Download SCN1A - The Neurology Report

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
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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
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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 WilliamsBeuren syndromes.
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Nonrecurrent Copy Number Variants

Nonrecurrent copy number variants are rare, occur
throughout the genome, and are not sequencedependent.

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
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Laboratory Methods
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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
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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
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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.
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Gene Discovery: Types of Analysis
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9
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).
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10
Diagnostic Pathways: Exome Sequencing
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11
Dravet Syndrome

Also known as severe myoclonic encephalopathy of
infancy (SMEI), Dravet syndrome is related to earlyonset 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.
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12
Research to Determine Cause and Effect

Heterologous expression systems

Primary neuron cultures

Transgenic knockout mouse models

Induced pluripotent stem cell technology
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13
Gene Identification Studies

Linkage studies

Association studies

Candidate-gene association studies

Genome-wide association studies

Next-generation sequencing
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14
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.
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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.
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Research for Gene Discovery

Familial aggregation studies

Twin studies

Multiplex family studies
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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
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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
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19
Timing of Mutations in Dravet Syndrome
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20
Promise of Epilepsy Genetics
Gene identification using exome sequencing may be
studied by evaluating consanguineous families or small
families with many linkage peaks.
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21
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
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References
1.
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.
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References
12. 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 sodiumchannel 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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