• Epileptic encephalopathy;
  • SCN1A;
  • CDKL5;
  • ARHGEF15;
  • CLCN4


  1. Top of page
  2. Summary
  3. Subjects and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure
  8. References


The management of epilepsy in children is particularly challenging when seizures are resistant to antiepileptic medications, or undergo many changes in seizure type over time, or have comorbid cognitive, behavioral, or motor deficits. Despite efforts to classify such epilepsies based on clinical and electroencephalographic criteria, many children never receive a definitive etiologic diagnosis. Whole exome sequencing (WES) is proving to be a highly effective method for identifying de novo variants that cause neurologic disorders, especially those associated with abnormal brain development. Herein we explore the utility of WES for identifying candidate causal de novo variants in a cohort of children with heterogeneous sporadic epilepsies without etiologic diagnoses.


We performed WES (mean coverage approximately 40×) on 10 trios comprised of unaffected parents and a child with sporadic epilepsy characterized by difficult-to-control seizures and some combination of developmental delay, epileptic encephalopathy, autistic features, cognitive impairment, or motor deficits. Sequence processing and variant calling were performed using standard bioinformatics tools. A custom filtering system was used to prioritize de novo variants of possible functional significance for validation by Sanger sequencing.

Key Findings

In 9 of 10 probands, we identified one or more de novo variants predicted to alter protein function, for a total of 15. Four probands had de novo mutations in genes previously shown to harbor heterozygous mutations in patients with severe, early onset epilepsies (two in SCN1A, and one each in CDKL5 and EEF1A2). In three children, the de novo variants were in genes with functional roles that are plausibly relevant to epilepsy (KCNH5, CLCN4, and ARHGEF15). The variant in KCNH5 alters one of the highly conserved arginine residues of the voltage sensor of the encoded voltage-gated potassium channel. In vitro analyses using cell-based assays revealed that the CLCN4 mutation greatly impaired ion transport by the ClC-4 2Cl/H+-exchanger and that the mutation in ARHGEF15 reduced GEF exchange activity of the gene product, Ephexin5, by about 50%. Of interest, these seven probands all presented with seizures within the first 6 months of life, and six of these have intractable seizures.


The finding that 7 of 10 children carried de novo mutations in genes of known or plausible clinical significance to neuronal excitability suggests that WES will be of use for the molecular genetic diagnosis of sporadic epilepsies in children, especially when seizures are of early onset and difficult to control.

For pediatric neurologists, some of the most challenging patients with epilepsy are those with early onset (≤6 months of age) seizures that are difficult to control with medications, or mixed seizure types that change over time, and with cognitive impairment, motor deficits, or autism as comorbid features. Intractable seizures, especially when they begin early in life, disrupt brain development, causing regression of cognitive, language, and motor skills (Wirrell et al., 2005). However, good seizure control does not guarantee good cognitive and behavioral outcome, in part because some epilepsy disorders have co-occurring intellectual disability and/or autism (Tuchman & Cuccaro, 2011). Taken together, these patients are at considerable risk for long-term disability, reduced quality of life, and even early death (Berg et al., 2004). Although many of these cases likely have an underlying genetic basis, the genetic heterogeneity of epilepsy makes Sanger-sequencing approaches for genetic testing impractical, with 265 epilepsy genes already identified (Lemke et al., 2012) and many thousands of genes potentially involved in brain development and function (Kang et al., 2011).

Fortunately, the rapid development of next-generation sequencing (NGS)—notably whole exome sequencing (WES)—now enables researchers to interrogate all known protein-coding genes in a single experiment. There is now great optimism that this technology will lead to the identification of new genes and new mutations that cause neurologic disease (Bamshad et al., 2011; Bras & Singleton, 2011; The Epi4K Consortium, 2012). In particular, the application of NGS in parent–offspring trios and quartets has been successful in identifying candidate pathogenic de novo mutations in patients with neurodevelopmental disorders (Vissers et al., 2010; de Ligt et al., 2012; Neale et al., 2012; O'Roak et al., 2012; Rauch et al., 2012; Sanders et al., 2012; Xu et al., 2012), including epilepsy (Veeramah et al., 2012).

In this study we explore the applicability of WES within a trio framework to identify de novo variants for molecular genetic diagnosis of children with sporadic cases of epileptic encephalopathies with no known etiology. These 10 patients, from a single community-based pediatric neurology practice, also had a mix of other clinical manifestations, including developmental delay, autistic features, cognitive impairment, and motor deficits.

Subjects and Methods

  1. Top of page
  2. Summary
  3. Subjects and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure
  8. References


We identified 10 children being treated at the Tucson Center for Neurosciences who had epilepsy that was difficult to control as well as one or more complicating factors in their clinical history, either before or after seizure onset (Table 1). They presented with seizures between the ages of 3 weeks and 8.5 years (median age of onset = 6 months). At the time of the study, the probands had been followed for between 10 months and 17.5 years (median follow-up 7 years, 3 months). Many of the children have had mixed seizure types, often changing over time. Three of the probands (A, B, H) had clinical features of West syndrome with primary presentation of infantile spasms, but without a specific etiology, and two of the probands (D, E) were suspected of having Dravet syndrome on clinical grounds. As a group, their seizures were intractable to medical therapy, or inadequately controlled on antiepileptic medication, or required several medications or trials of medications to achieve adequate control. Six children have persistently refractory seizures (probands A, B, C, D, E, and G) and in three others seizures are infrequent but not completely controlled (probands F, I, and J). Seizures are currently controlled in only one patient (proband H), who had late-onset infantile spasms, was treated with adrenocorticotropic hormone (ACTH) and subsequently stabilized on two antiepileptic medications. Proband H was also the only child with congenital anomalies (hypospadias and ventriculoseptal defect) and significant prematurity (born at 34 weeks of gestation), requiring a lengthy neonatal intensive care unit (NICU) stay.

Table 1. Clinical summary of patients examined by WES
Proband (sex)AgeAge at seizure onsetGestational, birth, and neonatal HxInitial seizure type(s)Subsequent seizure historyEEG findings and evolutionNeurodevelomental history and examOther
  1. AD(H)D, attention deficit/hyperactivity disorder; FTT, failure to thrive; GTCS, generalized tonic–clonic seizure; HTN, hypertension; LGA, large for gestational age; L, left; R, right; VSD, ventriculoseptal defect; WES; whole exome sequencing.

A (F)6 years3 weeksFull term; nuchal cord, brief O2Multifocal clonic, generalized clonic, myoclonic, and generalized tonicInfantile spasms from 5 months to 5 years; complex partial seizures at 3 years; subsequently myoclonic, tonic, GTCS. Now: refractoryNormal at 3 weeks; hypsarrhythmia at 5 months; then diffuse slowing, frequent multifocal spikes, bursts of generalized spike/polyspike-and-wave, runs of slow spike-wave and episodic diffuse suppressionEpileptic encephalopathy; nonverbal, limited comprehension; autism; hypotonia, but increased ankle tone; poor coordination; unsteady wide-based gait; L BabinskiPresentation consistent with West syndrome
B (M)14 years10 weeksGestational diabetes (insulin) and HTN; full term, C-sectionMultifocal myoclonic and generalized myoclonicInfantile spasms at 8 months, responsive to pyridoxine; myoclonic at 3 years; at 4 years: myoclonic/tonic; later atonic and GTCS. Now: refractoryMultifocal sharp waves and generalized spike-wave to hypsarrhythmia to pattern of Lennox-Gastaut syndrome (multifocal spikes, runs of slow spike-wave, generalized spike-wave bursts and periods of diffuse suppression)Epileptic encephalopathy; acquired microcephaly (<2nd‰); severe delay with episodic regression; nonverbal, limited comprehension; hypotonia, with increased ankle tone; incoordination, gait instabilityPresentation consistent with West syndrome PET scan – relative hypometabolism L temporal and parietal lobes
C (M)14 months4 monthsDetached placenta, mild fetal distress, C-sectionComplex partial seizuresComplex partial seizures and secondarily GTCS, some prolonged, not fully responsive to medications. Now: refractory8 months: bilateral independent high-amplitude spikes, L > R; bursts of generalized spike and polyspike-waveMicrocephaly (2nd‰), delayed milestones, diffuse hypotonia, dystonic posturing of arms; smiles, follows, reaches; unable to sit, roll over. No regressionMRI: corpus callosum hypoplasia, increased FLAIR signal in white matter
D (F)14 years5 monthsFull term after preterm labor, LGA, C-section (breech)Prolonged febrile GTCSGeneralized tonic-clonic; myoclonic; tonic. Dravet syndrome suspected. Now: refractoryNormal at 5 months; generalized spike-and-wave at 2.5 yearsEpileptic encephalopathy; normal at 1 year, then progressive delay; poor coordination, unsteady gait, speaks 2–3 word phrases, no reading, math 
E (M)4 years6 monthsPreeclampsia and gestational diabetes (insulin). Full term, nuchal cord, brief O2Vaccine-related febrile GTCS and hemiclonicGTCS and hemiclonic febrile and nonfebrile, some prolonged. Dravet syndrome suspected. Now: refractoryNormal at 6 months; bifrontal spikes at 4 yearsEpileptic encephalopathy; normal motor development; language delay with limited progression, some regression (uses 2–3-word phrases); decreased coordination(+) FH febrile seizure (maternal uncle)
F (M)13 years6 monthsFull termNonfebrile GTCSGTCS or hemiclonic, prolonged and often in clusters with illness; relatively good control with valproate; occasional, brief clonic (facial). Now: partly controlledFrequent multifocal spikes, almost continuous during sleep, even with good seizure controlEpileptic encephalopathy; normal until 6 months; mild motor and severe language delay; regression at 3 years – reduced social interaction and attention; nonverbal with limited comprehension; autism; hypotonia 
G (M)18 years6 monthsFull term; Apgar 4Infantile spasms, nonresponsive to ACTH, controlled with vigabatrinComplex partial seizures at 3.5 years, with recurrence of clusters of flexor spasms at 14 years. Now: refractory6 months: hypsarrhythmia (resolved on vigabatrin); subsequently bihemispheric slowing, multifocal sharp wavesEpileptic encephalopathy; mild motor delay, moderate to severe speech-language delay; 1st-grade level; movements slow with poor coordination; emotional lability and compulsive behaviorsPET: R > L frontal lobe hypometabolism
H (M)7 years23 monthsBorn at 34 weeks, birthweight 3 lb 11 oz, NICU × 19 day; VSD, hypospadiasEpileptic spasms with focal features – head down and to L, eyes to L.Epileptic spasms; complex partial seizures, followed by spasms, controlled after ACTH and maintenance on valproate and ethosuximide. Now: controlledBitemporal independent spikes, L > R; bursts of generalized spike and polyspike-wave, sometimes followed by suppressionEpileptic encephalopathy; mild motor delay – walked at 17 months; severe language delay, mild autism, poor coordination, stereotypies; ADD, behavior problems (tantrums)MRI: L temporal atrophy, ?neuron migration defect; PET: L temporal hypometabolism
I (F)7.5 years3.5 yearsFull termComplex partial seizures, with R clonicSimple and complex partial: R face, R hemiclonic, hemisensory, some in clusters, postictal confusion; excellent response to prednisone, maintenance on valproate and zonisamide. Now: partly controlledFrequent, L centrotemporal spikes, almost continuous during sleep; intermittent R centrotemporal synchronous spikesNormal milestones and exam; ADHD and significant behavior problems after seizure onset.PET: bitemporal hypometabolism R > L; seizure worse on carbamazepine; (+) FH intractable epilepsy (maternal 1st cousin)
J (M)17 years8.5 yearsGestational diabetes (diet-treated); born at 37 weeks; severe FTTComplex partial seizures, giggling with decreased awareness, eye deviationComplex partial seizures. Now: partly controlled (brief and infrequent, every few weeks, on valproate and zonisamide)Diffuse slowing, multifocal spikes bifrontal and L occipital; clinical seizure originating from L frontal regionAcquired microcephaly (<2nd‰); motor regression, progressing to spastic quadriparesis; choreoathetosis, dystonic posturing; severe ID, nonverbal; short statureMRI progressed from normal to delayed myelination to periventricular leukomalacia

Seven of the 10 patients have an epileptic encephalopathy causing regression in developmental abilities (probands A, B, D, E, F, G, and H). Two showed severe developmental delay with cognitive impairment without a clear regression in skills or cognition (probands C and J). One child was showing regression in academic skills with increasing seizure frequency, but this stabilized and reversed with better seizure control (proband I). In all probands, electroencephalography revealed diffuse or multifocal bihemispheric abnormalities. Brain magnetic resonance imaging (MRI) studies were normal or showed mild nonspecific abnormalities. None of the probands had an etiologic diagnosis, and all were considered to be sporadic (i.e., had no first-degree relatives with similar neurologic problems). Blood was collected from each proband and their parents. At the time of sample collection, the probands ranged in age from 14 months to 18 years. Informed consent for participation of each trio was obtained from both parents, and the study was approved by the University of Arizona Institutional Review Board.

Whole exome sequencing, processing, and analysis

Whole exome sequencing was performed on all 10 families by array capture of 64 MB of exome target sequence using the Illumina TruSeq Enrichment methodology followed by paired-end sequencing on an Illumina HiSeq 2000 (San Diego, CA, U.S.A.). Sequences were aligned to the human genome (hg19) using Burrows-Wheeler Aligner (Li & Durbin, 2010). Base quality recalibration, indel realignment, and variant calling were performed using Genome Analysis Toolkit as previously described (McKenna et al., 2010). Variants were annotated using Annotate Variation (Wang et al., 2010). Gene annotations were made against the UCSC KnownGenes database. Previously known variants were annotated with their allele frequencies (using all ethnic affiliations) from the 1000 Genomes Project February 2012 release ( and the NHLBI GO Exome Sequencing Project (ESP) 5,400 samples release ( Nonsynonymous variants were annotated with Polyphen2 (Adzhubei et al., 2010), MutationTaster (Schwarz et al., 2010), and EvoD (Kumar et al., 2012) scores.

Candidate de novo variants that were predicted to alter protein function (i.e., nonsynonymous, stop-gain, stop-loss, frameshift, and splice-junction mutations) were identified in cases where only the proband, and neither parent, carried the nonreference allele. To winnow our list of candidates to a set of true de novo variants, we implemented a prioritizing system that hierarchically placed each candidate into one of seven “levels” according to sequence or genome architecture features that ranged from lowest (level 1) to highest (level 7) probability of being a false positive (Fig. 1). All candidate de novo variants that were novel (approximately 3 candidates per trio, range 0–5 per trio, total 29) or were found at a frequency of <0.001 in the ESP and <0.01 in the 1000 Genomes project (approximately 1 candidate per trio, range 0–2 per trio, total 9) (i.e., level 1 variants) were submitted for validation testing by Sanger sequencing and/or restriction fragment length polymorphism (RFLP) assays. We also submitted for validation a subset of candidate variants in lower levels (11 candidates total) that were chosen on the basis of variant properties and whether they were in genes previously implicated in severe epilepsies.


Figure 1. Automated hierarchical filtering system to identify real de novo mutations. All variants that fit a de novo inheritance pattern (variant allele present in the proband but neither parent) are first identified (genotypes aa/aa/ab or aa/aa/bb). When <30% of the reads in the proband contain the alternate allele, variants are placed into level 7 (Kong et al., 2012) because of a likely false heterozygous call. When >10% of the reads in either parent contain the alternate allele, variants are placed into level 6 (because of a likely false homozygous reference call). Remaining variants within 5 bp of an indel are assigned to level 5 due to possible misalignment. Remaining variants in proximity to at least one other Mendelian inheritance error (MIE; i.e., in the same gene), are assigned to level 4 because of either possible misalignment or poor region capture/sequencing. Remaining variants with a read depth (i.e., coverage) in the proband greater than two standard deviations from the mean are placed into level 3 as they likely lie in repetitive sequence. Remaining variants found in large population genetic variation screens (i.e., 1,000 Genomes and the 5,400 exomes of ESP) with a frequency above a certain threshold are assigned to level 2 because of likely nonpathogenicity or systematic sequencing artifacts. The remaining variants are assigned to level 1 and represent those putative de novo variants that are most likely to be real and are Sanger validated.

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We also screened for inherited variants with a frequency <1% in the ESP, both in cases of homozygous recessive or compound heterozygous state (i.e., when the same variant was present as two copies in the proband but only in one copy in both parents, and when two different variants were present in the same gene in the proband and were inherited from different parents, respectively). Candidate X-linked mutations in male probands present in the mother but not the father and never previously observed in the 1000 Genomes or ESP datasets were also examined.

RhoA activity in cells with mutant ARHGEF15 (Ephexin5) constructs

All plasmids were sequenced for correct complementary DNA. pCMV-SPORT6-ARHGEF15 was purchased (OpenBiosystems, Waltham, MA, U.S.A.). Ephexin5 mouse cDNA was described previously (Margolis et al., 2010). Human Ephexin5 R604C and mouse R612C were cloned using QuikChange Site Directed Mutagenesis (Stratagene, Clara, CA, U.S.A.). N-terminal Ephexin5 antibody was described previously (Margolis et al., 2010). LiCor Biosciences (Lincoln, NE, U.S.A.) Odyssey Infrared detection software was used to quantify Ephexin5 protein expression.

Human Embryonic Kidney 293 (HEK 293T) cells were transfected in a six-well dish for 36 h with 1 μg of indicated plasmids using the calcium phosphate method. Protein lysates (0.5 mg/ml) were subjected to the colorimetric-format G-LISA RhoA Activation Assay Kit (Cat# BK121; Cytoskeleton, Inc Denver, CO, U.S.A.) as per the manufacturer's instructions. All lysate samples were run in duplicate, and the GloMax Multi-plate Detection System (Promega Corporation, Madison, WI, U.S.A.) was used for luminometer readings. Final values for RhoA activation were calculated as a fold-change over the plasmid control transfection.

ClC-4 constructs and immunocytochemistry of transfected cells

Constructs for human ClC-4 in pCIneo and pTLN were described previously (Friedrich et al., 1999). The Gly544Arg mutation was introduced by polymerase chain reaction (PCR) and confirmed by sequencing the complete open reading frame (ORF). HeLa cells were transfected with pCIneo constructs using FuGENE6 (Roche, Basel, Switzerland) and analyzed after 2 days by confocal immunofluorescence as described (Stauber Jentsch, 2010). We used an affinity-purified polyclonal rabbit antibody raised against a C-terminal ClC-4 peptide (EEPPELPANSPHPLK, Poët et al., 2006).

Voltage-clamp analysis of Xenopus laevis oocytes

Xenopus laevis oocytes were injected with 25 ng complementary RNA transcribed from the pTLN constructs. After 2–3 days at 17°C they were examined by two-electrode voltage clamp (TEVC) using a protocol that started from a holding potential of −30 mV and clamped the oocytes to voltages between −80 and +80 mV in steps of 20 mV essentially as described (Friedrich et al., 1999).


  1. Top of page
  2. Summary
  3. Subjects and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure
  8. References

De novo variant discovery

The average exome sequence coverage per nucleotide per individual was 40X with a mean of 64% of the target exome sequence captured at a minimum of 20X. The number of candidate de novo variants per proband ranged from 53 to 149. After applying our hierarchical prioritizing system (Fig. 1), we validated 14 de novo variants (9 missense and 5 frameshift indels) across the 10 trios. Additional de novo candidate sequencing identified a nonsense variant in the gene CDKL5 with a 1:3 allele imbalance indicative of an early somatic mutation (we note that nine somatic mutations were observed in a previous whole exome sequencing study of 200 trios with autism; O'Roak et al., 2012). De novo variants were identified in 9 of the 10 trios, with an overall rate of 1.5 de novo variants per trio. Two trios possessed 2 de novo variants and two others possessed 3 de novo variants (Table 2). No de novo variants were identified in mitochondrial DNA (mtDNA).

Table 2. De novo mutations observed in 10 trios
TrioGeneMutation typecDNA changeProtein changeIn silico prediction
  1. Bold type represents genes with known or likely clinical significance.

  2. Fs, frameshift, damaging based on Polyphen2/Mutation Taster/EvoD; D, probably damaging (PP2), disease causing (MT), deleterious (EvoD); B, benign (PP2), polymorphism (MT), neutral (EvoD); np, no prediction.

A CDKL5 NonsenseC2494TQ832Xnp/D/np
B EEF1A2 Non-synonG208AG70SD/D/D
C CLCN4 Non-synonG1630AG544RD/D/D
D SCN1A KIAA1456 Non-synon Non-synonG3824C C277TG1275A L93FD/D/D D/D/D
E SCN1A OR10H2 MTMR11 Frameshift Non-synon Frameshift3580delA G392A 1528_1529insTI1194 fs R131H L511 fsnp/D/np B/B/D np/D/np
F KCNH5 Non-synonG980AR327HD/D/D
G ARHGEF15 HADHB Non-synon frameshiftC1810T 1141_1142insCTTTR604C A381 fsD/D/D np/D/np
I LPHN2 ZNF182 ZMYND8 Non-synon Non-synon FrameshiftG1400C A128G 1203delCR467T N43S P401 fsB/D/D B/B/B np/D/np
J THAP1 Frameshift6_7insAV2 fsnp/D/np

Genes previously associated with severe epilepsy

Two probands possess mutations in the voltage-gated sodium channel gene SCN1A, defects in which are known to cause Dravet syndrome (DS; Depienne et al., 2008), a rare and catastrophic form of intractable epilepsy that begins in infancy. Proband D carries a missense mutation in exon 21 that leads to a substitution of glycine with alanine at amino acid position 1,275 (p.Gly1275Ala). This amino acid position has previously been shown to be altered in a patient with DS, though with a G>T change rather than a G>C mutation, resulting in p.Gly1275Val substitution (Depienne et al., 2008). Proband E possesses a 1 bp deletion in exon 20 that is predicted to result in a substantially shorter amino acid sequence (i.e., 1,207 amino acids rather than 2,010) and thus nonsense-mediated decay. De novo nonsense or frameshift mutations are found in approximately 50% of DS patients with SCN1A mutations (Mulley et al., 2005).

A p.Gln832Stop premature stop-codon in exon 17 of cyclin-dependent kinase-like 5 (CDKL5) gene, which is known to cause an atypical form of Rett syndrome that results in early onset seizures as well as infantile spasms (Castrén et al., 2011), was found in proband A. Of particular interest is a previous report of a female with an atypical form of Rett syndrome with seizures beginning 10 days after birth who possessed a p.Gln834Stop premature stop-codon, just two amino acids downstream of proband A (Nectoux et al., 2006). Proband B possesses a nonsynonymous mutation, p.Gly70Ser, in EEF1A2, the gene encoding eukaryotic translation elongation factor 1, alpha-2. A recent WES survey of 100 individuals with intellectual disability identified the same de novo missense mutation in EEF1A2 in a child with onset of epilepsy at 4 months of age followed by severe psychomotor development delay and autistic features (trio 91 of de Ligt et al., 2012).

New epilepsy candidate genes with relevant neurobiologic functions

KCNH5, a gene not previously associated with any human disease, was found in proband F to contain a nonsynonymous mutation resulting in a p.Arg327His substitution in Kv10.2, the eaG-related voltage-gated potassium channel. The amino acid change occurs in the S4 transmembrane segment, disrupting the first of four conserved Arg residues that constitute essential features of the channel voltage sensor. voltage-gated potassium channels are essential regulators of neuronal excitability, and previous studies have shown that mutations in some members of this gene superfamily, such as KCNA1, KCNQ2, and KCNQ3 (Graves, 2006), can cause epilepsy. However, KCNH5 is the first member of the eaG-related subfamily H to be associated with this phenotype. Eag K+ channels are named for their loss-of-function convulsion-like phenotype in Drosophila (ether-a-gogo; Brüggemann et al., 1993). Their expression is selectively localized to layer IV of the cerebral cortex, in particular to the numerous excitatory interneurons (Saganich et al., 1999). Moreover, expression studies indicate a role for these channels in synaptic plasticity (Griffith et al., 1994).

Another ion transporter mutation was found in proband C, this time in the X chromosomal gene CLCN4 that encodes the voltage-dependent 2Cl/H+-exchanger ClC-4 (Scheel et al., 2005). The mutation is a nonsynonymous change causing a p.Gly544Arg substitution, and given that the proband is a male, any potential phenotypic effect is expected to manifest. CLCN4 expression is high in the nervous system where the exchanger may transport ions across intracellular membranes. The sequence change is within an intramembrane four-amino-acid-residue loop that connects intramembrane helices P and Q. Although this gene has not been previously implicated in any human disorder, mutations in several CLCN genes underlie human pathologies (Jentsch, 2008). These include Dent's disease that is caused by mutations in the endosomal 2Cl/H+-exchanger ClC-5 (Lloyd et al., 1996), a close homolog of ClC-4.

When transfected into HeLa cells, both wild-type (WT) and mutant ClC-4 localized to structures resembling the endoplasmic reticulum (ER; Fig. 2A). ER localization of ClC-4 has been observed previously upon heterologous overexpression (Okkenhaug et al., 2006), but native ClC-4 might rather be found on endosomes (Jentsch, 2008). When expressed in Xenopus oocytes, the Gly544Arg mutation almost abolished the outwardly rectifying ClC-4 currents that are mediated by electrogenic 2Cl/H+-exchange (Friedrich et al., 1999; Scheel et al., 2005; Fig. 2B), identifying it as a loss-of-function mutation.


Figure 2. Characterization of putative epilepsy-causing mutation in human CLC-4. (A) Overexpression of human ClC-4 wild-type and mutant protein (green) in HeLa cells show that the G544R mutation does not alter the subcellular localization of the protein. Nuclei are stained with DAPI in blue. (B) Current voltage-relationships of ClC-4 WT and ClC-4G544R expressed in Xenopus oocytes. The results were obtained with at least five oocytes from four batches of oocytes. Control measurements were done on uninjected oocytes. Error bars indicate standard error of the mean (some error bars are smaller than symbol size).

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Proband G possesses a de novo nonsynonymous mutation in the gene Rho Guanine Nucleotide Exchange Factor 15 (ARHGEF15) resulting in a p.Arg604Cys substitution in the protein product. ARHGEF15 is also known as Ephexin5. Although this gene has not previously been associated with a human disease, Ephexin5 is highly expressed in the brain (Sahin et al., 2005) and was recently shown to negatively regulate excitatory synapse formation during development (Margolis et al., 2010). In particular, Ephexin5 suppresses the function of EPH receptor B2 (EphB2), a mechanism believed to limit uncontrolled synapse formation. When EphB2 encounters its ligand Ephrin-B1/2, Ephexin5 is phosphorylated, ubiquinated, and finally degraded, thereby allowing synapse formation to occur. The process of degradation is mediated by ubiquitin protein ligase E3A (UBE3A). Of interest, 90% of patients diagnosed with Angelman syndrome, which is highly associated with seizures, lack expression of UBE3A (Dan, 2009). It has been suggested from Angelman mouse models that one mechanism of the disorder is due to a lack of degradation of Ephexin5 by UBE3A, which leads to a decrease in synaptic formation (Margolis et al., 2010).

The GEF exchange activity of Ephexin5 is required to restrict synapse formation. Because Ephexin5 was previously shown to promote the activation of the small GTPase RhoA, we hypothesized that a de novo mutation in Ephexin5 may disrupt its GEF exchange activity towards RhoA. To test this we cloned an Ephexin5 human cDNA containing the p.Arg604Cys substitution (R604C). We transfected HEK 293T cells with a control plasmid, a plasmid driving the expression of wild-type (WT) human Ephexin5, or a plasmid driving the expression of mutant R604C. We prepared extracts from transfected cells and subjected them to a colorimetric-based G-LISA RhoA activation assay. We find that RhoA activation by R604C is reduced by approximately 46% compared to WT (Fig. 3A). We also find that a homologous amino acid in mouse Ephexin5 at Arg612Cys (R612C) shows an approximately 47% reduction as compared with mouse WT protein (Fig. 3B). It is important to note that we did not detect differences in mutant protein expression compared to the WT protein (Fig. 3C,D), suggesting that the de novo mutation causes a deficit in GEF exchange activity, and not protein stability. We conclude that the R604C mutation may lead to decreased Ephexin5 GEF activity resulting in an increase in excitatory synapse number in the brain, which could be an underlying cause of the seizures observed in proband G.


Figure 3. De novo mutation at Arg604Cys disrupts Ephexin5's guanine nucleotide exchange activity. (A) Lysates from HEK 293T cells transfected with control plasmid, human WT-Ephexin5, or R604C were subjected to the G-LISA activation assay. Error bars indicate standard error of the mean (SEM) (n = 4) *p < 0.01, Student's t-test. (B) Mouse WT-Ephexin5, or R612C were assayed for RhoA activation similar to (A). Error bars indicate SEM (n = 3) *p < 0.05, Student's t-test. (C) Representative quantitative Western Blot using LiCor Odyssey IR software from lysates used in (A) and (B). Protein samples were run on an sodium dodecyl sulfate polyacrylamide gel electrophoresis gel and Ephexin5 protein was detected using a rabbit polyclonal antibody raised against the N-terminus. (D) Western blot quantification for all samples. Error bars indicate SEM (n = 4 per human conditions; n = 3 per mouse conditions), p > 0.05, Student's t-test.

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Other de novo variants of unclear functional significance

Proband G also possesses a frameshift de novo mutation in the gene HADHB, which is known to cause mitochondrial trifunctional protein (MTP) deficiency when found in simple homozygous or compound heterozygous form (Park et al., 2009). However, there were no other coding variants found in this trio in this gene.

Proband I possesses three nonsynonymous de novo mutations, the most interesting of which resulted in a p.Arg467Thr substitution in the protein Latrophilin-2 (LPHN2). Latrophilins were originally identified as G-protein–coupled receptors that bind α-Latrotoxin (black widow spider venom). Toxin binding at the synapse results in the mass release of neurotransmitters from synaptic vesicles (Silva Ushkaryov, 2010). Latrophilin-2 interacts with two SH3-and-multiple-ankyrin-repeat-domains proteins (SHANK1 and SHANK2; Kreienkamp, 2000), whereas Latrophilin-1 has recently been shown to bind neurexins (Boucard et al., 2012). Both SHANK and NRXN genes show strong associations with autism spectrum disorder and intellectual disability (Bourgeron, 2009). This proband also possesses a frameshift deletion at position 401 in the myeloid, Nervy, and DEAF-1 domain-type containing 8 Zinc Finger gene (ZMYND8). ZMYND8 is expressed at high levels in the brain and is involved in early development of the nervous system in Xenopus (Zeng et al., 2010). Although potentially interesting given their links to brain development, the functional mechanisms that might relate LPHN2 and ZMYND8 to epilepsy are not clear. It is noteworthy that this proband had later-onset seizures (3.5 years of age) that have been relatively well controlled by medication (Table 1).

Proband J was found to have a de novo frameshift mutation in the second codon of THAP domain-containing protein 1 (THAP1). THAP1 mutations, including frameshifts in the early part of the protein such as observed here, act in a dominant manner, causing torsion dystonia, DYT6 (Xiromerisiou et al., 2012). However, although proband J did demonstrate dystonic posturing, the THAP1 mutation has not been shown to be associated with epilepsy or any of the other clinical features exhibited by this proband (Table 1).

Four other de novo mutations in the genes ZNF182 (proband I), KIAA1456 (proband D), OR10H2 (proband E), and MTMR11 (proband E) were of unclear neurobiologic significance.

Simple recessive, compound heterozygous, and X-linked variants

We also scanned each trio's WES data for variants fitting a simple recessive or compound heterozygous pattern (with an allele frequency cutoff of 1% in ESP) as well as X-linked variants in male probands that were not previously observed in any public database. After removing likely alignment errors, we manually curated these lists using Genecards and OMIM, looking for genes with known roles in neurologic disorders, or known or hypothesized functions related to brain development or function and, where applicable, significant deleterious Polyphen2, MutationTaster, and EvoD (at least two of three) scores. Although none of the inherited variants met this strict criterion, proband H has two variants in the USP34 gene (p.R508S, p.H1671L) that are each predicted to be deleterious by two of the three in silico methods, although not in a concordant manner. This is of potential interest as this patient was the only proband in which we did not find a de novo variant. USP34 regulates Wnt signaling through Axins (Lui et al., 2011), and mouse mutations in its family members (UCHL1, Cartier et al., 2012 and USP14, Crimmins et al., 2006) cause nervous system phenotypes.


  1. Top of page
  2. Summary
  3. Subjects and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure
  8. References

Our results suggest that there is substantial value of applying WES in a trio framework to patients with epileptic encephalopathies of unknown etiology. Of the 15 true de novo variants, 4 represent mutations in genes (SCN1A, CDKL5, and EEF1A2) previously associated with severe epilepsies. Three of the true de novo variants represent mutations in genes (KCNH5, CLCN4, and ARHGEF15) not previously associated with epilepsies in humans but with a highly relevant biologic function. All seven highly functionally relevant de novo mutations caused substitutions at evolutionarily conserved amino acid positions and were predicted to be damaging by in silico methods (Table 2). Mutations in three genes may play a role in the proband's phenotype (Table 1) based on what is known about their biologic functions (LPHN2, ZMYND8, and THAP1).

Four candidate mutations are found in genes not currently screened in commercial Sanger-sequencing assays or in a proposed panel of 265 epilepsy-associated genes interrogated using NGS at high coverage (Lemke et al., 2012). Clearly, more confirmatory information is required for three of these genes (KCNH5, ARHGEF15, and CLCN4) before they can be accepted as truly pathogenic. Even the observation that the Gly544Arg mutation strongly impaired ClC-4 ion transport does not prove a role of CLCN4 in human epilepsy, especially because Clcn4−/− mice do not display seizures or any other obvious phenotype (Rickheit et al., 2010). Interestingly, recent work has found an enrichment of missense mutations in CLCN1 and CLCN2, but not of CLCN4 in individuals with complex idiopathic epilepsy syndromes (Chen et al., 2013). However it is important to note that the ClC-1 Cl- channel is predominantly expressed in skeletal muscle (Steinmeyer et al., 1991) and mutations in its gene cause myotonia (Koch et al., 1992; Jentsch, 2008), while previous work claiming a causative role of CLCN2 in epilepsy has been retracted (Haug et al., 2009). In addition to downstream functional studies, which are ongoing, large-scale sequencing efforts such as that being conducted by the Epi4K project (The Epi4K Consortium, 2012) should be useful for confirming the importance of these and related genes in epilepsy.

It is interesting to note that all seven of the probands in whose exomes we identified clear or promising candidate de novo mutations presented with seizures beginning within the first 6 months of life. On the other hand, we failed to identify plausible causal mutations in the three patients with seizures that presented later (i.e., at 23 month, 3.5 years, and 8.5 years). This suggests that patients with early onset epileptic encephalopathies may be more likely to carry de novo mutations that cause abnormal excitability and disrupt synaptic plasticity in the developing brain (Brooks-Kayal, 2010). Future studies of larger numbers of patients with a range seizure onset ages will help to determine whether the trend hinted at here is statistically significant, which could have important implications for diagnosis, prognosis, and treatment.

We have presented evidence suggesting that WES is likely to be of use for diagnosing sporadic epilepsies within a research setting. However, the application of this technology in the clinical setting still requires thorough investigation. Lyon and Wang (2012) detail many of the issues generic to most disorders. Two issues that are particularly relevant to the applicability of this approach are (1) the utility of NGS for diagnosis in the absence of one or both parents given the large number of genes involved in brain development and the growing evidence for the importance of de novo mutations; and (2) the challenge of communicating results to clinicians, patients, and parents when mutations in novel and potentially causal candidate genes are identified. These two issues will be aided in some respects by large gene discovery screens such as that being conducted by the Epi4K project (The Epi4K Consortium, 2012). Despite these challenges, the results presented here give cause for optimism that NGS screening will soon aid pediatric neurologists to diagnose children with epilepsies of unknown etiology.


  1. Top of page
  2. Summary
  3. Subjects and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure
  8. References

L.L.R. is funded by Autism Speaks and the Arizona Center for Biology of Complex Diseases. This work was partially supported by National Institute of Neurological Disorders and Stroke (NINDS) grant RO1 5R01NS045500 (M.E.G.) and by a Stuart H.Q. Victoria Quan Predoctoral Fellowship (J.S.). This work is dedicated to the memory of Shay Emma Hammer.


  1. Top of page
  2. Summary
  3. Subjects and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure
  8. References

None of the authors has any conflict of interest to disclose. We confirm that we have read the Journal's position on issues involved in ethical publication and affirm that this report is consistent with those guidelines. You will find the policy on ethical publication in Epilepsia instructions for authors at


  1. Top of page
  2. Summary
  3. Subjects and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure
  8. References
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