2018 ISBP Tel Aviv

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, hypersynchronousactivity in the brain

• Prevalence ~0.5-1%

• 50 million affected worldwide

2018 ISBP Tel Aviv

Epilepsy Classification

• Seizure type– Generalized

– Focal

• Age of onset– Infancy, childhood, adolescence, adult

• Etiology– Genetic

– Acquired CUREepilepsy.org

>50 syndromes described

2018 ISBP Tel Aviv

Epilepsy Classification

Focal Epilepsy

Generalized Epilepsy

(GGE)

Developmental & Epileptic

Encephalopathy*(DEE)

JMECAE

GEFS+

ADNFLEADPEAF

TLE

DravetOhtahara

Infantile spasmsLGS

2018 ISBP Tel Aviv

Genetics of Epilepsy

• Twin concordance

– GGE ~80%

– Focal ~36%

• Family studies

– Overall RR 2-5%

– Evidence for linkage in some families

– De novo mutations in severe cases

Helbig et al., 2008, Lancet Neurol

2018 ISBP Tel Aviv

Gene discovery in epilepsy

CHRNA4SCN1B

SCN1A

GABRG2

Microdeletions

1995 1997 1999 2001 2003 2005 2007 2009 2011 2013

15q13.316p13.1115q11.2

KCNQ2KCNQ3

GABRA1 LGI1 CACNA1H

SLC2A1

ARX CDKL5

2015

DNM1

KCNT1DEPDC5

TBC1D24

KCNC1

SCN8ASCN2AGRIN2A

HCN1CHD2

STXBP1

PRRT2

PCDH19

KCNA2

STX1BSLC6A1GABRA1GABRB3SIK1

ALG13GRIN2BPURA

KCNB1

ChannelopathyEra

Next-gen Sequencing Era

2018 ISBP Tel Aviv

Gene discovery in epilepsy

CHRNA4SCN1B

SCN1A

GABRG2

Microdeletions

1995 1997 1999 2001 2003 2005 2007 2009 2011 2013

15q13.316p13.1115q11.2

KCNQ2KCNQ3

GABRA1 LGI1 CACNA1H

SLC2A1

ARX CDKL5

2015

DNM1

KCNT1DEPDC5

TBC1D24

KCNC1

SCN8ASCN2AGRIN2A

HCN1CHD2

STXBP1

PRRT2

PCDH19

KCNA2

STX1BSLC6A1GABRA1GABRB3SIK1

ALG13GRIN2BPURA

KCNB1

ChannelopathyEra

2018 ISBP Tel Aviv

Gene discovery in epilepsy

CHRNA4SCN1B

SCN1A

GABRG2

Microdeletions

1995 1997 1999 2001 2003 2005 2007 2009 2011 2013

15q13.316p13.1115q11.2

KCNQ2KCNQ3

GABRA1 LGI1 CACNA1H

SLC2A1

ARX CDKL5

2015

DNM1

KCNT1DEPDC5

TBC1D24

KCNC1

SCN8ASCN2AGRIN2A

HCN1CHD2

STXBP1

PRRT2

PCDH19

KCNA2

STX1BSLC6A1GABRA1GABRB3SIK1

ALG13GRIN2BPURA

KCNB1

ChannelopathyEra

Next-gen Sequencing Era

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

GATGAGTG

TGCCATGC

~800 patientsVarious

epilepsies

*

Do any patients have pathogenic variants in ‘known’ epilepsy genes?

**

Do two or more patients have pathogenic variants in the same (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

Carvill et al., 2013, Nat Genet & unpublished

0

5

10

15

20

SCN

1A

STX

BP

1

CN

V

CD

KL5

SCN

2A

CH

D2

KC

NQ

2

SYN

GA

P1

DEP

DC

5

GA

BR

B3

GR

IN2

A

KC

NT1

SCN

8A

SLC

6A

1

GA

BR

G2

KIA

A2

02

2

CA

CN

A1

A

GA

BR

A1

KC

NB

1

MEF

2C

ALG

13

AR

HG

EF9

EEF1

A2

GN

B1

GR

IN2

B

IQSE

C2

KC

NA

2

MEC

P2

PC

DH

19

PN

PO

SLC

1A

2

SMC

1A

TBC

1D

24

UB

A5

UB

E3A

AR

ID1

B

CH

D8

CU

X2

DN

M1

DYN

C1

H1

GN

AO

1

KC

NH

1

KC

NH

5

KC

NQ

3

MB

D5

NB

EA

PN

KP

PP

P2

R5

D

SCN

1B

SLC

2A

1

STX

1B

USP

9X

39 Genetic diagnosis in ~45% of cases

<1% of cases

*

**

*** *

**

** * *

**

*

2018 ISBP Tel Aviv

CHD2: chromatin remodeler

Carvill et al., 2012, Nat Genet Thomas et al., 2015, Neurology

• ~1% of EE cohort

• Phenotype:• Onset 1-3 yrs• Myoclonic seizures• Photosensitivity• Eyelid myoclonus• Broader NDV phenotype

• CHD7 – CHARGE syndrome• CHD8 – syndromic autism• CHD4 – syndromic ID

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

WES de novo CUX2 variant in 2 patients with epilepsy + ID

CUX2

Targeted resequencing in large DEE cohort

2 patients with de novo variant

5 patients with de novo variant

Chatron et al. (2018) Ann Neurology

2018 ISBP Tel Aviv

CUX2 recurrent pathogenic variant

CUX2

• Developmental epileptic encephalopathy

• Multiple seizure types – onset 6 months

• Severe intellectual disability

Chatron et al. (2018) Ann Neurology

9 patientssame de novo variant

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

Helbig K et al (2018) AJHG

Missense variants confer gain of function

2018 ISBP Tel Aviv

GRIN2A mutations cause epilepsy aphasia

Carvill et al, 2013, Nat Genet also Lemke et al, Lesca et al

622 patients

4 GRIN2A mutations

All epilepsy aphasia

syndromes

2018 ISBP Tel Aviv

10-20% of epilepsy aphasia spectrum

(EAS) cases

GRIN2A mutations cause epilepsy aphasia

2018 ISBP Tel Aviv

Approach #2 – whole exome sequencing

Data AnalysisAGTTGTCA

De novo, Recessive, X-linkedanalysis

Exome Sequencing

We identified a genetic cause in 18.7% of our cohort

209 individuals with DEE

167 Trios20 Duos

22 Singletons

Prescreen DEE cohort

Targeted sequencing of 58 DEE genes

Muir, Mefford unpublished

2018 ISBP Tel Aviv

De novo analysis of exomes

Pathogenic or likely pathogenic de novo varaintsCandidate de novo variantExome Negative

NDV Gene Variant Diagnosis

ASH1L p.R1559X MAE

ATP1A3 p.870_871del IS

CIC p.P786fs MAE

COL4A1 c.4022-1G>A IS

DEAF1 p.R229P EE

DPF2 p.R179C EE

DYNC1H1 p.R4449S MAE

FBXO11 p.P905R MAE

HECW2 p.R1139K IS

HIVEP2 p.Q1726X LGS

IQSEC2 c.3016-3A LOS

IRF2BPL p.L713fs LGS

MTOR p.Q222E EOAE

SLC16A2 p.E134fs EE

SMARCA2 p.G1164W EOEE

DEE genes NDV genes

69 candidate genes in 58 individuals

13.9%

Muir, Mefford unpublished

2018 ISBP Tel Aviv

Increased cohort size facilitates discovery

Candidate Gene

De novoVariant

Diagnosis

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

MAST3 p.G510S IEE

SRGAP2 p.L159T EE

TANC2 p.E529fs EOEE

YWHAE p.M1fs CAE

ZMIZ1 p.T945fs EAS

Targeted sequencing of 44 candidate genes in >600 DEE patients

Entered genes into

Matchmaker Exchange

Muir, Mefford unpublished

2018 ISBP Tel Aviv

Gene discovery in epilepsy

CHRNA4SCN1B

SCN1A

GABRG2

Microdeletions

1995 1997 1999 2001 2003 2005 2007 2009 2011 2013

15q13.316p13.1115q11.2

KCNQ2KCNQ3

GABRA1 LGI1 CACNA1H

SLC2A1

ARX CDKL5

2015

DNM1

KCNT1DEPDC5

TBC1D24

KCNC1

SCN8ASCN2AGRIN2A

HCN1CHD2

STXBP1

PRRT2

PCDH19

KCNA2

STX1BSLC6A1GABRA1GABRB3SIK1

ALG13GRIN2BPURA

KCNB1

ChannelopathyEra

Next-gen Sequencing Era

2018 ISBP Tel Aviv

Diverse biological mechanisms

MetabolismALG13, PNKP, PNPO

Intracellular signalingARHGEF9SYNGAP1DEPDC5GNB1

MTORGNAO1PLCB1PCDH19

Synaptic vesicle traffickingDNM1STXBP1

GABRA1/B1/B3/G2GRIN2A/2BCACNA1AHCN1SCN1A/1B/2A/8AKCNQ2/3

Gene regulationChromatin remodeling

Txn factorsARXFOXG1

CDKL5UBE3AMECP2MBD5CHD2

mRNA regulationHNRNPU

KCNH5KCNT1SLC13A5SLC25A22SLC2A1SLC6A1

Neurotransmission

2018 ISBP Tel Aviv

Epilepsy genetics in the clinic

Panels/exomes

CNV testing

Genetic diagnosis

achieved in 40-50% of patients

2018 ISBP Tel Aviv

How do we solve the rest?

CGTGCAT CGTGCAT

CGTACAT

GENOME sequencing in trios to identify de novo

variants

RNA-seq to aid variant interpretation

Patient fibroblasts

iPSCs

Cell morphology

Electrophysiology

Functional studies

Drug discovery

Cortical spheroids

2018 ISBP Tel Aviv

WGS + RNAseq

WGS +

Aberrant splicing of disease gene

2018 ISBP Tel Aviv

WGS + RNAseq

Adapted from Cummings et al. (2017) SciTransMed

WGS +

Aberrant splicing of disease gene

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

*AVOID sodium Channel blockers

SCN1A (Dravet)

*USE sodium channel blockers*NO Keppra

SCN8A mutations

GLUT1 deficiency Ketogenic diet

Pyridoxine (Vit B6)ALDH7A1 (PDE)

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

Adapted from Baraban et al., 2013, Nat Comm

2018 ISBP Tel Aviv

(Human) epilepsy in a dish…

“Treat” with various potential drugs

2018 ISBP Tel Aviv

Precision Medicine in Epilepsy

KCNT1

GRIN2A

KCNQ2

mTOR pathway

quinidine

memantine

ezogabine

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 LabAlison Muir

Candace MyersAmy LaCroix

Michele MehaffeyEdith FuerteJohn NguyenAaron Rosen

NorthwesternGemma Carvill

U MelbourneIngrid SchefferLynette SadleirSam Berkovic

Amy SchneiderGeorgie Hollingsworth

2018 ISBP Tel Aviv

Epi4K: Exome sequencing

Trio analysis

Sequence mom + dad + child

Look for de novo change in child

Severe, de novo disorders

INFANTILE SPASMS (IS)• 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

356 Trios analyzed: 429 de novo variants in >400 genes

~15% solved

2018 ISBP Tel Aviv

Epi4K: Trio exome sequencing in EE

15% solved

* *