University of Southern Denmark

Clinical spectrum of STX1B-related epileptic disorders

Wolking Stefan May Patrick Mei Davide Moslashller Rikke S Balestrini Simona HelbigKatherine L Altuzarra Cecilia Desmettre Chatron Nicolas Kaiwar Charu Stoumlhr KatharinaWiddess-Walsh Peter Mendelsohn Bryce A Numis Adam Cilio Maria R Van PaesschenWim Svendsen Lene L Oates Stephanie Hughes Elaine Goyal Sushma BrownKathleen Sifuentes Saenz Margarita Dorn Thomas Muhle Hiltrud Pagnamenta Alistair TVavoulis Dimitris V Knight Samantha J L Taylor Jenny C Canevini Maria Paola DarraFrancesca Gavrilova Ralitza H Powis Zoumle Tang Shan Marquetand Justus ArmstrongMartin McHale Duncan Klee Eric W Kluger Gerhard J Lowenstein Daniel HWeckhuysen Sarah Pal Deb K Helbig Ingo Guerrini Renzo Thomas Rhys H Rees MarkI Lesca Gaetan Sisodiya Sanjay M Weber Yvonne G Lal Dennis Marini Carla LercheHolger Schubert JulianPublished inNeurology

DOI101212WNL0000000000007089

Publication date2019

Document versionFinal published version

Document licenseCC BY

Citation for pulished version (APA)Wolking S May P Mei D Moslashller R S Balestrini S Helbig K L Altuzarra C D Chatron N Kaiwar CStoumlhr K Widdess-Walsh P Mendelsohn B A Numis A Cilio M R Van Paesschen W Svendsen L LOates S Hughes E Goyal S Schubert J (2019) Clinical spectrum of STX1B-related epileptic disordersNeurology 92(11) e1238-e1249 httpsdoiorg101212WNL0000000000007089

ARTICLE OPEN ACCESS

Clinical spectrumof STX1B-related epileptic disordersStefan Wolking MD Patrick May PhD Davide Mei PhD Rikke S Moslashller PhD Simona Balestrini PhD

Katherine L Helbig MS Cecilia Desmettre Altuzarra MD Nicolas Chatron PhD Charu Kaiwar MD

Katharina Stohr MD Peter Widdess-Walsh MB Bryce A Mendelsohn PhD Adam Numis MD

Maria R Cilio PhDWimVan Paesschen MD Lene L SvendsenMD Stephanie Oates MD Elaine Hughes MD

Sushma Goyal MD Kathleen Brown MS Margarita Sifuentes Saenz MD Thomas Dorn MD

Hiltrud Muhle MD Alistair T Pagnamenta PhD Dimitris V Vavoulis PhD Samantha JL Knight PhD

Jenny C Taylor PhDMaria Paola Canevini MD Francesca Darra MD Ralitza H GavrilovaMD Zoe PowisMS

Shan Tang PhD Justus Marquetand MD Martin Armstrong PhD Duncan McHale PhD Eric W Klee PhD

Gerhard J Kluger MD Daniel H Lowenstein MD Sarah Weckhuysen PhD Deb K Pal PhD Ingo Helbig MD

Renzo Guerrini MD Rhys H Thomas PhD Mark I Rees PhD Gaetan Lesca PhD Sanjay M Sisodiya PhD

Yvonne G Weber MD Dennis Lal PhD Carla Marini PhD Holger Lerche MD and Julian Schubert PhD

Neurologyreg 2019921-12 doi101212WNL0000000000007089

Correspondence

Dr Lerche

holgerlerche

uni-tuebingende

AbstractObjectiveThe aim of this study was to expand the spectrum of epilepsy syndromes related to STX1B encoding thepresynaptic protein syntaxin-1B and establish genotype-phenotype correlations by identifying further disease-related variants

MethodsWe used next-generation sequencing in the framework of research projects and diagnostic testing Clinical dataand EEGs were reviewed including already published cases To estimate the pathogenicity of the variants we usedestablished and newly developed in silico prediction tools

ResultsWe describe 17 new variants in STX1B which are distributed across the whole gene We discerned 4 differentphenotypic groups across the newly identified and previously published patients (49 patients in 23 families) (1) 6sporadic patients or families (31 affected individuals) with febrile and afebrile seizures with a benign course generallygood drug response normal development and without permanent neurologic deficits (2) 2 patients with geneticgeneralized epilepsy without febrile seizures and cognitive deficits (3) 13 patients or families with intractable seizuresdevelopmental regression after seizure onset and additional neuropsychiatric symptoms (4) 2 patients with focalepilepsy More often we found loss-of-function mutations in benign syndromes whereas missense variants in theSNARE motif of syntaxin-1B were associated with more severe phenotypes

ConclusionThese data expand the genetic and phenotypic spectrumof STX1B-related epilepsies to a diverse range of epilepsies thatspan the International League Against Epilepsy classification Variants in STX1B are protean and contribute to manydifferent epilepsy phenotypes similar to SCN1A the most important gene associated with fever-associated epilepsies

From theUniversity of Tubingen (SWolking JM YGWHL JS)DepartmentofNeurologyandEpileptologyHertie Institute forClinicalBrainResearch TubingenGermany LuxembourgCentre forSystemsBiomedicine (PM) University of Luxembourg Esch-sur-Alzette Pediatric Neurology and Neurogenetics Unit and Laboratories (DM RG CM) Childrenrsquos Hospital AnnaMeyer University of Florence ItalyDanish Epilepsy Centre (RSM) Dianalund Institute for Regional Health Services (RSM) University of Southern Denmark Odense Department of Clinical and Experimental Epilepsy (SB) UCL Institute ofNeurology and Epilepsy Society UK London Division of Neurology (KLH IH) Childrenrsquos Hospital of Philadelphia PA Department of Pediatric Neurology (CDA) Centre de CompetencesMaladies RaresCHUBesanccedilonServicedeGenetique (NC)HospicesCivilsdesLyonBronGENDEVTeam(NC)NeurosciencesResearchCenterofLyonBronFranceNeuropediatricClinicandClinic forNeurorehabilitation(KS) Epilepsy Center for Children and Adolescents Schoen Klinik Vogtareuth Germany Beaumont Hospital (PW-W) Dublin Ireland Department of Pediatrics Division of Medical Genetics Institute ofHuman Genetics (BAM) Departments of Neurology and Pediatrics (AN) and Departments of Neurology and Pediatrics and Institute of Human Genetics (MRC) University of California San FranciscoDepartment ofNeurology (WVP) UniversityHospitals Leuven BelgiumDepartment of Pediatrics (LLS) HvidovreHospital Denmark KingrsquosCollegeHospital (SO EH SG DKP) London Evelina LondonChildrenrsquosHospital (SO EH SG) London UK Section of Genetics (KBMSS) Department of Pediatrics University of Colorado andChildrenrsquosHospital Colorado Aurora CliniqueBernoiseMontana (TD)Crans-Montana SwitzerlandDepartment ofNeuropediatrics (HM) UniversityMedical Center Schleswig-Holstein Christian-AlbrechtsUniversity Kiel GermanyNational Institute forHealth ResearchOxfordBiomedical ResearchCentreWellcomeCentre forHumanGenetics (ATP SJLK JCT) andDepartment ofOncology (DVV) University ofOxford UK Epilepsy Center (MPC) Health SciencesDepartmentSanPaoloHospital University ofMilan ChildNeuropsychiatry (FD) Department of Surgical Sciences Dentistry Gynecology and Pediatrics University of Verona Italy Departments ofNeurology andClinicalGenomics (RHG) andHealthSciencesResearchandClinicalGenomics (EWK CK)MayoClinicRochesterMNAmbryGenetics (ZP) AlisoViejoCADepartmentofClinicalNeuroscience (ST) KingrsquosCollegeLondon New Medicines (MA DM) UCB Pharma Slough UK Neuropediatric Clinic and Clinic for Neurorehabilitation (GJK) Epilepsy Center for Children and Adolescents Schoen Klinik VogtareuthGermany Research Institute for Rehabilitation Transition and Palliation (GJK) PMU Salzburg Austria Department of Neurology (DHL) University of California San Francisco Neurogenetics Group (SWeckhuysen) Center forMolecularNeurology VIB Antwerp Laboratory ofNeurogenetics (SWeckhuysen) Institute Born-Bunge University of Antwerp Department ofNeurology (SWeckhuysen) AntwerpUniversityHospital Antwerp BelgiumDepartment of BasicampClinical Neuroscience Institute of Psychiatry PsychologyampNeuroscience (DKP)MRCCentre forNeurodevelopmentalDisorders (DKP) KingrsquosCollege LondonUK Evelina LondonChildrenrsquosHospital (DKP) London UKDepartment ofNeuropediatrics (IH) UniversityMedical Center Schleswig-Holstein Christian-AlbrechtsUniversity Kiel GermanyInstitute of Neuroscience (RHT) Henry Wellcome Building Newcastle University Neurology Research Group (MIR) Institute of Life Science Swansea University Medical School Swansea UK Service deGenetique (GL) Hospices Civils des Lyon Bron GENDEV Team (GL) Neurosciences Research Center of Lyon Bron France NIHR University College London Hospitals Biomedical Research Centre (SMS)UCL Institute of Neurology London UK Cologne Center for Genomics (DL) University of Cologne Germany Stanley Center for Psychiatric Research (DL) and Program inMedical and Population Genetics(DL) Broad Institute of MIT and Harvard Cambridge Psychiatric and Neurodevelopmental Genetics Unit (DL) Massachusetts General Hospital and Harvard Medical School Boston

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 Wellcome Trust

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 2019 The Author(s) Published by Wolters Kluwer Health Inc on behalf of the American Academy of Neurology 1

Published Ahead of Print on February 8 2019 as 101212WNL0000000000007089

Genetic generalized epilepsies (GGEs) and genetic epilepsieswith febrile seizures (FS) plus (GEFS+) are genetically andphenotypically heterogeneous epileptic disorders GGEs sharecommon clinical hallmarks such as seizure types generalizedepileptic discharges on EEG and typical onset between child-hood and adolescence Most GGE cases have a polygenic in-heritance but a few monogenic causes have been identifiedsuch as GABRA11 SLC2A12 CACNA1H3 as well as micro-deletions such as 15q1334 GEFS+ is a familial epilepsy syn-drome characterized by focal or generalized febrile and afebrileseizures and focal or generalized epileptiform discharges onEEG The clinical presentation may differ considerably amongaffected individuals within the same family56 Privatemutationshave been identified in genes encoding subunits of voltage-gated Na+ channels (SCN1A SCN1B)78 and the γ-amino-butyric acid type A receptor (GABRG2 GABRD)910 Most ofthe genes that are associated with GGE and GEFS+ are alsoimplicated in developmental and epileptic encephalopathies(DEEs) which are characterized by seizure onset in the firstyears of life often pharmacoresistant seizures cognitive re-gression and neurologic deficits For certain genes such asSCN1A a clear genotype-phenotype correlation is described inGEFS+ a greater proportion of missense variants is foundwhereas in Dravet syndrome nonsense mutations or largedeletions are more common11

Recently we reported variants in the STX1B gene as a cause offever-associated epilepsies of variable severity12 Two largefamilies showed a rather benign course of GEFS+ syndromewhereas 4 individuals had a more severe DEE phenotype Anadditional 18-year-old patient exhibiting epilepsy withmyoclonic-atonic seizures and moderate to severe intellectualdisability was reported recently13

STX1B encodes syntaxin-1B a presynaptic protein that is partof the SNARE complex mediating the process of calcium-dependent synaptic vesicle release14

We aimed to describe additional variants in STX1B charac-terize the related clinical and EEG phenotypic spectrum andestablish genotype-phenotype correlations

MethodsClinical dataWe identified 49 individuals harboring heterozygous variantsin STX1B (NM_0528743) The cases were pooled from14 different centers identified through research studies and

clinical diagnostic testing The cohort consists of 7 familieswith 2 to 17 affected individuals and 16 sporadic patients Ofthese we previously reported 2 large families and 4 sporadicpatients121516 We systematically collected clinical in-formation such as age at onset seizure type neurologic andcognitive deficits neuroimaging outcome and antiepilepticdrug (AED) treatment through a standardized questionnaireWe reviewed original EEG recordings (S Wolking HL TDCM PW-W DM) for all but 3 patients (F3 F15 F18)EEGs for F1 and F2were described in previous publications1516

Standard protocol approvals registrationsand patient consentsWritten informed consent to participate in this study wasobtained from all patients or caregivers

GeneticsWe used different next-generation sequencing datasets frommulticenter research projects or diagnostic testing services toidentify the reported STX1B variants The methods and thecenters are available fromDryad (doi105061dryadcf0hj73)and in our previous report12 All patients are of Europeandescent

To evaluate the effect of the missense variants we used well-established in silico prediction tools SIFT (sorting tolerantfrom intolerant)17 PolyPhen-2 (polymorphism phenotypingv2)18 and MutationTaster19 We compared patient STX1Bmissense variants to variants identified in almost 150000control individuals stored in the Genome Aggregation Data-base (gnomAD gnomadbroadinstituteorg)20 Mutationdensity was calculated by counting the number of variantpositions in gnomAD divided by the window length withina window of 10 nucleotides using a sliding window approachover the coding sequence of the STX1B transcript NM_0528743 We determined paralog conservation as describedby Lal et al21 In short we aligned the canonical proteinsequences of the 7 paralog genes of the Ensembl SYNTAXINprotein family STX2 STX1A STX11 STX1B STX19 STX3and STX4 and scored the conservation at each alignmentposition using JalView22 Then we determined the mean andthe standard deviation paralog conservation for each singleprotein of the protein family and a z score (para_zscore)calculated for each residue position We defined paralogconservation as para_zscore gt0

Data availabilityData not published in this article will be shared as anonymizeddata by request from any qualified investigator

GlossaryDEE = developmental and epileptic encephalopathy FS = febrile seizuresGEFS+ = genetic epilepsies with febrile seizures plusGGE = genetic generalized epilepsy gnomAD = Genome Aggregation Database GTCS = generalized tonic-clonic seizureJME = juvenile myoclonic epilepsy PolyPhen-2 = polymorphism phenotyping v2 SIFT = sorting tolerant from intolerantSNARE = SNAP (soluble NSF attachment protein) receptor

2 Neurology | Volume 92 Number 11 | March 12 2019 NeurologyorgN

ResultsPhenotypic descriptionsReviewing the clinical characteristics of all patients we coulddistinguish 4 different phenotypic groups (numbering of thefamilies was defined as follows F1ndashF6 as in our previousstudy12 F7ndashF23 new families sorted by phenotype) (1)Three families and 3 sporadic patients with FS with orwithout additional generalized and more rarely focal afebrileseizures with a relatively benign course generally good drugresponse normal development and mild or no neuropsy-chiatric symptoms corresponding to GEFS+ (F1 2 7ndash10)(2) Two unrelated patients with generalized myoclonic andabsence seizures without FS and without major cognitivedeficits corresponding to juvenile myoclonic epilepsy (JME)a common subtype of GGE (F11 12) (3) Thirteen unrelatedpatients with intractable seizures occurrence of de-velopmental stagnation or regression after seizure onset andadditional neuropsychiatric deficits compatible with DEE(F3ndash6 F13ndash21) (4) Two patients with some form of focalepilepsy (F22 23) The main clinical characteristics of allfamilies and sporadic patients are summarized in table 1Moredetailed clinical data are available from Dryad doiorg105061dryadcf0hj73

Group 1 GEFS+ (31 patients in families F1 F2 F7ndash10)FS were present in the majority of patients The first seizurestarted between 10 months and 5 years (median 20 months)There was a large variety of afebrile seizure types includinggeneralized tonic-clonic seizures (GTCS) (n = 13) focalimpaired awareness seizures (n = 3) atonic seizures (n = 8)tonic seizures (n = 4) and absence seizures (n = 5) Ninepatients had infrequent seizures until adulthood that did notrequire treatment For most cases cognition was intact andneurologic deficits were not frequent in this cohort (table 1)Asymptomatic variant carriers were identified in 2 families (4in F1 3 in F2) which is of importance for genetic testing andcounseling in mild cases Moreover in F1 4 phenocopieswere identified (2 patients with a GGE phenotype notmatching GEFS+ 1 individual with a single FS and 1 patientwith falls and head nodding in early childhood that were laterinterpreted as nonepileptic15)The treatment outcome in thisgroup was overall positive More than 50 of patients wereseizure-free without medication (17) the remainder wereseizure-free with AED treatment (11) Only patient F7 hadongoing seizures with valproic acid EEGs showed focal andgeneralized epileptiform discharges Available ictal recordingsin F81 showed focal onset seizure

Group 2 GGE (2 patients F11 F12)The first seizures for patient F11 were at age 18 years (gen-eralized myoclonic seizures) followed by GTCS at 20 yearsand absences at 21 years GTCS occurred in clusters of 3 or 4seizures every 3 months On neuropsychological examinationthe patient showed slight executive dysfunction however IQand memory functions were normal F12 had his first seizuresat 11 years featuring both afebrile myoclonic seizures and

GTCS AWechsler Adult Intelligence test at 19 years revealedan IQ of 85 plusmn 3 On examination neither patient showeddeficits Treatment was difficult requiring multiple AED trialsin both patients (table 1)

For F11 the first EEG at age 21 showed an unusual pattern offrequent bursts of generalized slow waves associated withmyoclonic jerks of both shoulders Later typical generalizedbursts of spikes and sharp waves were documented underphotic stimulation (he was the only photosensitive patient inthe whole cohort as far as this was examined) For F12 thefirst available EEG was performed at age 19 and showed oc-casional brief bilateral epileptic discharges A video-EEG atage 24 demonstrated 4-Hz generalized spike-wave activityVideo-EEGmonitoring at age 31 showed no epileptic activity

Group 3 DEE (15 patients F3ndash6 F13ndash21)We retrieved detailed phenotypes for 12 of 15 patients Epi-lepsy onset was between 0 months and 35 years (median 15months) Six patients (F5 F14 F16 F17 F20) had FS withonset between 13 months and 2 years F14 had only a singleFS after vaccination Afebrile seizures comprised GTCS (n =9) myoclonic (n = 10) atonic (n = 8) tonic seizures (n = 3)atypical absences (n = 3) infantile spasms (n = 1) and hy-perkinetic focal seizures (n = 1)

Except for F6 initial development was normal in all patientswith severe global developmental delay or even regressioncoinciding with seizure onset In F19 and F21 global de-velopmental delay and seizures were present since birthNeurologic examination showed anomalies in most patientswith ataxia being the most frequent finding

Seizures were pharmacoresistant in all patients except F18undergoing on average 9 AED trials Only F5 and F21 ach-ieved seizure freedom with ongoing treatment Of note F4responded with a significant seizure reduction to the com-bined therapy of lamotrigine and valproic acid after severalunsuccessful AED trials After the administration of bromideF14 showed a significant reduction in seizure frequency

EEGs were available for review in 11 of 15 patients Inter-ictally the EEGs in all cases showed multifocal epileptic dis-charges that were predominantly located in the frontal ortemporal region In addition we detected generalized dis-charges in 10 of 11 patients presenting as generalized spike-wave or polyspike-wave discharges In 4 of 11 patients EEGsshowed frequent bursts of generalized rhythmic activity last-ing several seconds Ictal EEG recordings were available in 7of 11 patients In 4 patients typical tonic seizures with gen-eralized beta activity with increasing amplitude and decreasingfrequency were recorded EEG curves are available fromDryad (additional figures) doi105061dryadcf0hj73

Group 4 Focal epilepsy (2 patients F22 F23)F22 had right temporal lobe epilepsy with a single FS reported at6 years Afebrile impaired awareness seizures with automatisms

NeurologyorgN Neurology | Volume 92 Number 11 | March 12 2019 3

Table 1 Condensed phenotypes of patients and families F1ndashF23

ID No VariantFS AaO(y)

NonfebrileseizuresAaO (y)seizuretype

Intellectualdisability

Neurologicexamination EEG

Outcome(currentAEDs)

Geneticepilepsy withfebrile seizuresplus

F1 17 c166CgtT pQ56 15-7 3patientswo FS

17-6 2exceptionswith onsetinadulthoodGTCS (6) AS(4) Abs (4)TS (2)

Aspergersyndrome (2)

Normal 3-4s GSW (5)FSW (1)

All Sfmost wotreatment

F2 8 c133_134insGGATGTGCATTGpK45delinsRCMIE andc135_136ACgtGApL46M

12-3 2patientswo FS

12-17GTCS (5)FIAS (3) AS(3)

Dyslexia (1)dyscalculia (1)learningdisability (1)

Macrocephaly(1)

FSW (7) GSW (7) All Sf 4wotreatment

F7 1 c23_26dupTGCGpS10Afs7 de novo

08NAGTCS

13 GTCS Normal Mildhypotonia

NA Os (VPA)

F8 3 c852dup pT285Dfs75 09-14 5 (1) GTCSTS

Learningdisability (1)

Normal FSW (1) All Sf 2undertreatment(VPA PB)

F9 1 c733CgtT pR245 2 FIASGTCS

3 FIAS Normal Normal FSW Sf (OXC)

F10 1 c420CgtG pY140 2 ASGTCS

NA Mild Milddysmorphicfeatures

FSW Sf (VPACLB)

Geneticgeneralizedepilepsy

F11 1 c628GgtA pE210K NA 18 GTCSMyo Abs

Impairedexecutivefunctions

Normal GSWphotosensitive

Os (LEVCLB)

F12 1 c277AgtT pK93 NA 11 GTCSMyo

Normal Normal GSW Sf (VPALEV TPM)

Developmentaland epilepticencephalopathy

F3 1 c140CgtA pS47 denovo

uk uk uk uk uk uk

F4 1 c657TgtA pV216E NA 35 GTCSTS Myo Abs

Moderatecognitiveimpairment

Ataxiadysarthriamacrocephaly

FSW GSW Os (VPALTG)

F5 1 c678GgtC pG226R denovo

11 17 GTCSMyo AbsAS TS

Developmentalregression

Ataxia 35s GSW FSW Sf (LEVSTP VPA)

F6 1 arr[hg19] 16p112(30332532-31104012)x1 de novo

NA 11 Myo ASGTCS

Developmentalregression

Milddysmorphicfeatures

FSW Os (CLBSTP)

F13 2 c563dupA pN189Afs5 NA 08-13 Myo(2) atyp Abs(2) GTCS (2)TS (2)

Developmentalregression (1)

Ataxia (1)dystonia (1)

FSW (2) GSW (2) Os (PHTVPA CBZVGB)

Continued

4 Neurology | Volume 92 Number 11 | March 12 2019 NeurologyorgN

occurred at the same age Sometimes she would describe an aurawith a rushing sensation in her head or blurred vision Occa-sionally secondary generalization would occur Interictal EEGsshowed right temporal epileptiform discharges Ictal recordingof one seizure depicted a seizure onset over the entire righthemisphere with subsequent evolution most prominent overthe right temporal region Neuropsychological testing andneurologic examination were unremarkable RepeatedMRIs showed nonspecific white matter lesions An [18F]-fluorodeoxyglucosendashPET at age 28 revealed hypometabolismin the right temporal lobe Seizures were pharmacoresistant tooxcarbazepine lacosamide and zonisamide

In F23 weekly seizures started in teenage years with staringepisodes accompanied by subtle twitching of the right arm Inaddition GTCS started at 24 years Seizures were controlled withlamotrigine At 45 years the patient developed stroke-like epi-sodes featuring left-sided hemicrania dilatation of the right pupil

hemihypesthesia and mild hemiparesis The episodes lasted be-tween 30 and 120 minutes and occurred about once a week AnMRI at age 46 years showed no abnormalities Previous EEGsshowed left temporal epileptiform discharges However themostrecent EEGs were normal A video-EEG recorded during one ofthe stroke-like episodes was without EEG correlate Of note inthe patientrsquos fifth decade of life several autoimmune diseases werediagnosed type 1 diabetes mellitus celiac disease and hypo-gammaglobulinemia Treatment with IV immunoglobulins led toa decrease in the frequency of the stroke-like episodes

Molecular geneticsOf the 17 newly identified variants in STX1B 8 are missense(pVal88Phe pCys144Phe pGlu210Lys pLeu221PropAla246Pro pSer258Gln pArg261Gln pIle282Thr)5 frameshift (pSer10Alafs7 pGln52Argfs2 pGlu128-Glyfs2 pAsn189Alafs5 pThr285Aspfs75) and 3 stopgain variants (pLys93 pTyr140 pArg245) One patient

Table 1 Condensed phenotypes of patients and families F1ndashF23 (continued)

ID No VariantFS AaO(y)

NonfebrileseizuresAaO (y)seizuretype

Intellectualdisability

Neurologicexamination EEG

Outcome(currentAEDs)

F14 1 c845TgtC pI282T denovo

13 2 Abs MyoAS TS

Developmentalregression

Ataxiaaphasia

GPSW GSW Os (VPABR ESM)

F15 1 c773GgtA pS258N uk uk uk uk uk uk

F16 1 c662TgtC pL221P 2 3 GTCSAbs MyoAS CPS

Developmentalstagnation

Mild ataxia GPSW GPS Os (VPALTG LEVCLB)

F17 1 c155delA pQ52Rfs2de novo

13 4 GTCSMyo Abs AS

Developmentalregression

Dysarthriaataxia

GSW Os (LEVVPA)

F18 1 c431GgtT pC144F denovo

NA 0816 Abs Developmentalstagnation

Ataxiatremordysarthria

uk Sf (NA)

F19 1 c736 GgtC pA246P denovo

NA Since birthIS

Severelyimpaired

Severe motorand speechimpairment

Hypsarrhythmia Os (CBD)

F20 2 c(_242)_(3565_)del 2 (2) 2 AS (1) Abs(1) Myo (1)GTCS

Intellectualimpairment

Ataxia GPSW GSW Os (VPA)

F21 1 c383del pQ128Gfs2 NA 02Myoapnea andcyanosis

Developmentalstagnation

Hypotonia GPSW Sf (CLB)

Focal epilepsy

F22 1 c782GgtA pR261Q 6 6 GTCSFIAS

Normal Normal FSW (righttemporal)

Os (OXCLCM ZNS)

F23 1 c262GgtT pV88F NA 12 GTCSFIAS

Normal Normal FSW (lefttemporal)

Sf (LTG)

Abbreviations AaO = age at onset Abs = absence seizure AED = antiepileptic drug AS = atonic seizure atyp = atypical BR = bromide CBD = cannabidiolCBZ = carbamazepine CLB= clobazam CPS = complex partial seizure ESM= ethosuximide FIAS = focal impaired awareness seizure FS = febrile seizures FSW= focal sharp waves GPS = generalized polyspikes GPSW = generalized polyspikes and sharpwaves GSW = generalized sharp waves GTCS = generalizedtonic-clonic seizure IS = infantile spasms LCM = lacosamide LEV = levetiracetam LTG = lamotrigine Myo = generalized myoclonic seizure NA = notapplicable Os = ongoing seizures OXC = oxcarbazepine PB = phenobarbitone PHT = phenytoin Sf = seizure free STP = stiripentol TPM = topiramate TS =tonic seizure uk = unknown VGB = vigabatrin VPA = valproic acid wo = without ZNS = zonisamide

NeurologyorgN Neurology | Volume 92 Number 11 | March 12 2019 5

had a deletion of the entire gene (F20) Syntaxin-1B con-sists of an N-terminal helical domain containing the HAHB and HC domains the SNARE motif and the C-terminaltransmembrane domain (figure 1A) When in close proximitywith SNAP-25 and synaptobrevin syntaxin-1B forms theSNARE complex that catalyzes membrane fusion in Ca2+-triggered exocytosis Syntaxin-1B can adopt 2 conformationsopen and closed Both conformations are important for exo-cytosis the open conformation is necessary for the formationof the SNARE complex whereas the closed conformationinitiates the synaptic vesicle fusion reaction2324 Figure 1Ashows the new variants combined with those previouslyreported in a schematic view of the syntaxin-1B protein Incomparison to the location of loss-of-function variants mis-sense variants were more frequent in the second part of thegene containing (1) the SNARE motif conveying interactionwith the other 2 components of the SNARE complex and (2)

the transmembrane domain which is responsible for anchoringsyntaxin-1B into the cell membrane None of these variants wasreported so far in the gnomAD database currently the largestcollection of exomes and genomes publicly available20 Ingeneral STX1B is intolerant to missense variants (missense zscore gt363) and haploinsufficiency (pLI score of 094) in theExome Aggregation Consortium browser20 Evaluation bydifferent in silico prediction tools (SIFT17 PolyPhen-218

MutationTaster19) showed a damaging effect for 6 of 8 variantsin 3 of 3 prediction tools (table 2) We calculated the muta-tional density of missense variants in the general populationfrom the Exome Aggregation Consortium using a slidingwindow approach and could show that the variants are locatedin regions of different mutational density (figure 2B) We alsocalculated a paralog conservation score distribution for STX1B(para_zscore)21 and highlighted the patient variants (figure2C) This score gives the degree of conservation among paralog

Figure 1 STX1B gene with variants and pedigrees of newly identified variants

(A) Putative domain structure of syntaxin-1B derived from that for syntaxin-1A24 as the isoforms share 836 of their amino acid sequences (using thealignment programClustalO39) Shown are the functional domains and depiction of variants Missense variants are colored in black other variants are shownin gray The boxed variants represent developmental and epileptic encephalopathies (B) Pedigrees of sporadic patientsfamilies with newly identifiedvariants F21 was adopted and there was no information about the biological parents Abs = absence seizure AS = atonic seizure atyp = atypical CPS =complex partial seizure FE = focal epilepsy FIAS = focal impaired awareness seizure GEFS+ = genetic epilepsies with febrile seizures plus GGE = geneticgeneralized epilepsy GTCS = generalized tonic-clonic seizure IS = infantile spasms Myo = generalizedmyoclonic seizure TMR = transmembrane region TS =tonic seizure

6 Neurology | Volume 92 Number 11 | March 12 2019 NeurologyorgN

genes in the human Syntaxin gene family Disease variants areenriched in paralog-conserved residues21 In our paralog anal-ysis the SNARE motif turned out to be the most paralog-conserved and gnomAD variantndashdepleted region of the proteinwith the highest para_zscores (figure 3 A and B) Furthermorein a direct comparison of patient and gnomADmissense variantdistribution across the linear protein sequence we observeda significant enrichment of patient variants in the SNAREmotif(p = 0036) We ascertained missense variants in an unbiasedmanner so that SNARE motif enrichment by chance is im-probable The amino acid positions of most of the detectedmissense variants showed high or intermediate para_zscoresThere was no significant difference between the para_zscoresfrom missense variant positions in patients compared to thosein gnomAD (figure 3C) We also detected a few disease-associated variants in regions with a high mutational density ingnomAD and low para_zscores (pArg261Gln pIle282Thr)which were located in the transmembrane domain or the partlinking the SNARE motif with the transmembrane domain ofthe protein

Segregation of variants in families andfamily historiesThe segregation status for all variants in the respective pedi-grees together with a classification of the phenotypes isshown in figure 1B Previously published pedigrees can befound in Schubert et al12 We validated inheritance in 6 of thetotal 23 variants in families with more than one affected (4new and 2 published) They were mostly cosegregating withtheir phenotypes but also included both asymptomatic variantcarriers and phenocopies (F1 2 8 13 16 20) In 3 patientsa positive family history of epilepsy was available but addi-tional family members were not available for genetic testing

(F4 11 23) The available phenotypes of the other affectedindividuals are depicted in figure 1B

We showed de novo occurrence for 8 of the remaining 14 var-iants which were all detected in sporadic patients (F3 5 6 7 1417 18 19) whereas in 6 patients the respective family memberswere not available for segregation analysis (F9 10 12 15 21 22)

DiscussionThe current study expands the number of reported patientswith STX1B-related epileptic syndromes and describes 17 newvariants Four different phenotypes can be discerned (1)a benign epilepsy syndrome with febrile and afebrile seizurescorresponding to GEFS+ (2) a GGE phenotype (3) a DEEsyndrome with refractory seizures and moderate to severedevelopmental deficits and (4) a focal epilepsy phenotypeSTX1B-related epilepsies thus show a remarkable phenotypicheterogeneity that is in its extent reminiscent of other epilepsy-related genes such as SCN1A11 SCN2A25 KCNQ226 andSTXBP127 In comparison to STXBP1 which is closely relatedto STX1B (since the respective proteins interact with eachother in the synaptic transmitter release machinery) andexhibits in most cases an early onset of epilepsy presenting asOhtahara syndrome or West syndrome28 STX1B-related DEEshows a later onset of epilepsy after the first birthday Excep-tions to this rule are F19 (pA246P) and F21 (pQ128Gfs2)who presented with a seizure onset within the first days of lifeand are more reminiscent of STXBP1-related syndromes It isof interest that STX1B- and STXBP1-relatedDEEs both featureataxia and other movement disorders28 However in the vastmajority of STXBP1-related epilepsy syndromes moderate to

Table 2 Prediction scores and para_zscore of newly identified missense variants

GRCh37hg19position

Alternateallele

CDSposition

Proteinposition

Aminoacids SIFT PolyPhen-2

MutationTaster

para_zscore

1631004164 G 845 282 IT Deleterious(001)

Benign (0104) D (10) minus202

1631004455 T 782 261 RQ Deleterious(001)

Probably damaging(0981)

D (10) minus038

1631004464 T 773 258 SN Tolerated (006) Possibly damaging (0491) D (10) minus071

1631004501 G 736 246 AP Deleterious (0) Possibly damaging (0919) D (10) 028

1631004681 G 662 221 LP Deleterious (0) Probably damaging (1) D (10) minus071

1631004715 T 628 210 EK Deleterious (0) Probably damaging(0987)

D (10) 094

1631008304 A 431 144 CF Deleterious (0) Probably damaging(0998)

D (10) 028

1631012267 A 262 88 VF Deleterious(001)

Probably damaging(0913)

D (10) 094

Abbreviations PolyPhen-2 = polymorphism phenotyping v2 SIFT = sorting tolerant from intolerantReported missense variants with in silico prediction scores The table shows the genomic location of the variants in ascending order the amino acid changeand the predicted effect of these variants to the protein function by using SIFT PolyPhen-2 andMutationTaster as well as the paralog conservation score TheGenome Aggregation Database was used as a source for minor allele frequencies

NeurologyorgN Neurology | Volume 92 Number 11 | March 12 2019 7

severe developmental delay is present27ndash29mdashmilder pheno-types as in our cohort are not observed In comparison toother common DEE disorders the median time of seizureonset differentiates neonatal or early infantile for SCN2A andKCNQ2 (a later onset with these genes is rarer and associatedwith different functional consequencesmdashgain- vs loss-of-function mutations252630) and later infantile for SCN1A andSTX1B Also in other aspects parallels can be drawn withSCN1A-related disorders Thus in 5 STX1B-related DEE casesFS and the presence of myoclonic seizures were reminiscent ofDravet syndrome31 However the presence of tonic seizures inthese patients would be unusual forDravet syndrome A patient

with generalized tonic-clonic atonic and myoclonic seizureswith onset at 1 year and severe developmental delay wasreported before this study with a 12 Mb de novo 16p112deletion13 including the STX1B gene The phenotype wasclassified as epilepsy with myoclonic-atonic seizures a syn-drome that has been associated with SCN1A in severalpatients32 The previously reported patient showed a remark-able resemblance to F6 and F20 who also harbor a comparabledeletion Another similarity with SCN1A is that our cohortfeatures several patients and families with GEFS+ syndrome7

and one patient with mesial temporal lobe epilepsy and FS (forwhich common polymorphisms in SCN1A have been described

Figure 2 Mutational density and paralog conservation scores of STX1B missense variants

(A) Putative domain structure of syntaxin-1B (figure 1A) (B) Variation density in STX1B amino acid positions according to the Genome Aggregation Database(gnomAD) depicting the reported missense variants (C) Paralog conservation score (para_zscore) of the STX1B amino acid positions depicting the reportedmissense variants (positive values are considered as paralog conserved) Previously reported variants12 are colored in gray new variants in black TMR =transmembrane region

8 Neurology | Volume 92 Number 11 | March 12 2019 NeurologyorgN

as a genetic risk factor33) and at least one GEFS+ multiplexfamily with mesial temporal lobe epilepsy has been reported34

Of interest is our patient with episodes reminiscent of hemi-plegic migraine SCN1A mutations are a recognized cause offamilial hemiplegic migraine35 We acknowledge that the as-sociation with a migraine-like syndrome in a single patientcould also be coincidental In contrast classic GGE syndromessuch as represented by the 2 patients with JME in our cohortare not typically described in patients with SCN1A mutationsOf note both patients with JME had an atypical response ofpharmacoresistant seizures In the DEE group 7 of 13 (F3 F5F6 F14 F17 F18 F19) variants were de novo This supportsthe likelihood of a causative role in the context of the observedphenotypes Three missense variants showed a high paralogconservation (pC144F pG226R pA246P) increasing theprobability of a functional role An exception to this rule waspI282T (F14) a variant that was discovered using a genepanel approach Possibly despite the de novo status of thisvariant another as yet unknown genetic alteration in a gene notcaptured by the gene panel might be more relevant in this caseAlthough variant pV216E (F4) showed a slightly negativeparalog score this variant lies in a highly conserved region Thepatient had a positive family history and previous functionaldata ie rescue experiments in STX1B knockdown zebra fishlarvae12 support the pathogenicity of this variant In F13(pN189Afs5) both affected siblings had a similar phenotypeThis suggests an inheritance from the mother who has notbeen tested while the father was tested negative Since themother and other maternal family members were reportedlynot affected germline mosaicism could be possible Howeverother genetic causes cannot be entirely excluded because thevariant was found in a gene panel of 85 epilepsy genes In F15(pS258N) the prediction tools suggest a benign variant Sinceno family members were tested and the paralog score wasrather low the role of this variant has to be considered carefullyHowever the variant is located at the end of the SNAREmotifwhich supports a causative role In F16 (pL221P) the familyhistory suggests an inherited defect The variant was identifiedby exome sequencing and no other convincing variants werefound The unaffected mother also carries the variant but otheraffected family members were not available for testing Pre-diction tools indicate a deleterious variant and the variant islocated in themiddle of the SNAREmotif although the paralogscore was rather low a case of incomplete penetrance istherefore possible or the variant could represent a predisposingfactor for epilepsy In F20 the gene deletion was inherited fromthe father who had a milder phenotype suggesting the pres-ence of other genetic modifiers In F21 (pQ128Gfs2) theparents were not tested and the variant was identified in a genepanel of 115 candidate genes so that other genetic causescannot be excluded In the GEFS+ group in 3 of 6 cases (F1F2 F8) a positive family history with cosegregating geneticfindings support the causative role of the reported variants F7(pS10Afs7) was confirmed to be de novo For F9 (pR245)and F10 (pY140) the parents were not tested Since all

Figure 3 Mutational density and paralog conservationscore of different gene regions

(A) Distribution of mutational density of 4 STX1B gene regions The SNAREmotif shows the lowest mutational density the transmembrane region(TMR) the highest density (B) Paralog conservation score (para_zscore)distribution of 4 STX1B gene regions The SNARE motif shows the highestparalog conservations the TMR the lowest conservation (C) Comparison ofparalog conservation score for all GenomeAggregation Database (gnomAD)variants and patient variants

NeurologyorgN Neurology | Volume 92 Number 11 | March 12 2019 9

variants in this group predict a complete loss of function weconsider them as disease-causing and the mechanism for theGEFS+ group seems to be clearly a haploinsufficiency In theGGE group neither case had parents who were tested F11(pE210K) has a positive family history that is suggestive of aninherited disease The causative role of this variant is under-scored by the in silico findings that the variant is predicteddeleterious lies in a region of very low mutational density andshows high paralog conservation pK93 (F12) leads toa haploinsufficiency in line with the variants causing GEFS+Furthermore this variant was identified by whole-genome se-quencing and no other convincing variants were found Of in-terest in a recent genome-wide association study of commonepilepsies a significant signal in the region of STX1B wasdetected in the JME cohort36 In the FE group the role ofSTX1B should be considered more carefully In F22 (pR261Q)and F23 (pV88F) parents were not tested For pR261Q theestablished prediction tools point toward a deleterious variantwhile the position shows medium mutational density and lowparalog conservation However whole-genome sequencingshowed no other likely pathogenic variants For pV88F theused prediction tools show a more coherent picture of a dele-terious variant the paralog score was high and whole-exomesequencing found no other convincing variants

Regarding genotype-phenotype correlation it is notable thatmissense variants in the functionally relevant SNARE motifwere identified in 6 of 7 patients or families (86) associatedwith DEE The seventh patient (pE210K) had refractoryJME In contrast all but one of the GEFS+ patients or familieshad truncating variants (5 of 6 83) predicting nonsense-mediated decay and complete loss of function of one alleleindicating haploinsufficiency as the disease mechanism

A possible explanation could be that missense variants in crucialprotein regions such as the SNAREmotif may lead to a deficientgene product that interferes with protein-protein interactions andnormal presynaptic vesicle fusion (dominant-negative effect)whereas missense variants in the first part of the gene may affectassembly of syntaxin-1B proteins and therefore have similarconsequences as a truncation with nonsense-mediated decayThe loss-of-function mechanisms could also be compensatedmdashat least in partmdashby syntaxin-1A and thus be less damaging37

We demonstrate that STX1B variants are linked to 4 differentepilepsy phenotypes De novo dominant mutagenesis is the eti-ology of most DEEs whereas the more common epilepsies arelikely complex genetic disorders This work suggests a role forultrarare STX1B variants in the pathogenesis of a broad range ofboth common and rare epilepsies There are plausible explan-ations for genotype-phenotype correlations such that hap-loinsufficiency causes rather benign phenotypes whereasmissense variants in functionally critical regions cause more se-vere phenotypes Other unknown factors which may includegenetic modifier individual genetic background and epigeneticeffects could also have an important role and explain phenotypicheterogeneity reduced penetrance and variable phenotypes

among carriers of truncating mutations Functional studies ona protein and cellular neuronal level will shed more light on therelevance and degree of severity of the described variants andcontribute to a better understanding of the dysfunction ofsyntaxin-1B and synaptic transmission in epilepsy and strengthenour current interpretation of genotype-phenotype correlations

Author contributionsS Wolking study concept and design phenotyping and acqui-sition of data analysis and interpretation writing of the manu-script P May analysis and interpretation writing of themanuscript D Mei genotyping and acquisition of data RSMoslashller phenotyping and acquisition of data critical revision ofthe manuscript for important intellectual content S Balestriniphenotyping and acquisition of data KL Helbig acquisition ofdata C Desmettre Altuzarra phenotyping and acquisitionof data N Chatron phenotyping and acquisition of dataC Kaiwar phenotyping and acquisition of data K Stohr phe-notyping and acquisition of data P Widdess-Walsh pheno-typing and acquisition of data BA Mendelsohn phenotypingand acquisition of data A Numis phenotyping and acquisitionof data MR Cilio phenotyping and acquisition of data W VanPaesschen phenotyping and acquisition of data LL Svendsenphenotyping and acquisition of data S Oates phenotyping andacquisition of data E Hughes phenotyping and acquisition ofdata S Goyal phenotyping and acquisition of data K Brownacquisition of data M Sifuentes Saenz phenotyping and ac-quisition of data T Dorn phenotyping and acquisition of dataH Muhle phenotyping and acquisition of data AT Pagna-menta acquisition of data critical revision of the manuscript forimportant intellectual content DV Vavoulis acquisition of dataSJL Knight acquisition of data critical revision of the manu-script for important intellectual content JC Taylor acquisitionof data critical revision of the manuscript for important in-tellectual content MP Canevini phenotyping and acquisitionof data F Darra phenotyping and acquisition of data RHGavrilova phenotyping and acquisition of data Z Powis ac-quisition of data S Tang acquisition of data J Marquetandphenotyping and acquisition of data M Armstrong acquisitionof data D McHale acquisition of data EW Klee phenotypingand acquisition of data critical revision of the manuscript forimportant intellectual content GJ Kluger phenotyping andacquisition of data critical revision of the manuscript for im-portant intellectual content DH Lowenstein phenotyping andacquisition of data critical revision of the manuscript for im-portant intellectual content S Weckhuysen phenotyping andacquisition of data critical revision of the manuscript for im-portant intellectual content DK Pal phenotyping and acqui-sition of data critical revision of the manuscript for importantintellectual content I Helbig phenotyping and acquisition ofdata critical revision of the manuscript for important intellectualcontent R Guerrini phenotyping and acquisition of data criticalrevision of the manuscript for important intellectual contentanalysis and interpretation RH Thomas phenotyping andacquisition of data critical revision of the manuscript for im-portant intellectual content analysis and interpretation MIRees phenotyping and acquisition of data critical revision of the

10 Neurology | Volume 92 Number 11 | March 12 2019 NeurologyorgN

manuscript for important intellectual content analysis and in-terpretation G Lesca phenotyping and acquisition of datacritical revision of the manuscript for important intellectualcontent SM Sisodiya phenotyping and acquisition of datacritical revision of the manuscript for important intellectualcontent analysis and interpretation YG Weber phenotypingand acquisition of data critical revision of the manuscriptfor important intellectual content D Lal analysis and in-terpretation writing the manuscript C Marini phenotypingand acquisition of data critical revision of the manuscript forimportant intellectual content analysis and interpretationH Lerche study concept and design analysis and interpretationwriting of the manuscript study supervision J Schubert studyconcept and design analysis and interpretation writing of themanuscript study supervision

AcknowledgmentThe authors thank the families and the study participants fortheir permission to undertake the research and the Epi4KConsortium for contributing genetic data of one of the casesfrom Allen et al 201338 and the EuroEPINOMICS-RESMAEWorking Group for contributing one of the cases

Study fundingThis work was supported by the German Research Foundation(DFG grants Le103016-1 We48964-1 He54157-1 andKr50932-1) by the German Federal Ministry of Education andResearch (BMBF in the frame of the ERA-NET NEURONprogram project SNAREopathies grant 01EW1809A) andby the Medical Faculty of the University of Tubingen via theClinician Scientist Program (418-0-0) and the Fortune Pro-gram (2306-0-0) This work was also supported by thefoundation ldquono epileprdquo by the National Institute for HealthResearch (NIHR) Biomedical Research Centre Oxford withfunding from the Department of Healthrsquos NIHR BiomedicalResearch Centrersquos funding scheme Epilepsy Research UKproject grant P1104 Part of this work was undertaken atUniversity College London Hospitals which received a pro-portion of funding from the NIHR Biomedical ResearchCentre funding scheme The work was also supported bya Wellcome Trust Strategic Award (WT104033AIA) theMuir Maxwell Trust and the Epilepsy Society UK The workwas also supported by the National Institute of NeurologicalDisorders and Stroke (The Epilepsy PhenomeGenomeProject NS053998 Epi4KNS077364 NS077274 NS077303and NS077276) This work was also supported by grants fromthe Canadian Institutes of Health Research (201503MOP-342469 DKP) European Union Program of the SeventhFramework Program Development of Strategies for In-novative Research to improve diagnosis prevention andtreatment in children with difficult to treat epilepsy ldquoDE-SIRErdquo (EU grant agreement FP7 602531) NIH Research(DKP) Medical Research Council (DKP) WaterlooFoundation (DKP) Charles Sykes Epilepsy Research Trust(DKP) NIHR Specialist Biomedical Research Centre forMental Health of South London and Maudsley NHS Foun-dation Trust (DKP)

DisclosureThe authors report no disclosures relevant to the manuscriptGo to NeurologyorgN for full disclosures

Publication historyReceived by Neurology April 5 2018 Accepted in final form November4 2018

References1 Cossette P Liu L Brisebois K et al Mutation of GABRA1 in an autosomal dominant

form of juvenile myoclonic epilepsy Nat Genet 200231184ndash1892 Striano P Weber YG Toliat MR et al GLUT1 mutations are a rare cause of familial

idiopathic generalized epilepsy Neurology 201278557ndash5623 Chen Y Lu J Pan H et al Association between genetic variation of CACNA1H and

childhood absence epilepsy Ann Neurol 200354239ndash2434 Helbig I Mefford HC Sharp AJ et al 15q133 microdeletions increase risk of idio-

pathic generalized epilepsy Nat Genet 200941160ndash1625 Scheffer IE Berkovic SF Generalized epilepsy with febrile seizures plus a genetic

disorder with heterogeneous clinical phenotypes Brain 1997120479ndash4906 Singh R Scheffer IE Crossland K Berkovic SF Generalized epilepsy with febrile

seizures plus a common childhood-onset genetic epilepsy syndrome Ann Neurol19994575ndash81

7 Escayg AMacDonald BTMeislerMH et al Mutations of SCN1A encoding a neuronalsodium channel in two families with GEFS+2 Nat Genet 200024343ndash345

8 Wallace RH Scheffer IE Parasivam G et al Generalized epilepsy with febrile seizuresplus mutation of the sodium channel subunit SCN1B Neurology 2002581426ndash1429

9 Wallace RH Marini C Petrou S et al Mutant GABA(A) receptor gamma2-subunit inchildhood absence epilepsy and febrile seizures Nat Genet 20012849ndash52

10 Dibbens LM Feng HJ Richards MC et al GABRD encoding a protein for extra- orperi-synaptic GABAA receptors is a susceptibility locus for generalized epilepsiesHum Mol Genet 2004131315ndash1319

11 Zuberi SM Brunklaus A Birch R Reavey E Duncan J Forbes GH Genotype-phenotype associations in SCN1A-related epilepsies Neurology 201176594ndash600

12 Schubert J Siekierska A Langlois M et al Mutations in STX1B encoding a pre-synaptic protein cause fever-associated epilepsy syndromes Nat Genet 2014461327ndash1332

13 Vlaskamp DRM Rump P Callenbach PMC et al Haploinsufficiency of the STX1Bgene is associated with myoclonic astatic epilepsy Eur J Paediatr Neurol 201620489ndash492

14 Smirnova T Miniou P Viegas-Pequignot E Mallet J Assignment of the humansyntaxin 1B gene (STX) to chromosome 16p112 by fluorescence in situ hybridiza-tion Genomics 199636551ndash553

15 Lerche H Weber YG Baier H et al Generalized epilepsy with febrile seizures plusfurther heterogeneity in a large family Neurology 2001571191ndash1198

16 Weber YG Jacob M Weber G Lerche H A BFIS-like syndrome with late onset andfebrile seizures suggestive linkage to chromosome 16p112ndash16q121 Epilepsia 2008491959ndash1964

17 Kumar P Henikoff S Ng PC Predicting the effects of coding non-synonymous variantson protein function using the SIFT algorithm Nat Protoc 200941073ndash1081

18 Adzhubei IA Schmidt S Peshkin L et al A method and server for predicting dam-aging missense mutations Nat Methods 20107248ndash249

19 Schwarz JM Cooper DN Schuelke M Seelow D MutationTaster2 mutation pre-diction for the deep-sequencing age Nat Methods 201411361ndash362

20 Lek M Karczewski KJ Minikel EV et al Analysis of protein-coding genetic variationin 60706 humans Nature 2016536285ndash291

21 Lal DMay P Samocha K et al Gene family information facilitates variant interpretationand identification of disease-associated genes bioRxiv [online serial] Epub 2017 Jan 1Available at biorxivorgcontentearly20170705159780abstract

22 Waterhouse AM Procter JB Martin DMA Clamp M Barton GJ Jalview version 2a multiple sequence alignment editor and analysis workbench Bioinformatics 2009251189ndash1191

23 Khvotchev M Dulubova I Sun J Dai H Rizo J Sudhof TC Dual modes of Munc18-1SNARE interactions are coupled by functionally critical binding to syntaxin-1 Nterminus J Neurosci 20072712147ndash12155

24 Gerber SH Rah JC Min SW et al Conformational switch of syntaxin-1 controlssynaptic vesicle fusion Science 20083211507ndash1510

25 WolffM Johannesen KM Hedrich UBS et al Genetic and phenotypic heterogeneitysuggest therapeutic implications in SCN2A-related disorders Brain 20171401316ndash1336

26 Weckhuysen S Ivanovic V Hendrickx R et al Extending the KCNQ2 encephalop-athy spectrum clinical and neuroimaging findings in 17 patients Neurology 2013811697ndash1703

27 Carvill GL Weckhuysen S McMahon JM et al GABRA1 and STXBP1 novel geneticcauses of Dravet syndrome Neurology 2014821245ndash1253

28 Di Meglio C Lesca G Villeneuve N et al Epileptic patients with de novo STXBP1mutations key clinical features based on 24 cases Epilepsia 2015561931ndash1940

29 Saitsu H Kato M Mizuguchi T et al De novo mutations in the gene encodingSTXBP1 (MUNC18-1) cause early infantile epileptic encephalopathy Nat Genet200840782ndash788

NeurologyorgN Neurology | Volume 92 Number 11 | March 12 2019 11

30 Miceli F Soldovieri MV Ambrosino P et al Early-onset epileptic encephalopathycaused by gain-of-function mutations in the voltage sensor of Kv72 and Kv73 po-tassium channel subunits J Neurosci 2015353782ndash3793

31 Dravet C Guerrini R Dravet Syndrome Montrouge John Libbey Eurotext 201132 Tang S Pal DK Dissecting the genetic basis of myoclonic-astatic epilepsy Epilepsia

2012531303ndash131333 Kasperaviciute D Catarino CB Matarin M et al Epilepsy hippocampal sclerosis and

febrile seizures linked by common genetic variation around SCN1A Brain 20131363140ndash3150

34 Abou-Khalil B Ge Q Desai R et al Partial and generalized epilepsy with febrileseizures plus and a novel SCN1A mutation Neurology 2001572265ndash2272

35 Dichgans M Freilinger T Eckstein G et al Mutation in the neuronal voltage-gatedsodium channel SCN1A in familial hemiplegic migraine Lancet 2005366371ndash377

36 The International League Against Epilepsy Consortium on Complex EpilepsiesGenome-wide mega-analysis identifies 16 loci and highlights diverse biologicalmechanisms in the common epilepsies Nat Commun 201895269

37 Zhou P Pang ZP Yang X et al Syntaxin-1 N-peptide and Habc-domain performdistinct essential functions in synaptic vesicle fusion EMBO J 201332159ndash171

38 Allen AS Berkovic SF Cossette P et al De novo mutations in epileptic encephalo-pathies Nature 2013501217ndash221

39 McWilliam H Li W Uludag M et al Analysis Tool Web Services from the EMBL-EBI Nucleic Acids Res 201341W597ndashW600

12 Neurology | Volume 92 Number 11 | March 12 2019 NeurologyorgN

DOI 101212WNL0000000000007089 published online February 8 2019Neurology

Stefan Wolking Patrick May Davide Mei et al -related epileptic disordersSTX1BClinical spectrum of

This information is current as of February 8 2019

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