© 2013 Direct One Communications, Inc. All rights reserved. 1 From Molecules to Cells, Networks, and Seizures: How Does a Gene Cause Epilepsy? Belinda

© 2013 Direct One Communications, Inc. All rights reserved. 1

From Molecules to Cells, Networks, and Seizures: How Does a Gene Cause Epilepsy?

Belinda Oyinkan Marquis, MD

State University of New York Downstate Medical Center, Brooklyn, New York

A REPORT FROM THE 66th ANNUAL MEETING OF THE AMERICAN EPILEPSY SOCIETY


Gene Discovery in Epilepsy

Genome-scanning technologies have uncovered an unexpected number of variants (deletions, duplications, insertions, inversions, and translocations), collectively known as copy number variants (CNVs), in the human genome.1

Microdeletions or microduplications of segments range from a few hundred base pairs to several hundred megabases (Mb).

Changes in copy number are revealed by comparing two or more genomes1

CNVs are involved in genetically complex epilepsies and may be inherited or occur de novo2


Recurrent Copy Number Variants

Three large, recurrent microdeletions at 15q13.3, 16p13.11, and 15q11.2 are each present in 0.5%–1% of patients with epilepsy.3

Microdeletions of 15q13.3 are associated with idiopathic generalized epilepsies (odds ratio, 68)4 and are risk factors for related mental disorders (intellectual disability, autism, schizophrenia)5

Known medical conditions related to microdeletion disorders are Angelman, Prader-Willi, and Williams-Beuren syndromes.


Nonrecurrent Copy Number Variants

Nonrecurrent copy number variants are rare, occur throughout the genome, and are not sequence-dependent.

Large (> 2 Mb), rare copy number variants, including deletion at 16p13.11, are enriched in patients with diverse epilepsy syndromes.6

Rare copy number variants occur in 10% of patients with various types of epilepsy3 and 8% of patients with epileptic encephalopathies.7


Laboratory Methods


Laboratory Methods:

Comparative Gene Hybridization (CGH)

CGH is the most robust method for performing genome-wide scans to find novel copy number variants.

CGH uses labeled fragments from a genome of interest (proband).

Fragments are hybridized with a second differentially labeled genome to arrays spotted with cloned DNA fragments to reveal copy number differences between two genomes.1

CGH has revealed many more copy number variants in humans than expected.8


Laboratory Methods:

Single-Nucleotide Polymorphisms (SNPs)

SNPs are DNA arrays that detect polymorphisms within a population.

An array contains the target nucleic acid sequence; one or more labeled allele-specific oligonucleotide probes are applied, and a detection system that records and interprets the hybridization signal is used.9

SNPs are useful for detecting unilateral disomy and consanguinity


Laboratory Methods:

Analysis of Copy Number Variants

Sanger method

» Involves a DNA primer, DNA polymerase, and deoxynucleopeptides

» Sample DNA is denatured and copied, sequenced, and analyzed.

» Time-consuming and expensive

Next-generation sequencing

» Captures exomes, the part of the genome formed by exons

» Information-rich extraction is then run through automated next-generation sequencing.


Gene Discovery: Types of Analysis


Exome Sequencing for Diagnosis

Next-generation gene panels are available commercially to sequence many genes but are not all-inclusive.

For a specific diagnosis, clinicians should investigate a particular gene (eg, SCN1A for Dravet syndrome, MECP2 for Rett syndrome).

For a nonspecific epilepsy-plus syndrome or multiple causative genes, consider testing for copy number variants or gene-panel analysis.

For an interesting phenotype and a supportive family history, consider whole-exome sequencing (if financially feasible).


Diagnostic Pathways: Exome Sequencing


Dravet Syndrome

Also known as severe myoclonic encephalopathy of infancy (SMEI), Dravet syndrome is related to early-onset seizures and associated with cognitive impairments and 15% mortality by adolescence.10

Most often caused by a de novo loss of function mutation in the neuronal sodium-channel gene SCN1A, which leads to haploinsufficiency of NaV1.1 channels.11

» Voltage-gated sodium channels are critical in initiating and propagating action potentials and are crucial regulators of neuronal excitability.

» Mutations in SCN1A cause genetically distinct epilepsy syndromes.


Research to Determine Cause and Effect

Heterologous expression systems

Primary neuron cultures

Transgenic knockout mouse models

Induced pluripotent stem cell technology


Gene Identification Studies

Linkage studies

Association studies

Candidate-gene association studies

Genome-wide association studies

Next-generation sequencing


The Epi4K Consortium

The National Institute of Neurological Disorders and Stroke (NINDS) issued a Funding Opportunity Announcement to create the Epi4K Consortium.12

This “center without walls” is a collaborative study to sequence at least 4,000 subjects with epilepsy.

The Consortium consists of three cores, four scientific projects, and a steering committee 0f primary investigators and representatives from NINDS.


The Epi4K Consortium

Three scientific projects will analyze epileptic encephalopathies, multiplex families and pairs, and epilepsy prognosis from seven large-scale genetic studies conducted around the world.

The remaining project in the Epi4K Consortium will apply cutting-edge analytic techniques related to the detection of copy number variants.


Research for Gene Discovery

Familial aggregation studies

Twin studies

Multiplex family studies


Practical Use of Gene Discoveries

Monogenic epilepsies often are related to dominant inheritance and can display genetic heterogeneity with pleiotropic expression.

De novo mutagenesis has unexpectedly shown significant importance in various epilepsies, particularly Dravet syndrome.

» Parental germ-line mutations

» Postzygotic mutations

» Mosaicism


Mosaicism and Dravet Syndrome

In Dravet syndrome, somatic mosaicism found when mildly affected parents have one or more affected offspring.

Germ-line mosaicism inferred in cases of unaffected parents having multiple affected offspring.13

Gametal mutations linked to negligible recurrence risk, whereas germ-line mosaicism linked to a high risk of recurrence.13–20


Timing of Mutations in Dravet Syndrome


Promise of Epilepsy Genetics

Gene identification using exome sequencing may be studied by evaluating consanguineous families or small families with many linkage peaks.


Promise of Epilepsy Genetics

Next-generation exome will significantly impact diagnosis and management21

» Novarino et al22 identified inactivating mutations in BCKDK in consanguineous families with autism, epilepsy, and intellectual disability.

Exome sequencing may allow greater:

» Understanding of novel disease-causing genes in genetically enriched families

» Identification of known causes of disease

» Correction of diagnosis and prognosis

» Direction of treatment


References1. Feuk L, Carson A, Scherer S. Structural variation in the human genome. Nature. 2006;7:85–97.

2. Scheffer IE, Berkovic SF. Copy number variants—an unexpected risk factor for the idiopathic generalized epilepsies. Brain. 2010:133:7–8.

3. Mefford HC, Muhle H, Ostertag P, et al. Genome-wide copy number variation in epilepsy: novel susceptibility loci in idiopathic generalized and focal epilepsies. PLoS Genet. 2010;6:e1000962.

4. Dibbens LM, Mullen S, Helbig I, et al. Familial and sporadic 15q13.3 microdeletions in idiopathic generalized epilepsy: precedent for disorders with complex inheritance. Hum Mol Genet. 2009;18:3626–3631.

5. Mefford HC, Mulley JC. Genetically complex epilepsies, copy number variants and syndrome constellations. Genome Med. 2010;2:71.

6. Heinzen EL, Radtke RA, Urban TJ, et al. Rare deletions at 16p13.11 predispose to a diverse spectrum of sporadic epilepsy syndromes. Am J Hum Genet. 2010;86:707–718.

7. Mefford HC, Yendle SC, Hsu C, et al. Rare copy number variants are an important cause of epileptic encephalopathies. Ann Neurol. 2011;70:974–985.

8. Mills RE, Walter K, Stewart C, et al. Mapping copy number variation by population-scale genome sequencing. Nature. 2011;470:59–65.

9. Sherry ST, Ward MH, Kholodov M, et al. dbSNP: the NCBI database of genetic variation. Nucleic Acids Res. 2001;29:308–311.

10. Genton P, Velizarova R, Dravet C. Dravet syndrome: the long-term outcome. Epilepsia. 2011;52:44–49.

11. Claes L, Del-Favero J, Ceulemans B, Lagae L, Van Broeckhoven C, De Jonghe P. De novo mutations in the sodium-channel gene SCN1A cause severe myoclonic epilepsy of infancy. Am J Hum Genet. 2001;68:1327–1332.


References12. The Epi4K Consortium. Epi4K: gene discovery in 4,000 genomes. Epilepsia. 2012;53:1457–1467.

13. Vadlamudi L, Dibbens LM, Lawrence KM, et al. Timing of de novo mutagenesis—a twin study of sodium-channel mutations. N Engl J Med. 2010;363:1335–1340.

14. Gennaro E, Santorelli FM, Bertini E, et al. Somatic and germline mosaicisms in severe myoclonic epilepsy of infancy. Biochem Biophys Res Commun. 2006;341:489–493

15. Marini C, Mei D, Cross JH, Guerrini R. Mosaic SCN1A mutation in familial severe myoclonic epilepsy of infancy. Epilepsia. 2006;47:1737–1740.

16. Marini C, Scheffer IE, Nabbout R, et al. SCN1A duplications and deletions detected in Dravet syndrome: implications for molecular diagnosis. Epilepsia. 2009;50:1670–1678.

17. Depienne C, Arzimanoglou A, Trouillard O, et al. Parental mosaicism can cause recurrent transmission of SCN1A mutations associated with severe myoclonic epilepsy of infancy. Hum Mutat. 2006;27:389–398.

18. Morimoto M, Mazaki E, Nishimura A, et al. SCN1A mutation mosaicism in a family with severe myoclonic epilepsy in infancy. Epilepsia. 2006;47:1732–1736.

19. Selmer KK, Eriksson AS, Brandal K, Egeland T, Tallaksen C, Undlien DE. Parental SCN1A mutation mosaicism in familial Dravet syndrome. Clin Genet. 2009;76:398–403.

20. Rodda JM, Scheffer IE, McMahon JM, Berkovic SF, Graham HK. Progressive gait deterioration in adolescents with Dravet syndrome. Arch Neurol. 2012;69:873–878.

21. Dixon-Salazar TJ, Silhavy JL, Udpa N, et al. Exome sequencing can improve diagnosis and alter patient management. Sci Transl Med. 2012;4:138ra78.

22. Novarino G, El-Fishawy P, Kayserili H, et al. Mutations in BCKD-kinase lead to a potentially treatable form of autism with epilepsy. Science. 2012;338:394–397.

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© 2013 Direct One Communications, Inc. All rights reserved. 1 From Molecules to Cells, Networks, and Seizures: How Does a Gene Cause Epilepsy? Belinda