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Gene discoveries in epilepsy & its co-morbidities using exome sequencing - Leanne Dibbens
Gene discoveries in epilepsy & its co-morbidities using exome sequencing - Leanne Dibbens
Gene discoveries in epilepsy & its co-morbidities using exome sequencing - Leanne Dibbens
Gene discoveries in epilepsy & its co-morbidities using exome sequencing - Leanne Dibbens
Gene discoveries in epilepsy & its co-morbidities using exome sequencing - Leanne Dibbens
Gene discoveries in epilepsy & its co-morbidities using exome sequencing - Leanne Dibbens
Gene discoveries in epilepsy & its co-morbidities using exome sequencing - Leanne Dibbens
Gene discoveries in epilepsy & its co-morbidities using exome sequencing - Leanne Dibbens
Gene discoveries in epilepsy & its co-morbidities using exome sequencing - Leanne Dibbens
Gene discoveries in epilepsy & its co-morbidities using exome sequencing - Leanne Dibbens
Gene discoveries in epilepsy & its co-morbidities using exome sequencing - Leanne Dibbens
Gene discoveries in epilepsy & its co-morbidities using exome sequencing - Leanne Dibbens
Gene discoveries in epilepsy & its co-morbidities using exome sequencing - Leanne Dibbens
Gene discoveries in epilepsy & its co-morbidities using exome sequencing - Leanne Dibbens
Gene discoveries in epilepsy & its co-morbidities using exome sequencing - Leanne Dibbens
Gene discoveries in epilepsy & its co-morbidities using exome sequencing - Leanne Dibbens
Gene discoveries in epilepsy & its co-morbidities using exome sequencing - Leanne Dibbens
Gene discoveries in epilepsy & its co-morbidities using exome sequencing - Leanne Dibbens
Gene discoveries in epilepsy & its co-morbidities using exome sequencing - Leanne Dibbens
Gene discoveries in epilepsy & its co-morbidities using exome sequencing - Leanne Dibbens
Gene discoveries in epilepsy & its co-morbidities using exome sequencing - Leanne Dibbens
Gene discoveries in epilepsy & its co-morbidities using exome sequencing - Leanne Dibbens
Gene discoveries in epilepsy & its co-morbidities using exome sequencing - Leanne Dibbens
Gene discoveries in epilepsy & its co-morbidities using exome sequencing - Leanne Dibbens
Gene discoveries in epilepsy & its co-morbidities using exome sequencing - Leanne Dibbens
Gene discoveries in epilepsy & its co-morbidities using exome sequencing - Leanne Dibbens
Gene discoveries in epilepsy & its co-morbidities using exome sequencing - Leanne Dibbens
Gene discoveries in epilepsy & its co-morbidities using exome sequencing - Leanne Dibbens
Gene discoveries in epilepsy & its co-morbidities using exome sequencing - Leanne Dibbens
Gene discoveries in epilepsy & its co-morbidities using exome sequencing - Leanne Dibbens
Gene discoveries in epilepsy & its co-morbidities using exome sequencing - Leanne Dibbens
Gene discoveries in epilepsy & its co-morbidities using exome sequencing - Leanne Dibbens
Gene discoveries in epilepsy & its co-morbidities using exome sequencing - Leanne Dibbens
Gene discoveries in epilepsy & its co-morbidities using exome sequencing - Leanne Dibbens
Gene discoveries in epilepsy & its co-morbidities using exome sequencing - Leanne Dibbens
Gene discoveries in epilepsy & its co-morbidities using exome sequencing - Leanne Dibbens
Gene discoveries in epilepsy & its co-morbidities using exome sequencing - Leanne Dibbens
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Gene discoveries in epilepsy & its co-morbidities using exome sequencing - Leanne Dibbens

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Epilepsy is a common disorder comprising more than 30 different syndromes. Around 70% of epilepsy cases are thought to be genetic in origin. The genetic and phenotypic heterogeneity seen in epilepsy …

Epilepsy is a common disorder comprising more than 30 different syndromes. Around 70% of epilepsy cases are thought to be genetic in origin. The genetic and phenotypic heterogeneity seen in epilepsy can make it difficult to diagnose and to treat. By studying rare large families with monogenic forms of epilepsy we have been able to carry out linkage analysis to identify a chromosomal region harbouring the causal variant in a family. By exome sequencing affected individuals and extracting the unique variants from within the linkage interval we have been able to chase down the putative causal variant. The identification of independent mutations in the same gene in patients who are phenotypically similar validates our identification of the casual gene. Using this approach we have recently identified two new genes in autosomal dominant forms of focal epilepsy, a severe form of nocturnal frontal love epilepsy (NFLE) with intellectual disability and psychiatric features and a syndrome known as FFEVF. The findings will lead to improved molecular diagnostic tests in epilepsy and reveal new pathways involved in the pathogenesis of epilepsy.

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  • 1. Gene discoveries in epilepsy & its co-morbidities using exome sequencing Leanne Dibbens PhD Epilepsy Research Program Adelaide, Australia
  • 2. 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
  • 3. Epilepsy Epilepsy - more than 30 different syndromes - affects 2% of the population at some stage in life Symptomatic Other - epilepsy is secondary Tumour Trauma Cardiovascular 70% Genetic Bio-bank 16 000 cases (sporadic & familial) Generalized or Focal
  • 4. Spectrum of Severity Genetic epilepsies Benign Infantile Epilepsies (KCNQ2 & Q3, PRRT2) Dravet Syndrome (SCN1A) Epilepsy, Female Limited (EFL) Genetic Generalised Epilepsies (GGE) SCN1B, SCN1A, GABRG2 & GABRA1 (PCDH19)
  • 5. “Monogenic” Epilepsies X-Linked PCDH19 Epilepsy, Female Limited Dibbens et al. Nat Gen 2008 de novo Autosomal Recessive SCARB2, GOSR2 Progressive Myoclonus Epilepsy Berkovic et al. AJHG 2008 Autosomal Dominant PRRT2 Heron et al. Nat Gen 2011
  • 6. Focal Epilepsies Account for ~ 60% epilepsy Proportion with structural lesions Not regarded as genetic
  • 7. 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
  • 8. Family with ADNFLE: WES Genome wide linkage analysis (SNP array - Bahlo group) suggestive linkage LOD 2.71 9q34.3 Australian family WES 2.4 Mb 99 genes ADNFLE WES 9q34.3 (het variants)
  • 9. Exome Sequencing Exome Capture: SureSelect Human All Exon 50Mb kit (Agilent Technologies, Santa Clara, CA). Princess Alexandra Hospital Brisbane, Australia 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
  • 10. Variants in linkage region Granddaughter # inDBSNPO rNot chrom osom e position refe renceBase sam pleGenotype allelesMaq none 9 137811078C Y C/T none 9 138255708G R A/G none 9 138462717G R A/G none 9 138462950G R A/G none 9 138876495G R A/G none 9 138957723C A A/A none 9 138995059C S C/G none 9 139094899T C C/C none 9 139229814T G G/G none 9 139602178G S C/G none 9 139790858A T T/T accession NM_020822 NM_181701 NM_014866 NM_014866 NM_003792 NM_018998 NM_000954 NM_207309 NM_014434 NM_138462 NM_024757 functionGVS m issense m issense intron intron utr-3 nonse nse intron intron intron intron intron am inoAcids ARG,C YS ARG,TRP none none none GLU,stop none none none none none proteinPo sition scorePhastCons consScoreGERchim pAlle le P 928/1257 0.984 2.24 C 184/699 0.001 -9.36 G NA 0 1.1 NA 0.005 0 NA 0.108 -0.222 G 84/567 1 4.5 unknown NA 0 -3.01 C NA 0 -4.7 T NA 0 -0.399 T NA 0.32 1.31 G NA 0.001 -4.17 A gene List KCNT1 QSOX2 SEC16A SEC16A EDF1 FBXW 5 PTGDS UAP 1L1 NDO R1 ZMYND19 EHMT1 accession NM_015447 NM_003086 NM_015160 NM_014866 NM_014866 NM_198946 NM_000954 NM_138778 NM_138462 NM_024757 functionGVS intron m issense nonse nse intron intron intron intron m issense intron intron am inoAcids none GLU,ASP TYR ,stop none none none none ASP,GLY none none proteinPo sition scorePhastCons consScoreGERchim pAlle le P NA 0 -2.36 A 672/1470 0.012 3.43 C 270/526 1 2.86 C NA 0 1.1 NA 0.005 0 NA 0 -2.62 unknown NA 0 -3.01 C 224/453 1 5.06 unknown NA 0.32 1.31 G NA 0 0.953 unknown gene List CAMSAP1 SNAP C4 PMPCA SEC16A SEC16A LCN6 PTGDS W DR85 ZMYND19 EHMT1 Grandmother # inDBSNPO rNot chrom osom e position refe renceBase sam pleGenotype allelesMaq none 9 137856438A R A/G none 9 138396577C G G/G none 9 138431400C M A/C none 9 138462717G R A/G none 9 138462950G R A/G none 9 138759426C Y C/T none 9 138995059C S C/G none 9 139579397T Y C/T none 9 139602178G S C/G none 9 139731503T A A/A Threshold of mutation detection set at 15% Mutation absent in 2nd individual (III.4): 0/6 reads
  • 11. KCNT1 mutation: segregation Australian family +/+ +/+ +/+ +/+ +/+ ADNFLE +/+ +/m +/m +/m +/+ +/m +/+ +/+ +/+ +/+ m KCNT1 Arg928Cys mutation Mutation absent in 111 Australian controls, dbSNP & EVS +/m +/m
  • 12. KCNT1: Further mutational analysis • KCNT1 mutation analysis by High Resolution Melting (HRM) • 117 unrelated familial & sporadic cases NFLE
  • 13. Additional KCNT1 mutations Italian Sephardic Jewish +/m Y796H +/+ +/+ R398Q +/m +/m +/m +/+ +/m +/m +/m NFLE m KCNT1 mutation Australian +/+ +/+ M869I 4 families have same phenotype: ADNFLE, ID and psychiatric features Severe form of ADNFLE de novo mutation +/m Heron et al Nat Gen 2013
  • 14. KCNT1 mutations Na+ regulated K+ channel NH2 (amino) terminus p.Arg398Gln Tyr796His (B) RCK NAD+ binding domain electrophysiology animal models COOH (carboxy) terminus Met896Ile (D) Arg928Cys (A) Heron et al Nature Genetics 2012
  • 15. Focal Epilepsy with Variable Foci • Autosomal dominant inheritance • Seizures originating from different brain regions • 8 families linkage to 22q12 (5.3 Mb; >100 genes) • WES in two families 22q12 (het variants) Australian Family Australian Family Dutch Family
  • 16. Unique putative variants in Ch 22 linkage interval Aust Family 2 1 2 2 1 1 1 2 2 1 1 2 2 1 2 1 2 1 2 2 2 Chrom. pos. (22q12) 27868137 28025765 28215313 28493438 28493438 28746153 29061481 29217908 29672432 29672432 29814555 30181151 30438394 30480928 30809099 30809102 30876430 30876430 30957012 31086882 31224485 Variant type Gene Accession 3’-UTR missense missense synonymous synonymous missense missense missense missense missense missense missense missense nonsense missense missense missense missense missense 3’-UTR missense KREMEN1 EWSR1 NEFH UQCR10 UQCR10 MTMR3 SF3A1 SEC14L4 MORC2 MORC2 SMTN EIF4ENIF1 PRR14L DEPDC5 SLC5A1 SLC5A1 C22orf42 C22orf42 SLC5A4 RFPL3 FBXO7 NM_001039570 NM_005243 NM_021076 NM_013387 NM_013387 NM_021090 NM_005877 NM_174977 NM_014941 NM_014941 NM_134270 NM_019843 NM_173566 NM_014662 NM_000343 NM_000343 NM_001010859 NM_001010859 NM_014227 NM_001098535 NM_012179 Nucleotide change c.1377+37G>C c.1855G>T c.1684C>G c.51G>A c.51G>A c.2505T>G c.2255C>A c.824G>T c.136T>C c.136T>C c.257T>A c.1250C>T c.5431A>T c.21C>G c.622A>T c.625G>T c.530G>C c.530G>C c.1072G>T c.954+63C>T c.1537C>G Amino Acid change none p.Gly619Trp p.Pro562Ala none none p.Phe835Leu p.Ala752Glu p.Arg275Met p.Ser46Pro p.Ser46Pro p.Leu86Gln p.Ser417Phe p.Thr1811Ser p.Tyr7* p.Thr208Ser p.Val209Leu p.Cys177Ser p.Cys177Ser p.Asp538Tyr none p.Arg513Gly
  • 17. DEPDC5 mutations cause FFEVF Australian Family m DEPDC5 mutation (m/+) Focal Epilepsy (m/+) Frontal 2 +/+ +/+ m/+ +/+ m/+ Temporal Parietal 2 m/+ +/+ +/+ +/+ +/+ +/+ +/+ +/+ m/+ m/+ m/+ +/+ m/+ m/+ m/+ m/+ m/+ +/+ Autism spectrum disorder Panayiotopoulos syndrome Intellectual disability 7/8 published FFEVF families have DEPDC5 mutation from Australia (1/2) The Netherlands(1), Spain(2), Canada(3)
  • 18. DEPDC5 in familial focal epilepsy • 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)
  • 19. DEPDC5 in familial focal epilepsy: Mutations in 12% small families DEPDC5 mutations in 10/82 (12%) pedigrees with familial focal epilepsy de novo mutation Focal seizure type Nocturnal frontal Frontal Fronto-temporal Temporal Parietal Occipital Multifocal Unclassified
  • 20. Mutations in DEPDC5 • Dishevelled, Egl-10 and Pleckstrin (DEP) domain containing (protein) 5 • DEP domain in G protein signalling FC1,FC2,FC3: Val164delPhe P:Ala452Val D1:Arg555* N:Ser1104Leu O:Ser1073Arg A1:Tyr7* J:Arg487* S1:Trp1369* M:Arg1268* K:Arg843* L&G:Trp1466* S2:Gln1536* DEP NH2 100 381 COOH 1170 1251 1604 aa H:c.193+1G I:c.279+1G>A Dibbens et al Nat Gen 2013
  • 21. DEPDC5 protein localization in neurospheres from human iPS cells NeuN: neuronal marker GFAP: astrocyte marker SOX2: cell body marker DEPDC5 protein in neurons Massimo Pandolfo, Belgium
  • 22. mTOR signalling DEPDC5 Bar Peled et al Science 2013
  • 23. 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
  • 24. Genetics of the Epilepsies Rare families: Monogenic inheritance Multiple single gene disorders ~60 genes now identified Susceptibility variants Majority of cases have complex inheritance Many gene variants + CNVs Modified by environmental factors <5% families
  • 25. 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 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 Patients and their families Funding: National Health Medical Research Council of Australia Program Grant, CDF and ECF Fellowships
  • 26. Seizures associated with DEPDC5 mutations • 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% • Focal epilepsies are genetic
  • 27. DEPDC5 in mTOR signaling from commentary on Bar Peled et al Science 2013
  • 28. Gene Discovery Strategy 1. Whole genome linkage analyses on pedigrees 2. Capture exons of genome = exome of affecteds (Sure Select 50Mb) 3. Whole Exome Sequencing (WES) (SOLiD v4) Chromosome 22 4. Align WES data, filter 5. Interrogate WES data in linkage interval 22q12 linkage interval 6. Autosomal dominant inheritance (het mutations)
  • 29. Focal Epilepsy Syndrome: FFEVF Familial Focal Epilepsy with Variable Foci (FFEVF) Family members have seizures originating from different brain regions (m/+) m/+ m/+ • 8 families published with linkage to 22q12 (5.3 Mb, >100 genes) m/+ WES m/+ m/+ m/+ m/+ +/+ • Exome sequencing in two families Psychiatric disorder m/+ m/+ m/+ m/+ +/+ Australian Family m DEPDC5 mutation
  • 30. Depdc5 expression in mouse RT-PCR mouse tissue
  • 31. A model of the population distribution of susceptibility alleles for more common epilepsies with complex inheritance seizure threshold insufficient number of susceptibility alleles to cause epilepsy healthy (=controls) Number of susceptibility alleles enough susceptibility alleles to cause common polygenic epilepsy “monogenic” epilepsy Mulley et al Hum Mol Genet 2005
  • 32. 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
  • 33. Family 1 with FFEVF (Australian) 4 3 3 3 MPS 4 2 2 3 Unclassified seizures Temporal lobe epilepsy Suggestive linkage Centro-parietal lobe epilepsy LOD score of 2.94 Frontal lobe epilepsy Probable syncope Probable Panayiotopoulos syndrome Intellectual disability/autism 2 Chromosome 22q12
  • 34. 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% Seizure types in 95 DEPDC5 32% positive individuals in (12% nocturnal) 7 large families 24% unclassified epilepsy 2% multifocal epilepsy 1% ASD no seizures 14% 1% 24% 2%
  • 35. Epilepsy Epilepsy - more than 30 different syndromes - affects 2% of the population at some stage in life Causes of Epilepsy Symptomatic Epilepsies Tumour Trauma Other ~ 200 genetic syndromes where epilepsy is secondary Cardiovascular ~70% Genetic Epilepsies Bio-bank 16000 cases (familial and sporadic)
  • 36. DEPDC5 mutations cause FFEVF Australian Family A1 m DEPDC5 mutation (m/+) Focal Epilepsy (m/+) Frontal 2 +/+ +/+ m/+ +/+ m/+ Temporal Parietal 2 m/+ +/+ +/+ +/+ +/+ +/+ +/+ +/+ m/+ m/+ m/+ +/+ m/+ m/+ m/+ m/+ m/+ +/+ Autism spectrum disorder Panayiotopoulos syndrome Intellectual disability 7/8 published FFEVF families have DEPDC5 mutation from Australia (1/2) The Netherlands(1), Spain(2), Canada(3)
  • 37. Family with ADNFLE: WES Genome –wide Linkage analysis (SNP array) suggestive linkage LOD 2.71 9q34.3 WES ADNFLE WES

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