Leanne Dibbens PhD Epilepsy Research Program

Adelaide, Australia

Gene discoveries in epilepsy

& its co-morbidities

using exome sequencing

Aims of the Epilepsy Research Group

• To identify the molecular genetic mechanisms

underlying epilepsy and related disorders

• To translate our findings into better outcomes for

patients with epilepsy

Epilepsy

Cardiovascular

Trauma

Tumour

Symptomatic

Epilepsy - more than 30 different syndromes

- affects 2% of the population at some stage in life

Other - epilepsy is secondary

70%

Genetic

Bio-bank 16 000 cases

(sporadic & familial)

Generalized or Focal

Spectrum of Severity

Dravet Syndrome

Genetic Generalised Epilepsies (GGE)

Epilepsy, Female Limited (EFL)

Genetic epilepsies

Benign Infantile Epilepsies

(KCNQ2 & Q3, PRRT2) (SCN1A)

(PCDH19) SCN1B, SCN1A, GABRG2 & GABRA1

“Monogenic” Epilepsies

Autosomal Dominant Autosomal Recessive

X-Linked

de novo

PCDH19

Epilepsy, Female Limited Dibbens et al. Nat Gen 2008

SCARB2, GOSR2

Progressive Myoclonus Epilepsy Berkovic et al. AJHG 2008

PRRT2 Heron et al. Nat Gen 2011

Focal Epilepsies

Account for ~ 60% epilepsy

Proportion with structural lesions

Not regarded as genetic

ADNFLE

• Autosomal Dominant Nocturnal Frontal Lobe Epilepsy

• Seizures occur during sleep

• Most patients have normal intellect

• ~10% of cases due to mutations in CHRNA4 & CHRNB2

• Cause of remaining cases unknown

Family with ADNFLE: WES

ADNFLE

WES

WES

Genome wide linkage analysis

(SNP array - Bahlo group)

suggestive linkage LOD 2.71

9q34.3

9q34.3

2.4 Mb

99 genes

(het variants)

Australian family

Exome Sequencing

Exome Capture:

SureSelect Human All Exon 50Mb kit

(Agilent Technologies, Santa Clara, CA).

MPSequencing:

SOLiD v4 instrument

(Applied Biosystems, Carlsbad, CA).

Alignment: UCSC Genome Browser hg18 BWA

Variant calling: SAMtools and annotated using SeattleSeq

(http://snp.gs.washington.edu/SeattleSeqAnnotation/)

• Data for Ch9 linkage interval extracted from annotation file

• Variants in dbSNP, intronic & intergenic variants filtered out

Princess Alexandra Hospital

Brisbane, Australia

Variants in linkage region

# inDBSNPOrNotchromosome position referenceBasesampleGenotypeallelesMaq accession functionGVS aminoAcids proteinPositionscorePhastConsconsScoreGERPchimpAlle le geneList

none 9 137811078C Y C/T NM_020822 missense ARG,CYS 928/1257 0.984 2.24 C KCNT1

none 9 138255708G R A/G NM_181701 missense ARG,TRP 184/699 0.001 -9.36 G QSOX2

none 9 138462717G R A/G NM_014866 intron none NA 0 1.1 - SEC16A

none 9 138462950G R A/G NM_014866 intron none NA 0.005 0 - SEC16A

none 9 138876495G R A/G NM_003792 utr-3 none NA 0.108 -0.222 G EDF1

none 9 138957723C A A/A NM_018998 nonsense GLU,stop 84/567 1 4.5 unknown FBXW5

none 9 138995059C S C/G NM_000954 intron none NA 0 -3.01 C PTGDS

none 9 139094899T C C/C NM_207309 intron none NA 0 -4.7 T UAP1L1

none 9 139229814T G G/G NM_014434 intron none NA 0 -0.399 T NDOR1

none 9 139602178G S C/G NM_138462 intron none NA 0.32 1.31 G ZMYND19

none 9 139790858A T T/T NM_024757 intron none NA 0.001 -4.17 A EHMT1

Granddaughter

# inDBSNPOrNotchromosome position referenceBasesampleGenotypeallelesMaq accession functionGVS aminoAcids proteinPositionscorePhastConsconsScoreGERPchimpAlle le geneList

none 9 137856438A R A/G NM_015447 intron none NA 0 -2.36 A CAMSAP1

none 9 138396577C G G/G NM_003086 missense GLU,ASP 672/1470 0.012 3.43 C SNAPC4

none 9 138431400C M A/C NM_015160 nonsense TYR,stop 270/526 1 2.86 C PMPCA

none 9 138462717G R A/G NM_014866 intron none NA 0 1.1 - SEC16A

none 9 138462950G R A/G NM_014866 intron none NA 0.005 0 - SEC16A

none 9 138759426C Y C/T NM_198946 intron none NA 0 -2.62 unknown LCN6

none 9 138995059C S C/G NM_000954 intron none NA 0 -3.01 C PTGDS

none 9 139579397T Y C/T NM_138778 missense ASP,GLY 224/453 1 5.06 unknown WDR85

none 9 139602178G S C/G NM_138462 intron none NA 0.32 1.31 G ZMYND19

none 9 139731503T A A/A NM_024757 intron none NA 0 0.953 unknown EHMT1

Grandmother

Threshold of mutation detection set at 15%

Mutation absent in 2nd individual (III.4): 0/6 reads

KCNT1 mutation: segregation

ADNFLE

+/m

+/m +/m +/m

+/m +/m

+/+

+/+

+/+

+/+ +/+ +/+

+/+

+/+

+/+

+/+ +/+

m KCNT1 Arg928Cys mutation

Mutation absent in 111 Australian controls, dbSNP & EVS

Australian family

KCNT1: Further mutational analysis

• KCNT1 mutation analysis by High Resolution Melting (HRM)

• 117 unrelated familial & sporadic cases NFLE

NFLE

+/m

+/+

m KCNT1 mutation

+/m

Additional KCNT1 mutations

+/m +/+

+/m

+/m

+/m +/m

+/+

Y796H

+/+

+/m

+/+

M869I

R398Q

Italian

Australian

Sephardic Jewish

de novo

mutation

4 families have same phenotype:

ADNFLE, ID and psychiatric features

Severe form of ADNFLE Heron et al Nat Gen 2013

KCNT1 mutations

NH2

(amino)

terminus

COOH

(carboxy)

terminus

p.Arg398Gln

Tyr796His (B)

Arg928Cys (A)

RCK NAD+ binding domain

Met896Ile (D)

Heron et al Nature Genetics 2012

Na+ regulated K+ channel

animal models

electrophysiology

• 8 families linkage to 22q12

(5.3 Mb; >100 genes)

• WES in two families

Australian Family Dutch Family

Australian Family

Dutch Family

(het variants)

22q12

• Autosomal dominant inheritance

• Seizures originating from different brain regions

Focal Epilepsy with Variable Foci

Aust

Family

Chrom. pos. (22q12)

Variant type Gene Accession Nucleotide change

Amino Acid change

2 27868137 3’-UTR KREMEN1 NM_001039570 c.1377+37G>C none

1 28025765 missense EWSR1 NM_005243 c.1855G>T p.Gly619Trp

2 28215313 missense NEFH NM_021076 c.1684C>G p.Pro562Ala

2 28493438 synonymous UQCR10 NM_013387 c.51G>A none

1 28493438 synonymous UQCR10 NM_013387 c.51G>A none

1 28746153 missense MTMR3 NM_021090 c.2505T>G p.Phe835Leu

1 29061481 missense SF3A1 NM_005877 c.2255C>A p.Ala752Glu

2 29217908 missense SEC14L4 NM_174977 c.824G>T p.Arg275Met

2 29672432 missense MORC2 NM_014941 c.136T>C p.Ser46Pro

1 29672432 missense MORC2 NM_014941 c.136T>C p.Ser46Pro

1 29814555 missense SMTN NM_134270 c.257T>A p.Leu86Gln

2 30181151 missense EIF4ENIF1 NM_019843 c.1250C>T p.Ser417Phe

2 30438394 missense PRR14L NM_173566 c.5431A>T p.Thr1811Ser

1 30480928 nonsense DEPDC5 NM_014662 c.21C>G p.Tyr7*

2 30809099 missense SLC5A1 NM_000343 c.622A>T p.Thr208Ser

1 30809102 missense SLC5A1 NM_000343 c.625G>T p.Val209Leu

2 30876430 missense C22orf42 NM_001010859 c.530G>C p.Cys177Ser

1 30876430 missense C22orf42 NM_001010859 c.530G>C p.Cys177Ser 2 30957012 missense SLC5A4 NM_014227 c.1072G>T p.Asp538Tyr

2 31086882 3’-UTR RFPL3 NM_001098535 c.954+63C>T none

2 31224485 missense FBXO7 NM_012179 c.1537C>G p.Arg513Gly

2 31239669 3’-UTR SYN3 NM_003490 c.1743+10G>C none

Unique putative variants in Ch 22 linkage interval

Focal Epilepsy

m DEPDC5 mutation

Panayiotopoulos syndrome

Frontal

Temporal

Parietal

Intellectual disability

Autism spectrum disorder +/+

+/+

+/+

m/+

m/+ m/+

+/+

2

m/+ +/+

m/+

2

+/+ +/+

+/+

+/+

+/+ +/+ m/+ m/+

m/+

m/+ m/+ +/+

(m/+)

m/+

(m/+)

Australian Family

DEPDC5 mutations cause FFEVF

7/8 published FFEVF families have DEPDC5 mutation

from Australia (1/2) The Netherlands(1), Spain(2), Canada(3)

• Do DEPDC5 mutations also account for cases of

familial focal epilepsy in families which are

too small for a clinical diagnosis of FFEVF?

• 82 small families with focal epilepsy

- analysed for mutations in DEPDC5 (HRM analysis)

DEPDC5 in familial focal epilepsy

DEPDC5 in familial focal epilepsy:

Mutations in 12% small families

Nocturnal frontal

Frontal

Temporal

Parietal

Multifocal

Occipital

Fronto-temporal

Unclassified

Focal seizure type

DEPDC5 mutations

in 10/82 (12%)

pedigrees with

familial focal epilepsy

de novo

mutation

DEP

100 381 1170 1251 1604 aa

NH2 COOH

A1:Tyr7*

S1:Trp1369*

S2:Gln1536*

D1:Arg555* FC1,FC2,FC3:

Val164delPhe

H:c.193+1G

I:c.279+1G>A

J:Arg487* K:Arg843*

L&G:Trp1466*

N:Ser1104Leu

O:Ser1073Arg

P:Ala452Val

M:Arg1268*

Mutations in DEPDC5

• Dishevelled, Egl-10 and Pleckstrin (DEP) domain containing (protein) 5

• DEP domain in G protein signalling

Dibbens et al Nat Gen 2013

DEPDC5 protein localization

in neurospheres from human iPS cells

Massimo Pandolfo, Belgium

NeuN: neuronal marker

GFAP: astrocyte marker

SOX2: cell body marker

DEPDC5 protein

in neurons

mTOR signalling

DEPDC5

Bar Peled et al

Science 2013

Translation to the Clinic

• Genetics - greater role in focal epilepsy than previously thought

• DEPDC5 mutations not just found in rare large families

• Gene testing allows a molecular diagnosis, aids in prognosis

• Potential for improved therapies

Genetics of the Epilepsies

Susceptibility variants <5% families

Rare families: Monogenic inheritance

Multiple single gene disorders

~60 genes now identified

Majority of cases have complex inheritance

Many gene variants + CNVs

Modified by environmental factors

Acknowledgements

Epilepsy Group UniSA, Adelaide

Sarah Heron Michael Ricos

Bree Hodgson Robert Schultz

Xenia Iona Yeh Sze Ong

Marta Bayly Chiaoxin Lim

Bev Johns

Universite de Bruxelles, Belgium

Massimo Pandolfo

Simona Donatello

Satyan Chintawar

Patients and their families

Clinical Collaborators & teams

Eva Andermann, Montreal, Canada

Fred Andermann, Montreal, Canada

Patrick Cossette, Montreal, Canada

Jose Serratosa, Madrid, Spain

Rosa Guerrero Lopez, Madrid, Spain

Francesca Bisulli, Bologna, Italy

Laura Licchetta, Bologna, Italy

Sara Kivity, Petach Tikvah, Israel

Arn van den Maagdenberg, Holland

Ingrid Scheffer, Melbourne Australia

Sam Berkovic, Melbourne Australia

Funding: National Health Medical Research Council of

Australia Program Grant, CDF and ECF Fellowships

• Mean age of seizure onset 12.5 years (range 6 weeks - 52 yrs)

• FLE, TLE most common seizure types (70% combined)

• Penetrance of DEPDC5 mutations ~ 65%

Seizures associated with

DEPDC5 mutations

• Focal epilepsies are genetic

DEPDC5 in mTOR signaling

from commentary on Bar Peled et al Science 2013

Gene Discovery Strategy

2. Capture exons of genome

= exome of affecteds

(Sure Select 50Mb)

3. Whole Exome

Sequencing (WES)

(SOLiD v4)

1. Whole genome linkage

analyses on pedigrees

5. Interrogate WES data

in linkage interval

Chromosome 22

22q12 linkage interval

4. Align WES data, filter

6. Autosomal dominant

inheritance

(het mutations)

Focal Epilepsy Syndrome: FFEVF

Familial Focal Epilepsy with Variable Foci (FFEVF)

Family members have seizures originating from

different brain regions

Psychiatric disorder

Australian Family

• 8 families published with

linkage to 22q12

(5.3 Mb, >100 genes)

• Exome sequencing in two

families

WES

m DEPDC5 mutation

+/+

m/+ m/+ m/+ m/+ +/+

m/+ m/+ m/+ m/+

m/+

(m/+) m/+

m/+

Depdc5 expression in mouse

mouse tissue

RT-PCR

A model of the population distribution of

susceptibility alleles for more common epilepsies

with complex inheritance

insufficient number

of susceptibility alleles

to cause epilepsy

healthy

(=controls)

enough

susceptibility

alleles to cause

common polygenic

epilepsy

Number of susceptibility alleles “monogenic”

epilepsy

seizure threshold

Mulley et al Hum Mol Genet 2005

Mutations in DEPDC5 cause FFEVF

Australian family: 5 variants selected for follow-up analyses:

DEPDC5 Tyr7X, two variants in SLC5A1, variant in SLC5A4

& variant in MORC2

2/5 variants validated by Sanger sequencing

DEPDC5 Tyr7X and SLC5A1 Val209Leu

both segregated with FFEVF phenotype

additional 5 FFEVF families analysed for SLC5A1 & DEPDC5

4/5 families with FFEVF have DEPDC5 mutation

DEPDC5 is the FFEVF gene

Family 1 with FFEVF (Australian)

4

3

3 3

4 2 2

2 3

Unclassified seizures

Temporal lobe epilepsy

Centro-parietal lobe epilepsy

Frontal lobe epilepsy

Probable syncope

Probable Panayiotopoulos syndrome

Intellectual disability/autism

Suggestive linkage Chromosome 22q12 LOD score of 2.94

MPS

Clinical Features associated

with DEPDC5 mutations

• Mean age of seizure onset: 12.5 years

• FLE, TLE most common seizure types

• Penetrance of DEPDC5 mutations ~ 65%

32%

(12% nocturnal)

1%

2% 24% 14%

Seizure types in 95 DEPDC5

positive individuals in

7 large families

24% unclassified epilepsy

1% ASD no seizures

2% multifocal epilepsy

Causes of Epilepsy

Genetic

Epilepsies

Cardiovascular

Trauma

Tumour

Other ~ 200 genetic syndromes

where epilepsy is secondary Symptomatic

Epilepsies

~70%

Epilepsy - more than 30 different syndromes

- affects 2% of the population at some stage in life

Epilepsy

Bio-bank 16000 cases

(familial and sporadic)

Focal Epilepsy

m DEPDC5 mutation

Panayiotopoulos syndrome

Frontal

Temporal

Parietal

Intellectual disability

Autism spectrum disorder +/+

+/+

+/+

m/+

m/+ m/+

+/+

2

m/+ +/+

m/+

2

+/+ +/+

+/+

+/+

+/+ +/+ m/+ m/+

m/+

m/+ m/+ +/+

(m/+)

m/+

(m/+)

Australian Family A1

DEPDC5 mutations cause FFEVF

7/8 published FFEVF families have DEPDC5 mutation

from Australia (1/2) The Netherlands(1), Spain(2), Canada(3)

Family with ADNFLE: WES

ADNFLE

WES

WES

Genome –wide Linkage

analysis (SNP array)

suggestive linkage LOD 2.71 9q34.3