Recent Discoveries of Epilepsy Related Genes: A Preview of the Future Pediatric State of the Art Symposium Genetics of Catastrophic Infantile Epilepsies: From Gene Discovery to Practical Clinical Applications December 9, 2013 Erin Heinzen, Pharm.D., Ph.D. Center for Human Genome Variation Duke University American Epilepsy Society | Annual Meeting 1
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Recent Discoveries of Epilepsy Related Genes: A Preview of the Future
Pediatric State of the Art Symposium
Genetics of Catastrophic Infantile Epilepsies: From Gene Discovery to Practical Clinical Applications
December 9, 2013
Erin Heinzen, Pharm.D., Ph.D. Center for Human Genome Variation
Duke University
American Epilepsy Society | Annual Meeting
1
Disclosure
NONE
American Epilepsy Society | 2013 Annual Meeting 2
Learning Objectives
1. To provide an overview of gene discovery in epilepsy
2. To discuss recent studies using next-generation sequencing to investigate the role of de novo mutations in epileptic encephalopathies
3. To discuss what is needed to better characterize these discoveries to be able to apply the knowledge to clinical practice
ERDS (Estimation by Read Depth with SNvs) Average read depth (RD) of every 2-kb window were calculated, followed
by GC corrections. A paired Hidden Markov model was applied to infer copy numbers of every window by utilizing both RD information and heterozygosity information.
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NGS Study Designs
Multiplex families
Case-control studies
Trio sequencing of
sporadic diseases
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Number of publications reporting de novo mutations as the cause of a disease
2. ROLE OF THE GENE IN PARTICULAR PATIENT POPULATIONS
TARGETED SEQUENCING IN LARGER PATIENT
POPULATIONS
FUNCTIONAL STUDIES
1. MOLECULAR CONSEQUENCES
2. PHARMACOLOGIC MODULATION
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Epileptic Encephalopathies
Infantile Spasms (IS) • 1 in 3000 live births and onset between 4-12 months of life • Characteristic chaotic interictal & EEG pattern of hypsarrhythmia,
the sine qua non of the syndrome • 50-60% of IS cases have developmental brain malformations,
tuberous sclerosis complex, chromosomal syndromes and metabolic conditions
• Patients may evolve into LG Lennox-Gastaut syndrome (LGS) • Onset between 1-8 years • Characterized by mixed seizure types and intellectual disabilities • Cause unknown in about 25-35% cases, symptomatic of structural
or metabolic abnormalities
Exome sequenced 149 IS probands, 115 LGS probands and their
unaffected parents
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De novo mutation calling
• GATK Unified Genotyper – Multi-sample calling within each trio identifying variants that met the following criteria:
• Candidate de novo mutations were absent from populations controls
• Sanger sequencing was used to confirm the was present and de novo
Epi4K and EPGP Investigators. Nature 2013.
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Distribution of de novo mutations in 264 EE probands
0
10
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30
40
50
60
70
80
90
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NumberofEEproab
nds
Numberofdenovomuta onsperEEproband
0 1 2 3 4 5 6
likelihood analysis to test for presence of risk factors in these data (adapted from the model established in Neale et al, Nature 2013)
Epi4K and EPGP Investigators. Nature 2013.
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Genic tolerance to functional variation
Hypothesis: Genes intolerant to standing functional variation are more likely to carry mutations that cause disease
ExomeVariantServer: database of variants in 6500 individuals
• Y = Number of ‘damaging’ coding variants >0.1% MAF
• Regressor (X) = Total number of coding variants
• Score (S) = Studentized residuals from regressing Y on X
= RVIS Petrovski S, et al. PLoS Genet. 2013 Aug;9(8):e1003709.
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Adapted from Petrovski S, et al. PLoS Genet. 2013 Aug;9(8):e1003709.
RVIS for OMIM disease genes
OMIM disease genes (n=2131)
Genes not reported in OMIM to cause disease (14,712)
OMIM haploinsufficiency genes (n=175)
OMIM recessive genes (n=817)
OMIM de novo genes (n=467)
OMIM dominant-negative (n=364)
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RVIS Percentiles
C
um
ula
tive
per
cen
t o
f O
MIM
dis
eas
e
g
enes
exp
lain
ed
Neurodevelopmental genes tend to have lower RVIS
Neurodevelopmental disease gene list from Goh et al. 2007. The Human Disease Network. PNAS 104(21): 8685-8690
Adapted from Petrovski S, et al. PLoS Genet. 2013 Aug;9(8):e1003709.
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Distribution of de novo mutations in intolerant genes in 264 EE probands
Number of de novo coding mutations observed in intolerant
genes per IS/LGS trios
likelihood analysis • significant excess of de novo mutations in the
∼4,000 “intolerant” genes • ~90 EE genes • Mutation effect size of ~81
Epi4K and EPGP Investigators. Nature 2013.
1234560 1 2 3 4 5
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0
Nu
mb
er
of
trio
s
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Test for excess mutation in individual genes as a function of the mutation rate and size
The mutation rate (M) of each gene was calculated using the tri-
nucleotide mutation rate matrix provided by Drs. Shamil Sunyaev and
Paz Polak.
P = 1 – Poisson cumulative distribution function (x-1, λ)
where, x is the observed de novo mutant number for the specific gene, λ
is calculated as 2S*M for genes on autosome or (2f + m)*M for genes on
chromosome X
(S is the number of trios; f and m are the number of sequenced female
and male probands, respectively). Genes on Y chromosome were not
part of these analyses.
Epi4K and EPGP Investigators. Nature 2013.
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Gene Chr Average effectively
sequenced length (bp)
Weighted
mutation rate
De novo
mutation
number
p-value†
SCN1A 2 6063.70 1.61x10-4 5 1.12x10-9 ***
STXBP1 9 1917.51 6.44x10-5 5 1.16x10-11 ***
GABRB3 15 1206.86 3.78x10-5 4 4.11x10-10 ***
CDKL5 X 2798.38 5.44x10-5 3 4.90x10-7 **
ALG13 # X 475.05 1.03x10-5 2 7.77x10-12 ***
DNM1 9 2323.37 9.10x10-5 2 2.84x10-4
HDAC4 2 2649.82 1.16x10-4 2 4.57x10-4
SCN2A # 2 5831.21 1.52x10-4 2 1.14x10-9 ***
SCN8A 12 5814.48 1.64x10-4 2 9.14x10-4 # Two de novo mutants occur at the same position.
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A protein–protein interaction network of genes with de novo mutations
Epi4K and EPGP Investigators. Nature 2013. 21
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What’s next?
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Epi4K and EPGP Investigators. Nature 2013.
Nakamura et al. AJHG 2013.
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Glycosylation defects in EE
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Epi4K Additional exome
sequencing of EE trios
EuroEPINOMICS Exome sequencing of EE
trios
NEW EE GENES AND ASSOCIATED
CLINICAL PHENOTYPES
Epi4K Targeted NGS of EE candidate
genes in ~600 EE trios
EPIGEN Consortium Targeted NGS of EE
candidate genes in 500 adult EE patients
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iPSC-derived neurons to unravel disease pathophysiology
• Differentiate human stem cells carrying mutations into inhibitory neurons, excitatory neurons, and glia cells.
• Create functioning network of neurons in vitro to study the effects of the mutations, and to test compounds that will reverse the pathophysiological changes.
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Multi Electrode Array (MEA)
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iPSC-derived neuron activity
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Acknowledgements
Epi4K Consortium Samuel F. Berkovic, David B. Goldstein, Daniel H. Lowenstein, Andrew S. Allen, Patrick Cossette, Norman Delanty, Dennis Dlugos, Evan E. Eichler, Michael P. Epstein, Tracy Glauser, Yujun Han, Erin L. Heinzen, Yuki Hitomi, Katherine B. Howell, Michael R. Johnson, Ruben Kuzniecky, Daniel H. Lowenstein, Yi-Fan Lu, Maura R. Z. Madou, Anthony G. Marson, Heather C. Mefford, Sahar Esmaeeli Nieh, Terence J. O'Brien, Ruth Ottman, Slavé Petrovski, Annapurna Poduri, Elizabeth K. Ruzzo, Ingrid E. Scheffer, Elliott H. Sherr & Christopher J. Yuskaitis
Epilepsy Phenome/Genome Project Bassel Abou-Khalil, Brian K. Alldredge, Jocelyn F. Bautista, Samuel F. Berkovic, Alex Boro, Gregory D. Cascino, Damian Consalvo, Patricia Crumrine, Orrin Devinsky, Dennis Dlugos, Michael P. Epstein, Miguel Fiol, Nathan B. Fountain, Jacqueline French, Daniel Friedman, Eric B. Geller, Tracy Glauser, Simon Glynn, Sheryl R. Haut, Jean Hayward, Sandra L. Helmers, Sucheta Joshi, Andres Kanner, Heidi E. Kirsch, Robert C. Knowlton, Eric H. Kossoff, Rachel Kuperman, Ruben Kuzniecky, Daniel H. Lowenstein, Shannon M. McGuire, Paul V. Motika, Edward J. Novotny, Ruth Ottman, Juliann M. Paolicchi, Jack M. Parent, Kristen Park, Annapurna Poduri, Ingrid E. Scheffer, Renée A. Shellhaas, Elliott H. Sherr, Jerry J. Shih, Rani Singh, Joseph Sirven, Michael C. Smith, Joseph Sullivan, Liu Lin Thio, Anu Venkat, Eileen P. G. Vining, Gretchen K. Von Allmen, Judith L. Weisenberg, Peter Widdess-Walsh & Melodie R. Winawer
EuroEPINOMICS Peter De Jonghe, Ingo Helbig, Anna-Elina Lehesjoki, Holgar Lerche, Arvid Suls, Sarah Weckhuysen, Johanna Jaehn, Bobby Koeleman, Hiltrud Muhle, Padhraig Gormley, Sarah Von Spiczak, Silke Appenzeller
EpiGen Consortium David Goldstein, Norman Delanty, Gianpiero Cavalleri, Chantal Depondt, Danielle Andrade
Funding National Institute of Neurological Disorders and Stroke