A Genomic Approach to Epilepsy: From gene discovery to precision therapy
Heather C. Mefford, MD, PhD Associate Professor, Pediatrics Division of Genetic Medicine University of Washington
2018 ISBP Tel Aviv Epilepsy
• Recurrent, unprovoked seizures
• Excessive, hypersynchronous activity in the brain
• Prevalence ~0.5-1%
• 50 million affected worldwide
2018 ISBP Tel Aviv Epilepsy Classification
• Seizure type – Generalized – Focal
• Age of onset>50 syndromes described – Infancy, childhood, adolescence, adult
• Etiology
– Genetic CUREepilepsy.org – Acquired
2018 ISBP Tel Aviv Epilepsy Classification
Generalized Focal Epilepsy Epilepsy (GGE) JME ADNFLE CAE Developmental ADPEAF TLE GEFS+ & Epileptic Encephalopathy* (DEE) Dravet Ohtahara Infantile spasms LGS 2018 ISBP Tel Aviv Genetics of Epilepsy
• Twin concordance – GGE ~80% – Focal ~36%
• Family studies
– Overall RR 2-5% Helbig et al., 2008, Lancet Neurol – Evidence for linkage in some families – De novo mutations in severe cases
2018 ISBP Tel Aviv Gene discovery in epilepsy
STX1B SLC6A1 Next-gen GABRA1 GABRB3 Sequencing Era SIK1 KCNA2 ALG13 GRIN2B PURA KCNB1 KCNC1 DNM1 HCN1 Microdeletions CHD2 SCN8A 15q13.3 SCN2A 16p13.11 GRIN2A 15q11.2 DEPDC5 KCNT1 PRRT2 TBC1D24 STXBP1 PCDH19
GABRA1 LGI1 CACNA1H GABRG2 ARX CDKL5 Channelopathy SCN1A Era SLC2A1 KCNQ3 KCNQ2 SCN1B CHRNA4
1995 1997 1999 2001 2003 2005 2007 2009 2011 2013 2015
2018 ISBP Tel Aviv Gene discovery in epilepsy
STX1B SLC6A1 GABRA1 GABRB3 SIK1 KCNA2 ALG13 GRIN2B PURA KCNB1 KCNC1 DNM1 HCN1 Microdeletions CHD2 SCN8A 15q13.3 SCN2A 16p13.11 GRIN2A 15q11.2 DEPDC5 KCNT1 PRRT2 TBC1D24 STXBP1 PCDH19
GABRA1 LGI1 CACNA1H GABRG2 ARX CDKL5 Channelopathy SCN1A Era SLC2A1 KCNQ3 KCNQ2 SCN1B CHRNA4
1995 1997 1999 2001 2003 2005 2007 2009 2011 2013 2015
2018 ISBP Tel Aviv Gene discovery in epilepsy
STX1B SLC6A1 Next-gen GABRA1 GABRB3 Sequencing Era SIK1 KCNA2 ALG13 GRIN2B PURA KCNB1 KCNC1 DNM1 HCN1 Microdeletions CHD2 SCN8A 15q13.3 SCN2A 16p13.11 GRIN2A 15q11.2 DEPDC5 KCNT1 PRRT2 TBC1D24 STXBP1 PCDH19
GABRA1 LGI1 CACNA1H GABRG2 ARX CDKL5 Channelopathy SCN1A Era SLC2A1 KCNQ3 KCNQ2 SCN1B CHRNA4
1995 1997 1999 2001 2003 2005 2007 2009 2011 2013 2015
2018 ISBP Tel Aviv From gene discovery to precision therapy
Pediatric epilepsy
Gene discovery
Precision therapy
2018 ISBP Tel Aviv Approach #1 – targeted sequencing
50 genes
TGCCATGC GATGAGTG
~800 patients Various epilepsies
Do any patients have Do two or more patients have pathogenic variants in * pathogenic variants in the same * * ‘known’ epilepsy genes? (new) gene?
2018 ISBP Tel Aviv EE Cohort
Syndrome N Dravet 38 Epilepsy aphasia 56 Eyelid myoclonus 10 Early myoclonic 5 Early onset EE 43 Infantile spasms 97 Lennox Gastaut 45 Myoclonic astatic 96 Migrating focal sz of infancy 15 Ohtahara 4 ID+GGE 50 FE+ID 41 Other 300 TOTAL 799
2018 ISBP Tel Aviv EE is genetically heterogeneous
20 39 Genetic diagnosis in ~45% of cases 15 * * * <1% of cases * 10 * ** * * 5 * * * * * **
0
CNV
PNKP
CUX2
NBEA
UBA5
CHD2 CHD8
GNB1
PNPO
STX1B
MBD5
CDKL5
DNM1
ALG13 USP9X
SCN1B
KCNT1
SCN1A SCN2A SCN8A
KCNB1
UBE3A
KCNA2
KCNH1 KCNH5
MEF2C
KCNQ2 KCNQ3
MECP2
IQSEC2
SMC1A
SLC6A1 SLC1A2 SLC2A1
GNAO1
EEF1A2
STXBP1
ARID1B
GRIN2B
GRIN2A
DEPDC5
PCDH19
GABRB3
GABRA1
GABRG2
TBC1D24
PPP2R5D
SYNGAP1
ARHGEF9
DYNC1H1
CACNA1A KIAA2022
2018 ISBP Tel Aviv Carvill et al., 2013, Nat Genet & unpublished CHD2: chromatin remodeler
• ~1% of EE cohort
• Phenotype: • Onset 1-3 yrs • Myoclonic seizures • Photosensitivity • Eyelid myoclonus • Broader NDV phenotype
• CHD7 – CHARGE syndrome • CHD8 – syndromic autism Carvill et al., 2012, Nat Genet • CHD4 – syndromic ID Thomas et al., 2015, Neurology 2018 ISBP Tel Aviv PPP3CA
• Co-expressed with known EE genes
• Encodes calcineurin A – Dephosphorylates DNM1 – Regulatory step for endocytosis at nerve terminal
• 1 patient with a de novo mutation – 5 additional patients through collaborators
2018 ISBP Tel Aviv CUX2: transcription factor
Targeted resequencing in large DEE cohort 2 patients with de WES de novo novo variant CUX2 variant in CUX2 2 patients with epilepsy + ID 5 patients with de novo variant
Chatron et al. (2018) Ann Neurology
2018 ISBP Tel Aviv CUX2 recurrent pathogenic variant
CUX2 9 patients same de novo variant
• Developmental epileptic encephalopathy
• Multiple seizure types – onset 6 months
• Severe intellectual disability
Chatron et al. (2018) Ann Neurology
2018 ISBP Tel Aviv CACNA1E-related epilepsy
• Encodes pore-forming subunit of voltage-gated Ca++ channel at pre-synapse
• 30 patients with de novo variants identified
• Early onset seizures, profound DD • Congenital contractures, movement disorder
Helbig K et al (2018) AJHG
2018 ISBP Tel Aviv CACNA1E-related epilepsy
Missense variants confer gain of function
Helbig K et al (2018) AJHG
2018 ISBP Tel Aviv GRIN2A mutations cause epilepsy aphasia
622 patients
4 GRIN2A mutations
All epilepsy aphasia syndromes
Carvill et al, 2013, Nat Genet also Lemke et al, Lesca et al
2018 ISBP Tel Aviv GRIN2A mutations cause epilepsy aphasia
10-20% of epilepsy aphasia spectrum (EAS) cases
2018 ISBP Tel Aviv Approach #2 – whole exome sequencing
We identified a genetic cause in 18.7% of our cohort Prescreen DEE cohort 209 individuals with DEE Exome Sequencing Data Analysis AGTTGTCA Targeted sequencing of 58 DEE genes 167 Trios De novo, Recessive, X-linked 20 Duos analysis 22 Singletons
Muir, Mefford unpublished
2018 ISBP Tel Aviv De novo analysis of exomes
NDV Gene Variant Diagnosis
ASH1L p.R1559X MAE DEE genes NDV genes ATP1A3 p.870_871del IS 13.9% CIC p.P786fs MAE COL4A1 c.4022-1G>A IS DEAF1 p.R229P EE 69 candidate DPF2 p.R179C EE genes in 58 DYNC1H1 p.R4449S MAE individuals FBXO11 p.P905R MAE HECW2 p.R1139K IS HIVEP2 p.Q1726X LGS Pathogenic or likely pathogenic de novo varaints IQSEC2 c.3016-3A LOS Candidate de novo variant IRF2BPL p.L713fs LGS Exome Negative MTOR p.Q222E EOAE SLC16A2 p.E134fs EE SMARCA2 p.G1164W EOEE Muir, Mefford unpublished 2018 ISBP Tel Aviv Increased cohort size facilitates discovery
Candidate De novo Diagnosis Gene Variant AKAP6 p.T343fs EOEE CMPK2 p.T263M IEE CPSF1 p.A837S LGS CPSF1 p.R91fs EOAE DENND6B p.A122V MAE FBXW7 p.I490V EOAE GRIA2 p.V647L EOEE KCNV2 p.R405H EOEE Targeted sequencing of MAST3 p.G510S IEE Entered genes 44 candidate SRGAP2 p.L159T EE into genes in >600 Matchmaker TANC2 p.E529fs EOEE DEE patients YWHAE p.M1fs CAE Exchange ZMIZ1 p.T945fs EAS Muir, Mefford unpublished
2018 ISBP Tel Aviv Gene discovery in epilepsy
STX1B SLC6A1 Next-gen GABRA1 GABRB3 Sequencing Era SIK1 KCNA2 ALG13 GRIN2B PURA KCNB1 KCNC1 DNM1 HCN1 Microdeletions CHD2 SCN8A 15q13.3 SCN2A 16p13.11 GRIN2A 15q11.2 DEPDC5 KCNT1 PRRT2 TBC1D24 STXBP1 PCDH19
GABRA1 LGI1 CACNA1H GABRG2 ARX CDKL5 Channelopathy SCN1A Era SLC2A1 KCNQ3 KCNQ2 SCN1B CHRNA4
1995 1997 1999 2001 2003 2005 2007 2009 2011 2013 2015
2018 ISBP Tel Aviv Diverse biological mechanisms
Metabolism Intracellular signaling ALG13, PNKP, PNPO ARHGEF9 MTOR SYNGAP1 GNAO1 DEPDC5 PLCB1 GNB1 PCDH19
Synaptic vesicle trafficking DNM1 STXBP1 Gene regulation Chromatin remodeling CDKL5 mRNA regulation UBE3A HNRNPU Neurotransmission MECP2 MBD5 Txn factors GABRA1/B1/B3/G2 KCNH5 CHD2 ARX GRIN2A/2B KCNT1 FOXG1 CACNA1A SLC13A5 HCN1 SLC25A22 SCN1A/1B/2A/8A SLC2A1 KCNQ2/3 SLC6A1 2018 ISBP Tel Aviv Epilepsy genetics in the clinic
Genetic CNV testing diagnosis achieved in 40- 50% of patients
Panels/exomes
2018 ISBP Tel Aviv How do we solve the rest?
Patient iPSCs CGTGCAT CGTGCAT fibroblasts
CGTACAT Cortical spheroids GENOME sequencing in RNA-seq to aid trios to identify de novo variant interpretation Cell Functional variants morphology studies
Electrophysiology Drug discovery
2018 ISBP Tel Aviv WGS + RNAseq
WGS +
Aberrant splicing of disease gene
2018 ISBP Tel Aviv WGS + RNAseq
WGS +
Aberrant splicing of disease gene
Adapted from Cummings et al. (2017) SciTransMed
2018 ISBP Tel Aviv How do we solve the rest?
• Noncoding variation
• Epigenetics
• Alternative modes of inheritance
• Somatic mosaic mutations
2018 ISBP Tel Aviv Large-scale collaborations
• Epilepsy Genetics Initiative (EGI) – Repository for exome data – Regular re-analysis of data – CURE Foundation + NINDS
• Epi25 – International effort to combine available cohorts – >30,000 available epilepsy samples identified – Whole exome/genome sequencing
2018 ISBP Tel Aviv How do we go from a precision diagnosis to a precision therapy?
2018 ISBP Tel Aviv Precision Medicine in Epilepsy TODAY
SCN1A (Dravet) *AVOID sodium Channel blockers
*USE sodium SCN8A mutations channel blockers *NO Keppra
ALDH7A1 (PDE) Pyridoxine (Vit B6)
GLUT1 deficiency Ketogenic diet
2018 ISBP Tel Aviv From diagnosis to therapy
• Studies in cells
• Model organism experiments
• Patient cells: stem cell studies
• Eventually…clinical trials
2018 ISBP Tel Aviv Epilepsy in a fish…
“Treat” with various potential drugs
2018 ISBP Tel Aviv Adapted from Baraban et al., 2013, Nat Comm (Human) epilepsy in a dish…
“Treat” with various potential drugs
2018 ISBP Tel Aviv Precision Medicine in Epilepsy
KCNT1 quinidine
GRIN2A memantine
KCNQ2 ezogabine mTOR pathway rapamycin
2018 ISBP Tel Aviv SUMMARY
• Accelerated gene discovery in EE
• Significant genetic heterogeneity
• Genetics teaching us about biology
• Precision medicine in epilepsy: challenging but promising
2018 ISBP Tel Aviv Acknowledgments
Mefford Lab U Melbourne Alison Muir Ingrid Scheffer Candace Myers Lynette Sadleir Amy LaCroix Sam Berkovic Michele Mehaffey Amy Schneider Edith Fuerte Georgie Hollingsworth John Nguyen Aaron Rosen
Northwestern Gemma Carvill
2018 ISBP Tel Aviv Epi4K: Exome sequencing
INFANTILE SPASMS (IS) Trio analysis • Onset in first year of life • Characteristic EEG: hypsarrhythmia • Often evolves to LGS
LENNOX GASTAUT SYNDROME (LGS) • Onset before 4 years • Multiple, refractory seizure types • Moderate to severe ID Sequence mom + dad + child
Look for de novo change in child 356 Trios analyzed: 429 de novo variants in >400 genes Severe, de novo disorders ~15% solved
2018 ISBP Tel Aviv Epi4K: Trio exome sequencing in EE
15% solved
* *
2018 ISBP Tel Aviv