12/4/13

Outline The importance of genetic diagnosis in early onset • The genetic basis of early onset epilepsy Childhood Epilepsy Masterclass • Emerging/novel phenotypes 28th November 2013 • Novel diagnostic approaches- what can we do? • When/how does a genetic diagnosis make a difference? Dr Amy McTague • What does the future hold? MRC Research Fellow, Neurosciences Unit, UCL-Institute of Child Health

How much of early onset epilepsy is genetic?? Important genetic diagnoses- metabolic disorders

• SLC2A1 • Antiquitin

What do we mean by “genetic”? The genetic basis of early onset epilepsy

• Monogenetic • Early onset monogenic (or CNV-related) epileptic • Copy number variation encephalopathies where seizures are the prominent feature and imaging is normal • Recognisable genetic syndromes • Chromosomal disorders • Syndromes with severe developmental delay and epilepsy amongst other features • Polygenic/complex • Multifactorial- 2 hits

1 12/4/13

Ohtahara EMEE MPSI West Dravet/SMEI

How to classify genetic early onset epilepsy? Seizure types Tonic spasms Myoclonic Focal +/- autonomic Spasms Febrile hemi-clonic features, migrating Spasms

Other features DD Movement disorder DD Later gait disorder Dyskinesia Evolution to WS • Electroclinical syndrome EEG BS BS Migrating foci Hyps Normal initially • OMIM classification- EIEE1-18 Hyps BS

MRI MCD May be normal Atrophy Variety of structural Normal Hypomyelination Delayed/hypo malformations myelination WMH

Aetiology Structural Metabolic Genetic Multiple Genetic

Genetic causes: STXBP1 SLC25A22 KCNT1 ARX, CDKL5, SCN1A ARX SLC25A22, SCN1A SCN1A, SCN2A, (PCDH19) KCNQ2 PLCB1. TBC1D24 PNKP, ST3GAL3 SCN2A, CASK etc etc

The complexities of diagnosis in early onset The complexities of diagnosis in early onset epilepsy- genetic heterogeneity epilepsy-phenotypic pleiotropy

ARX

SCN2A CDKL5 Dravet West syndrome syndrome

West Syndrome SCN1A PLCB1 SCN1A

Non-specific MPSI ARGEF2 MAGI2 EIEE

EIEE Ohtahara EMEE MPSI West Dravet Others EIEE Gene Ohtahara EMEE MPSI West Dravet Others 1 ARX + + IE-DEE, XMESID, XLISS 1 ARX + + IE-DEE, 2 CDKL5 + Rett-like EIEE XMESID, ISSX-2 XLISS 3 SLC25A22 + + 4 STXBP1 + + Non-specific EIEE 2 CDKL5 + Rett-like EIEE 5 SPTAN1 + ISSX-2 6 SCN1A + + + SIMFE 7 KCNQ2 + Non-specific EIEE 3 SLC25A22 + + BFNS

8 ARHGEF9 Progressive EIEE 4 STXBP1 + + Non-specific with hyperekplexia EIEE 9 PCDH19 + EFMR 10 PNKP + Non-specific EIEE 5 SPTAN1 + MCSZ

11 SCN2A + Non-specific EIEE 6 SCN1A + + + SIMFE BFIS3

12 PLCB1 + + 7 KCNQ2 + Non-specific 13 SCN8A Non-specific EIEE EIEE BFNS 14 KCNT1 +

15 ST3GAL3 + 8 ARHGEF9 Progressive EIEE with 16 TBC1D24 + FIME, Non-specific EIEE hyperekplexia

17 GNAO1 + EIEE with MD 9 PCDH19 + EFMR

18 SZT2 Non-specific EIEE with dysmorphism

2 12/4/13

10 PNKP + Non-specific EIEE MCSZ STXBP1- not just Ohtahara syndrome? 11 SCN2A + Non-specific EIEE BFIS3

12 PLCB1 + +

13 SCN8A Non-specific EIEE • First described in a deletion in OS and then in 14 KCNT1 + larger OS cohorts- ~30% hit rate

15 ST3GAL3 + • De novo, heterozygous

16 TBC1D24 + FIME, Non-specific • Spectrum of phenotypes from West syndrome to EIEE ID without seizures- limited number of cases

17 GNAO1 + EIEE with MD • Movement disorder common- tremor, dyskinesia

18 SZT2 Non-specific EIEE with dysmorphism

Distinct genotype-phenotype correlations in Dravet syndrome early onset epilepsy: Dravet syndrome • 70-80% sequencing mutations in SCN1A • 1 in 40,000 children 40% truncating • febrile or afebrile generalised or hemiconvulsions 40% missense starting in the first year of life remainder splice site • seizure evolution- intractable generalised (myoclonic or atonic seizures, atypical absences)and focal • 2–3% Intragenic/whole gene deletions of SCN1A seizures +/- other contiguous (12.5% of mutation- negative DS) • normal early development, subsequent developmental delay and behavioral problems • Evolving spasticity and crouch gait

Difficulties in genetic diagnosis in Dravet What is the impact of a genetic diagnosis in DS?

Clinican’s views Parent’s views: • Usually de novo (90-95%) although often a family • Additional investigations were • 87% helpful in giving an avoided in 67% explanation history of FS/epilepsy • 69% altered treatment • 55% led to a change in • Germline/somatic mosaicism in ~7% DS families • 74% helped medication choice. treatment • Improved seizure control in 42% • 69% of this group fewer • ?explanation for intrafamilial phenotypic variability after medication change seizures • Lack of consistent genotype-phenotype correlation • 50% avoided inappropriate • 34% improvement in drugs developmental progress • SCN1A negative DS patients: PCDH19, GABRg2, • 41% other benefits: improved SCN1B access to therapies/ respite care/change of goals.

Brunklaus et al. Dev Med Child Neurol. 2013 Feb;55(2):154-61.

3 12/4/13

What is the impact of a genetic diagnosis in Emerging genotype-phenotype correlations DS? in early onset epilepsy:

• Earlier diagnosis- better treatment of status • PCDH19 • Avoidance of carbamazepine • KCNQ2 • Use of appropriate medications • CDKL5 • ?impact on long term cognitive outcome • CHD5 • KCNT1

Case example Key features of protocadherin 19 (PCDH19) related epilepsy • 12 months • Half-sister aged 4 • Explosive onset of years: clusters of GTC epileptic from 13 months, • seizures beginning by age 3 years encephalopathy with moderate learning • 67% of affected females have learning difficulties/ developmental difficulties and ASD of borderline intellect. regression • Mum- clusters of • Early development varies from normal to • Ix (MRI, CSF, seizures from 13 abnormal; developmental regression commonly autoimmune months to 14 years of occurs with seizure onset. aetiologies, age • obsessive features in 33%, aggressive behaviour mitochondrial disease) in 26%, ASD in 22% negative

PCDH19 related epilepsy • If girl with Dravet syndrome SCN1A negative, then test PCDH19 • Families with only girls affected – Unusual: X-linked - females affected, male carriers – Cellular interference • Spectrum of severity in females • Seizures may burn-out in teenage years • Clobazam can be helpful • Important diagnosis due to counselling implications

4 12/4/13

KCNQ2 related EIEE CDKL5 – not just an early onset Rett • 8/80 with EIEE of unknown syndrome cause had KCNQ2 mutations • infantile spasms by 5th month • Seizure onset first week of • peculiar seizure pattern with a tonic-vibratory life contraction, followed by a clonic phase with series • Tonic seizures with of spasms,gradually translating into repetitive autonomic features distal myoclonic jerks • EEG – SBS or multifocal • Poor gaze, hand stereotypies and bruxism • MRI- basal ganglia hyperintensities, some • But 30% will walk and epilepsy may resolve in transient 50% • Retigabine • Rett-like- consider MECP2, FOXG1, WDR45

CHD2 related epilepsy Migrating Partial Seizures of Infancy (MPSI) • Onset before 6 months

• Normal development before seizure onset

• Identified as candidate gene within CNV • Migrating focal motor seizures at onset • In 500 EIEE cases, 1.2% had de novo het • Characteristic semiology & EEG mutations in CHD2 • Onset 12 months -3 years (median 18 months) • Multifocal seizures - intractable to conventional AEDs • Photosensitive ++ • No identified aetiology • Myoclonic and atonic seizures amongst others • Profound psychomotor delay • 7/9 SCN1A-negative Dravet syndrome cases in • Rare ~100 cases reported to date recent series

The British Paediatric Neurology Surveillance Unit study Materials and Methods

• National survey of Migrating Partial Seizures in Infancy • 17 possible cases identified, 14 met criteria • Whole exome sequencing (Illumina/Hi-Seq) (n=5) • Rough prevalence of 0.11 per 100,000 • Novel clinical features identified • Direct Sanger sequencing (n=4) • Novel EEG features • Segregation studies with parental DNA • Novel radiological features

 Molecular genetic investigation

5 12/4/13

Results KCNT1

• sodium-acvated potassium • 3 paents idenfied with mutaons in KCNT1 channel subunit aka KCa4.1/ SLACK • Binds to KCNT2(SLICK) to • ~30% hit rate form heterotetrameric channel complexes • Slow hyperpolarisaon aer • 2 paents with previously idenfied mutaon repeve firing • Highly expressed in brain pVal271Phe RCK c.2800G>A; p.Ala934Thr NAD domains binding domain NH • 1 paent with novel mutaon 2 pAla934Thr COOH c.811G>T ; p.Val271Phe

The genec basis of MPSI MPSI GENE n GENE FUNCTION INHERITANCE REFERENCE (S) Na acvated K channel Clinical features of KCNT1 positive patients subunit De novo autosomal Barcia et al 2012 KCNT1 8 ion conductance/ dominant McTague et al 2013 Patient Age at MRI Novel EEG Novel Initial Later Age at Age developmental seizure findings clinical neurology neurology death signaling pathways. onset features exam exam GTPase-acvang (weeks) TBC1D24 2 regulaon of Autosomal recessive Milh et al 2013 membrane trafficking. 1 2 Atrophy - GI No Axial 5 yrs 4 - HM with dysmotility hypotonia months G-protein coupled enzyme, IP3/DAG WMI formaon, MRS low PLCB1 1 Autosomal recessive Poduri et al 2012 NAA intracellular transducon of many 2 4 Atrophy Electro Movement Yes- axial Peripheral - 5 yrs HM with decrementation disorder hypotonia, hypertonia extracellular signals. WMI (7.5 months) peripheral and alpha subunit of voltage gated sodium spasticity spasticity De novo autosomal Carranza et al 2011 SCN1A 3 channel evoking dominant Freilich et al 2011 neuronal acon 3 2 Normal Subtle BS - No Axial 19 - potenals. Normal (6 weeks) hypotonia months Alpha subunit of De novo autosomal MRS Hyps SCN2A 1 voltage gated sodium Dhamija et al 2013 dominant (6 months) channel Mitochondrial Autosomal recessive Poduri et al 2011 SLC25A22 1 glutamate transport

Copy number variation in EIEE The revolution in genetic technologies • Micro-deletion syndromes associated with epilepsy e.g Angelman’s (15q11.2-q13), • Mefford et al: 315 patients with infantile and childhood epileptic-7.9% to have CNVs, half of which were clearly pathogenic. • well recognised epilepsy–associated genes such as ARX • duplications of 16p11.2- previously reported in migrating partial seizures in infancy. • Microarray has a good yield in the investigation of EIEE

6 12/4/13

New approaches to genetic diagnosis- gene Next gen gene panels panel at GOSH 1. Digest DNA Sample is fragmented using restriction enzymes • Often difficult to predict genotype from phenotype 2. Hybridize probes Probe library is added and hybridized to the • Considerable cost to NHS to sequentially screen targeted fragments genes making them form a Halo shape. 3. Purify and ligate targets Probe/Fragment hybrids are retrieved with magnetic streptavidin • Develop a multiple gene panel beads. The circular molecules are then closed by ligation • In collaboration with diagnostic genetic colleagues 4. Amplify targeted fragments Only circular DNA targets are amplified. • Simultaneous screening of 29 genes Sample barcodes are introduced. Final product is ready for • Pilot study sequencing • Translation into clinical practice

Materials and Methods Data analysis • 48 children • NextGENe software (Softgenetics) • Early onset seizures, severe global • Array data: CGH Fusion (InfoQuant) developmental delay • Pre-screening with • Point mutations microarray/methylation (missense,nonsense ,insertions,deletions) studies (Angelman’s syndrome) (i) Confirmed by traditional Sanger sequencing • Copy number variants - exon-level aCGH (Roche (ii) Familial segregation studies Nimblegen) • Point mutations – next • Single exon-level copy number variants generation sequencing methods (Haloplex Confirmed by Multiplex ligation-dependent probe custom capture /Illumina) amplification (MLPA) or quantitative PCR

RESULTS Patient Sex Presentation Gene Mutation 1 F Microcephaly CDKL5 49kb Seizures (5 Duplication • Mutations identified in 5 patients (~10%) weeks) Exons 3-7 • Data quality high: >30x sequence data coverage, GDD 2 F Microcephaly MECP2 Nonsense 90% of targeted coding bases, no false positive Seizures mutation calls made GDD 3 F Microcephaly MECP2 3kb deletion GDD Exon 4 4 M Microcephaly SLC9A6 c.608delA Seizures GDD Dyskinesia 5 M Microcephaly EHMT1 c.1596delC GDD

7 12/4/13

PANEL ROUND 2 Panel Round 2

• expanded gene list • CDKL5; MECP2; PCDH19; ARHGEF9; ARX; • improved coverage ATRX; SLC16A2; SLC9A6; ADSL; CNTNAP2; • current hit rate 10-20% KIAA1279; NRXN1; PLCB1; POLG; PNKP; • mutations identified in FOXG1, UBE3A, KCNT1, SLC25A22; SLC2A1 [GLUT1]; UBE3A; ATP1A3; CDKL5, SCN2A to date CHRNA2; CHRNA4; CHRNB2; EHMT1; FOXG1; GABRG2; KCNT1; KCNQ2; LGI1; MAGI2; MBD5; MEF2C; PRRT2; SCN1A; SCN2A; SIP1 [ZEB2]; SPTAN1; STXBP1; SYNGAP1; TCF4; UBE2A; SCN1B; SCN8A; SMARCA2; ARID1A; ARID1B; SMARCA4; SMARCB1; SMARCE1.

Gene panels in childhood epilepsy- published experience Whole exome in early onset epilepsy- published experience Author Population No. of genes Technique Hit rate Comments Author No of Phenotype Hit rate Novel Comments Lemke 2012 33 patients with 265 Agilent 48% Vary from non- subjects genes childhood onset SureSelect/ screened epilepsy of SOLiD4 Dravet’s cases identified varying to non-specific Veeramah 10 triomes 3 WS, 2 7/10 de KCNH5 Small phenotypes MR with seizures 2013 DS, novo CLCN4 cohort Carvill 2013 500 patients with 65(19 known, 46 MIPs/HiSeq 10% Novel gene remainder causative ARHGEF15 Functional childhood onset candidate) Infantile spasms: discovery- non-specific mutation validation EE 5% CHD2, Ohtahara: 50% SYNGAP1 Epi4K 264 triomes Infantile 29 patients GABRB3 Little info on Expanded consortium spasms, with de ALG13 phenotypes phenotypic 2013 LGS novo No spectra of mutations functional SCN1A, SCN2A and SCN8A validation Kodera 2013 53 patients with 35 (30 known, 5 Agilent 22% High coverage EOEE candidate) SureSelect/ and long reads- Illumina able to detect small CNVs

Pathways in EIEE Gene panel or whole exome? Solute Stabilisation of transport membrane (glutamate Gene Whole transport) panel exome SPTAN1 Abnormal SLC25A22 Transcription synaptic (dys)regulation function ARX Limits number of variants Large number of putative STXBP1, for investigation mutations to investigate- CDKL5 ARGHEF9 FOXG1 PLCB1, MAGI2 Does not identify copy Exon level CGH number variations

Unbiased screening of Limited to genes in panel genome-novel gene but can add new genes Channelopathies Severe DNA repair identification early SCN1A, SCN2A, PNKP Highly targeted, good SCN8A, KCNQ2, onset Coverage limited coverage KCNT1 epilepsy CHD2

8 12/4/13

Conclusions Acknowledgements • Dr Manju Kurian • Professor Helen Cross • Professor Rod Scott • Dr Esther Meyer • A genetic diagnosis may limit investigations and • Dr Jo Ng direct treatment • Dr Adeline Ngoh • Colleagues at GOSH Genetics Service • Psychological/goal-setting benefits • Professor Nick Lench • Dr Richard Scott • Dr Jane Hurst • ?Benefits of a label • Helen Moody • Natalie Trump • Advancing understanding of common pathways/ • Diagnostic clinical scientists • Paediatric Neurology Colleagues at GOSH influence on networks Dr Sophie Varadkar, Dr Christin Eltze, Dr Sarah Aylett, Dr Krishna Das, Dr Robert Robinson, Dr Cheryl Hemingway, Dr Carlos De Sousa, Dr Lucinda • Development of novel treatments Carr, Dr Sanjay Bhate, Dr Prab Prabhakar, Dr Vijeya Ganesan, Dr Catherine de Vile • Caveats: limited ability to prognosticate, difficulties Please contact us regarding any patients with of diagnosis EIEE [email protected]

9