Current Literature
In Basic Science
Epi4K Phase I: Gene Discovery in Epileptic
Encephalopathies by Exome Sequencing
De Novo Mutations in Epileptic Encephalopathies.
Epi4K Consortium; Epilepsy Phenome/Genome Project, Allen AS, Berkovic SF, Cossette P, Delanty N, Dlugos D, Eichler EE,
Epstein MP, Glauser T, Goldstein DB, Han Y, Heinzen EL, Hitomi Y, Howell KB, Johnson MR, Kuzniecky R, Lowenstein DH, Lu
YF, Madou MR, Marson AG, Mefford HC, Esmaeeli Nieh S, O’Brien TJ, Ottman R, Petrovski S, Poduri A, Ruzzo EK, Scheffer
IE, Sherr EH, Yuskaitis CJ, Abou-Khalil B, Alldredge BK, Bautista JF, Berkovic SF, Boro A, Cascino GD, Consalvo D, Crumrine
P, Devinsky O, Dlugos D, Epstein MP, Fiol M, Fountain NB, French J, Friedman D, Geller EB, Glauser T, Glynn S, Haut SR,
Hayward J, Helmers SL, Joshi S, Kanner A, Kirsch HE, Knowlton RC, Kossoff EH, Kuperman R, Kuzniecky R, Lowenstein DH,
McGuire SM, Motika PV, Novotny EJ, Ottman R, Paolicchi JM, Parent JM, Park K, Poduri A, Scheffer IE, Shellhaas RA, Sherr
EH, Shih JJ, Singh R, Sirven J, Smith MC, Sullivan J, Lin Thio L, Venkat A, Vining EP, Von Allmen GK, Weisenberg JL, WiddessWalsh P, Winawer MR. Nature 2013;501:217–221.
Epileptic encephalopathies are a devastating group of severe childhood epilepsy disorders for which the cause is often
unknown. Here we report a screen for de novo mutations in patients with two classical epileptic encephalopathies:
infantile spasms (n = 149) and Lennox–Gastaut syndrome (n = 115). We sequenced the exomes of 264 probands, and
their parents, and confirmed 329 de novo mutations. A likelihood analysis showed a significant excess of de novo mutations in the ~4,000 genes that are the most intolerant to functional genetic variation in the human population (P =
2.9 × 10−3). Among these are GABRB3, with de novo mutations in four patients, and ALG13, with the same de novo mutation in two patients; both genes show clear statistical evidence of association with epileptic encephalopathy. Given the
relevant site-specific mutation rates, the probabilities of these outcomes occurring by chance are P = 4.1 × 10−10 and P
= 7.8 × 10−12, respectively. Other genes with de novo mutations in this cohort include CACNA1A, CHD2, FLNA, GABRA1,
GRIN1, GRIN2B, HNRNPU, IQSEC2, MTOR and NEDD4L. Finally, we show that the de novo mutations observed are enriched
in specific gene sets including genes regulated by the fragile X protein (P < 10−8), as has been reported previously for
autism spectrum disorders.
Commentary
The Epi4K Consortium was launched in 2011 in response
to a National Institute of Neurological Disorders and Stroke
(NINDS) Funding Opportunity Announcement soliciting applications for “Centers Without Walls for Collaborative Research
in the Epilepsies: Genetics and Genomics of Human Epilepsies.”
This large-scale collaborative effort leverages large sample
cohorts to increase statistical power and accelerate epilepsy
gene discovery. The ultimate goal of the Epi4K Center without
Walls is to sequence the genomes of at least 4,000 individuals
with epilepsy using next-generation sequencing (NGS) (1).
NGS technology has revolutionized our ability to efficiently
and inexpensively sequence individuals at the whole exome
or whole genome level. The cost of whole exome sequencing
is now under $1000 per sample. Whole exome sequencing
studies have been quite successful for identifying de novo
dominant mutations in individuals with profound neurodevelEpilepsy Currents, Vol. 14, No. 4 (July/August) 2014 pp. 208–210
© American Epilepsy Society
208
opmental phenotypes (2, 3). This approach relies on a simple
genetic model in which the causative mutation is assumed to
be present in the genome of the affected child but absent in
the unaffected parents. On average, each individual carries a
mutation in one of their genes that was not present in their
parents (4) and, thus, is denoted as a de novo mutation. The
occurrence of de novo mutations in the same gene in two or
more unrelated individuals with similar clinical phenotypes
supports a potential pathogenic contribution to the disease.
The first project tackled by the Epi4K Consortium focused
on discovery of de novo mutations in two well-defined epileptic encephalopathies: Lennox–Gastaut syndrome and infantile
spasms. Patients were ascertained by the NINDS-funded
Epilepsy Phenome/Genome Project, a multi-center collaborative effort designed to collect detailed, high-quality phenotype information for genetic studies (5). The Epi4K Consortium
performed whole exome sequencing on 264 patient–parent
trios, of which 149 were classified as infantile spasms and 115
were Lennox–Gastaut syndrome. They identified 329 confirmed de novo mutations. On average, each patient harbored
1.25 de novo mutations, which is consistent with other studies
reported in the literature (4, 6). The majority of the mutations
Epileptic Encephalopathies Exome Sequencing
(72%) were missense (encoding an amino acid change), while
7.5% resulted in premature stop codons. Relative to control
populations, the observed rate of loss-of-function mutations
was significantly elevated in the patients.
Nine genes were found to have de novo mutations in two
or more probands, of which five genes had an already known
association with epileptic encephalopathy. The known epileptic encephalopathy genes with mutations in multiple Epi4K
subjects were: SCN1A, STXBP1, SCN8A, SCN2A, and CDKL5. Additional novel genes with mutations in multiple individuals included: GABRB3, ALG13, CACNA1A, CHD2, FLNA, GABRA1, GRIN1,
GRIN2B, HNRNPU, IQSEC2, MTOR, and NEDD4L. To evaluate the
novel genes, the authors applied a more rigorous standard
that takes into account the local mutation rate, gene size, and
mutation intolerance. Using this approach, they found statistical evidence of association between epileptic encephalopathy
and GABRB3 and ALG13.
De novo missense mutations in GABRB3 were observed in
four individuals, including one with infantile spasms only and
three with infantile spasms that evolved to Lennox–Gastaut
syndrome. Although missense mutations in GABRB3 had
previously been associated with childhood absence epilepsy
(7), this report expands the phenotype range of this gene to
include epileptic encephalopathies. Future functional studies will be required to determine the underlying molecular
mechanisms and understand how mutations in GABRB3 lead
to seemingly disparate clinical syndromes.
ALG13 is an X-linked gene that encodes a subunit of the
uridine diphosphate-N-acetylglucosamine transferase. De
novo mutations in ALG13 had previously been associated with
severe intellectual disability with seizures and dysmorphic
features (8), and a glycosylation disorder with microcephaly,
seizures, and early lethality (9). In the current study, the same
missense mutation was observed in two unrelated patients,
one with infantile spasms only and one with infantile spasms
that evolved to Lennox–Gastaut syndrome. Interestingly, this
mutation was identical to the mutation previously reported by
de Ligt and colleagues in a patient with intellectual disability,
seizures, and dysmorphic features (8). Together, these reports
indicate that there is considerable phenotype heterogeneity
associated with mutations in ALG13. Again, functional studies
will be necessary to understand the mechanisms by which
ALG13 mutations result in variable developmental disorders.
The collective list of epileptic encephalopathy genes reported by Epi4K has substantial overlap with genes identified
in studies of other neurodevelopmental disorders, including
intellectual disability and autism (2, 3, 6). This high degree of
phenotype heterogeneity suggests that other factors, including environmental, stochastic, and genetic background effects,
interact with the mutation during development to shape the
distinct clinical phenotype that is expressed in an individual.
Epileptic encephalopathies also exhibit considerable locus
heterogeneity, with mutations in many different genes resulting in infantile spasms or Lennox–Gastaut syndrome. Based
on the observed locus heterogeneity, there are likely more
epileptic encephalopathy genes in the long list of single-hit
genes reported by the Epi4K Consortium in a supplementary
table. A protein–protein interaction analysis on all genes that
had at least one de novo mutation revealed a network of 71 in-
terconnected proteins, including six previously associated with
epileptic encephalopathy. Approximately 20% of the single-hit
genes fit in this network, strongly suggesting that they may
represent additional epileptic encephalopathy genes. Followup in larger patient cohorts will likely uncover additional mutations in some of these genes and validate them as epileptic
encephalopathy genes. Additionally, future functional studies
will provide further support and advance our understanding
of the underlying mechanisms.
The high degree of locus heterogeneity observed in epileptic
encephalopathies has practical implications for molecular
diagnostics. It highlights the need for a strategy shift away from
gene-by-gene or gene panel screening. Given that it is difficult
to predict the gene likely to be mutated, clinical exome sequencing is the most expeditious and cost–effective path to genetic
diagnosis once proximate causes have been ruled out and a
genetic basis is likely. There are challenges for integrating exome
sequencing into clinical practice, including interpretation and
counseling, diagnostic yield and insurance authorization. However, similar obstacles existed for earlier generations of genetic
testing, like single gene tests, and they were overcome with education, accumulating knowledge, and shifting norms. Ultimately,
rapid genetic diagnosis is another tool that will improve disease
management and may lead to more favorable long-term outcomes. Moreover, genetic diagnosis is valuable to the family for
establishing clinical and social support services, family planning,
and alleviating the stress of diagnostic uncertainty.
by Jennifer A. Kearney, PhD
References
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Epileptic Encephalopathies Exome Sequencing
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