 channel mutations cause malignant

migrating partial seizures of infancy Nat Genet 201244

1255ndash1259

31 Syrbe S Hedrich UB Riesch E et al De novo loss- or

gain-of-function mutations in KCNA2 cause epileptic

encephalopathy Nat Genet 201547393ndash399

32 Eunson LH Rea R Zuberi SM et al Clinical genetic

and expression studies of mutations in the potassium chan-

nel gene KCNA1 reveal new phenotypic variability Ann

Neurol 200048647ndash656

33 Sicca F Imbrici P DrsquoAdamo MC et al Autism with

seizures and intellectual disability possible causative role

of gain-of-function of the inwardly-rectifying K1 channel

Kir41 Neurobiol Dis 201143239ndash247

34 Wood ML Freites JA Tombola F Tobias DJ Atomistic

modeling of ion conduction through the voltage-sensing

domain of the Shaker K1 ion channel J Phys Chem B

20171213804ndash3812

35 Freites JA Tobias DJ Voltage sensing in membranes

from macroscopic currents to molecular motions

J Membr Biol 2015248419ndash430

36 Speca DJ Ogata G Mandikian D et al Deletion of the

Kv21 delayed rectifier potassium channel leads to neuro-

nal and behavioral hyperexcitability Genes Brain Behav

201413394ndash408


DOI 101212NXG000000000000020620173 Neurol Genet

Carla Marini Michele Romoli Elena Parrini et al mutations

geneKCNB1Clinical features and outcome of 6 new patients carrying de novo

This information is current as of December 12 2017

reserved Online ISSN 2376-7839Published by Wolters Kluwer Health Inc on behalf of the American Academy of Neurology All rightsan open-access online-only continuous publication journal Copyright Copyright copy 2017 The Author(s)

is an official journal of the American Academy of Neurology Published since April 2015 it isNeurol Genet

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Supplementary Material httpngneurologyorgcontentsuppl2017121236e206DC1

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References httpngneurologyorgcontent36e206fullhtmlref-list-1

This article cites 36 articles 6 of which you can access for free at

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phenotypes have been classified as epilepticencephalopathyndashearly infantile 26 (OMIMEIEE26 600397) Table 1 summarizes theclinical and genetic features of previouslyreported patients

Here we describe the electroclinical fea-tures and long-term follow-up of 6 novel pa-tients carrying de novo missense andnonsense KCNB1 mutations and provide anoverall view of the spectrum of epilepsy phe-notypes associated with KCNB1 mutationsranging from Lennox-Gastaut syndrome toremitting infantile spasms and to mild gener-alized epilepsy with eyelid myoclonia Oursmall cohort of patients also suggests thatthe KCNB1 EIEE26 manifests infantile-onset seizures with a tendency to attenuateor resolve completely over time in some pa-tients Concomitant impairment of cognitiveand motor functions constant and oftensevere persists over time and is at times com-bined with absent speech autism spectrumdisorder (ASD) and psychiatric problemsMutations in this gene which are not rareconfirm that genes coding for potassiumchannels are important genetic contributorsto epilepsies and more broadly to neurodeve-lopmental disorders

METHODS From a highly heterogenous cohort of 873 pa-

tients with pediatric epilepsies either refractory or benign with

a supposed genetic etiology recruited at the Meyer Childrenrsquos

Hospital or referred from other national or international epilepsy

centers for genetic testing we identified and studied 6 patients

carrying de novo variants of the KCNB1 gene (GenBank Acces-

sion Number NM_0049752) Three patients were examined at

the Neurology unit and 3 were referred for genetic testing to our

Neurogenetic Laboratory Collection and analysis of retrospective

clinical EEG neuropsychological neuroimaging data were per-

formed using a specific format filled by the treating specialist

aiming to obtain accurate and homogenous information We

classified seizure types and epilepsysyndromes according to the

International League Against Epilepsy guidelines1617

Molecular genetic testing Genomic DNA was extracted from

peripheral blood leukocytes using a QIAsymphony SP robot

(Qiagen Hilden Germany) according to the manufacturerrsquos pro-

tocol High-quality DNA was quantified using a QuantiFluor

Fluorometer (Promega Madison WI) Targeted resequencing of

a panel including 95 genes associated with epilepsy was per-

formed in all 6 patients Target enrichment and library prepara-

tion were performed using a custom-designed Nextera Rapid

Capture assay (Illumina San Diego CA) and sequencing was

performed on an Illumina MiSeq (Illumina) with a 2 3 150-bp

paired-end protocol as previously described18 Variants were

annotated and filtered using the ANNOVAR tool19 Variants

localized in intronic regions outside the 10-bp exon-flanking

boundaries and in the 59- and 39-UTR regions were excluded

Variants reported in the Exome Aggregation Consortium (ExAC)

database (exacbroadinstituteorg) andor in the 1000 Genomes

Project (in the National Heart Lung and Blood Institute Exome

Sequencing Project [ESP] ESP6500 database evsgswashington

eduEVS) in the Genome Aggregation Database (gnomAD)

(gnomadbroadinstituteorg) with a minor allele frequency 0

01 (1) were dropped out In silico prediction of mutationsrsquo

pathogenicity was obtained using the dbNSFP database (v33)

which provides functional prediction scores on more than 20

different algorithms (sitesgooglecomsitejpopgendbNSFP)

Putative causative variants were analyzed by Sanger sequencing to

confirm the next-generation sequencing (NGS) results in pro-

bands and investigated in the parents to check the inheritance

status We classified variants according to the international

Table 1 Clinical and genetic summary of previously published patients

Patient Ethnicity Phenotype Genotypes Genetic analysis Inheritance Reference

1 Chinese 2 y TC sz spasm drug resistantEEG GSW 3 y regression

pGly379Val WES De novo 15

3 Caucasian EOEE pPhe416Leu pArg312HispTyr533

NGS gene panel De novo 14

1 Caucasian ID severe speech impairment no epilepsy pVal378Leudeg WES De novo 13

2 Caucasian EOEE pGly381Arg pPhe416Leu WES De novo 12

1 Caucasian EE pVal378Aladeg WES De novo 11

2 Asian DD and severe infantile generalized sz pArg306Cys pGly401Arg WES De novo 10

1 Caucasian EE pGly379Arg^ WES De novo 9

3 Caucasian Sz onset 475 y EE hypotonia DDintermittent agitation

pS347Arg WES De novo 8

Sz onset 8 mo drug-resistant sz DDabsent speech stereotyped handwringing movements

pGly379Arg^ WES De novo

Sz onset first year of life TC atypical absence atonicinfantile spasms and focal dyscognitive

pThr374Ile WES De novo

Abbreviations ^ deg 5 recurrent mutations 5 nonsense mutation DD 5 developmental delay EE 5 epileptic encephalopathy EOEE 5 early-onset epilepticencephalopathy GSW 5 generalized spikes and waves ID 5 intellectual disability mo 5 months NGS 5 next-generation sequencing TC 5 tonic-clonicseizures sz 5 seizures WES 5 whole-exome sequencing y 5 years




Group20












ID 1 2 3 4 5 6


Sz onsetoffset










EEG


Bilateral SW R L NA






Treatment

















c1747CT pArg583


























































ment no RF-2013-02355240) (to RG)






















































288529ndash8538





76529ndash540



201476473ndash483






2015146399ndash410

















sia 198930389ndash399











2015101556ndash1566









445



1998279403ndash406















20154773ndash77










1255ndash1259















20171213804ndash3812







201413394ndash408





















Reprints






Group20












ID 1 2 3 4 5 6


Sz onsetoffset










EEG


Bilateral SW R L NA






Treatment

















c1747CT pArg583


























































ment no RF-2013-02355240) (to RG)






















































288529ndash8538





76529ndash540



201476473ndash483






2015146399ndash410

















sia 198930389ndash399











2015101556ndash1566









445



1998279403ndash406















20154773ndash77










1255ndash1259















20171213804ndash3812







201413394ndash408





















Reprints






















































ment no RF-2013-02355240) (to RG)






















































288529ndash8538





76529ndash540



201476473ndash483






2015146399ndash410

















sia 198930389ndash399











2015101556ndash1566









445



1998279403ndash406















20154773ndash77










1255ndash1259















20171213804ndash3812







201413394ndash408





















Reprints







288529ndash8538





76529ndash540



201476473ndash483






2015146399ndash410

















sia 198930389ndash399











2015101556ndash1566









445



1998279403ndash406















20154773ndash77










1255ndash1259















20171213804ndash3812







201413394ndash408





















Reprints























Reprints




clinical features and outcome of 6 new patients carrying de ...renzo guerrini, md, frcp...

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