2013;80;1078-1085 Published Online before print February 13, 2013
Tim T. Chen, Tara L. Klassen, Alica M. Goldman, et al.
variants in epilepsy
Novel brain expression of ClC-1 chloride channels and
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2013;80;1078-1085 Published Online before print February 13, 2013
Tim T. Chen, Tara L. Klassen, Alica M. Goldman, et al.
variants in epilepsy
Novel brain expression of ClC-1 chloride channels and enrichment of
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Tim T. Chen, PhD
Tara L. Klassen, PhD
Alica M. Goldman, MD,
Carla Marini, MD, PhD
Renzo Guerrini, MD
Jeffrey L. Noebels, MD,
Editorial, page 1074
Supplemental data at
Novel brain expression of ClC-1 chloride
channels and enrichment of CLCN1
variants in epilepsy
Objective: To explore the potential contribution of genetic variation in voltage-gated chloride
channels to epilepsy, we analyzed CLCN family (CLCN1-7) gene variant profiles in individuals
with complex idiopathic epilepsy syndromes and determined the expression of these channels in
human and murine brain.
Methods: We used parallel exomic sequencing of 237 ion channel subunit genes to screen indi-
viduals with a clinical diagnosis of idiopathic epilepsy and evaluate the distribution of missense
variants in CLCN genes in cases and controls. We examined regional expression of CLCN1 in
human and mouse brain using reverse transcriptase PCR, in situ hybridization, and Western
Results: We found that in 152 individuals with sporadic epilepsy of unknown origin, 96.7% had at
least one missense variant in the CLCN genes compared with 28.2% of 139 controls. Nonsy-
nonymous single nucleotide polymorphisms in the “skeletal” chloride channel gene CLCN1 and in
CLCN2, a putative human epilepsy gene, were detected in threefold excess in cases relative to
controls. Among these, we report a novel de novo CLCN1 truncation mutation in a patient with
pharmacoresistant generalized seizures and a dystonic writer’s cramp without evidence of var-
iants in other channel genes linked to epilepsy. Molecular localization revealed the unexpectedly
widespread presence of CLCN1 mRNA transcripts and the ClC-1 subunit protein in human and
murine brain, previously believed absent in neurons.
Conclusions: Our findings support a possible comorbid contribution of the “skeletal” chloride
channel ClC-1 to the regulation of brain excitability and the need for further elucidation of the
roles of CLCN genes in neuronal network excitability disorders. Neurology?2013;80:1078–1085
BCM 5 Baylor College of Medicine; dbSNP 5 database of single nucleotide polymorphism; ESM 5 ethosuximide; GTC 5
generalized tonic-clonic; HRP 5 horseradish peroxidase; IE 5 idiopathic epilepsy; ISH 5 in situ hybridization; LTG 5 lamo-
trigine; nsSNP 5 nonsynonymous single nucleotide polymorphism; PB 5 phenobarbital.
CLCN1 was the first voltage-gated ion channel gene shown to cause muscle excitability disease,
and ClC-1 chloride channel mutations remain the most frequent cause of inherited dominant
(Thomsen) and recessive (Becker) nondystrophic myotonia.1–3This disease link, coupled with
robust expression in skeletal myocytes4and lack thereof in brain tissue,5resulted in ClC-1 being
coined “the skeletal muscle chloride channel.” Voltage-dependent chloride currents resembling
heterologously expressed ClC-1 and ClC-2 have been described in neurons,6,7but their molec-
ular composition and contribution to network excitability remain unclear. Although CLCN2
mutations have been reported in patients with epilepsy,8–10the evidence for a functional role has
been challenged,11and seizures were not detected in Clcn2-deficient mice.12Interestingly, Clcn3
colocalizes with the GABA vesicular transporter VGAT in hippocampal pyramidal neurons
where it regulates loading of GABAergic synaptic vesicles.13Deletion of Clcn3 in mice reduces
the quantal size of inhibitory neurotransmission, causes seizures, and produces a pattern of early
hippocampal cell loss similar to that of temporal lobe epilepsy.14,15
From the Departments of Neurology (T.T.C., T.L.K., A.M.G., J.L.N.), Molecular and Human Genetics (J.L.N.), and Neuroscience (J.L.N.),
Baylor College of Medicine, Houston, TX; Pediatric Neurology Unit and Laboratories (C.M., R.G.), Children’s Hospital A. Meyer-University of
Florence, Florence; and IRCCS Stella Maris Foundation (R.G.), Calambrone (Pisa), Italy.
Go to Neurology.org for full disclosures. Funding information and disclosures deemed relevant by the authors, if any, are provided at the end of the article.
1078 © 2013 American Academy of Neurology
We hypothesized that if CLCN1 is expressed
in human brain, then loss-of-function muta-
tions that reduce chloride conductance and
cause hyperexcitability in skeletal muscle could
contribute to enhanced network excitability and
increased susceptibility to seizures or other neu-
rologic phenotypes. Using a combination of
molecular genetic, biochemical, anatomical, and
CLCN1 is a likely candidate gene in a proband
with idiopathic epilepsy (IE) and mild myotonic
motor features, a novel neurologic phenotype
that supports a functional role of CLCN chloride
channels in epilepsy.
METHODS Standard protocol approvals, registrations,
and patient consents. The Baylor College of Medicine (BCM)
Institutional Review Board approved this study. Written informed
consent for this research was obtained from all study participants
from BCM and Meyer University Hospital of Florence.
Study population structure. As described previously,16we
evaluated self-reported white Caucasian and white Hispanic
adults with clinically confirmed generalized or localization-related
epilepsy of unknown origin and the absence of identifiable famil-
ial, laboratory, or imaging risk factors for seizures, along with
neurologically unaffected control individuals of equivalent ages
examined at BCM affiliated hospitals. See e-Methods on the
Neurology®Web site at www.neurology.org for inclusion criteria,
table e-1 for cohort details, and table e-2 for additional informa-
tion on diagnosis and clinical phenotype.
Channotype analysis of exomic sequencing data. Using our
Sanger exomic sequencing data,16we performed channotype anal-
ysis and compiled personal structural variant profiles for all indi-
viduals in the sporadic IE and neurologically unaffected control
cohorts. Genetic profiles were linked to a clinical phenotype data-
base for genotype–phenotype studies.
Pedigree analysis. Parental genomic DNAwas extracted from
blood (DNeasy Blood Kit, Qiagen, Hilden, Germany). PCR
amplification of the mutation-containing exon 23 of the CLCN1
gene (NM_000083) was performed using primers targeting intron
22/23 and 39 UTR yielding a 480-bp amplicon (table e-3). The 30
cycle PCR protocol had a 1-minute 60°C annealing step and
1-minute extension time. PCR products were size-resolved on a
2% agarose/Ethidium bromide gel, and gel extracted (Gel Extrac-
tion Kit, Qiagen).PCRproducts were sequenced in bothdirections
(Genewiz, South Plainfield, NJ).
Human and mouse brain tissue. De-identified human fro-
zen postmortem brain tissue samples were obtained from the
Department of Pathology (BCM) from previously healthy individ-
uals who died accidently. Mice were killed by cervical dislocation,
andwholebrain,skeletal muscle,andheartwerepromptly removed
under sterile, RNase-free conditions, dissected, and flash frozen.
Reverse transcriptase PCR to detect CLCN transcripts.
Tissues were homogenized with a Tissue Tearor. RNA was ex-
tracted using Trizol (Life Technologies, Philadelphia, PA) follow-
ing the manufacturer’s standard protocol. cDNA template was
created using Phusion reverse transcriptase PCR (Finnzyme, New
England Biosystems, Waltham, MA) according to the supplied
protocol. PCR amplification of transcripts was performed (Hot-
star, Qiagen) using primers spanning exon/exon boundaries
(table e-3) with 35 (mouse) or 40 (human) cycles of a 1-minute
58°C annealing step and 1-minute second extension time ampli-
fication, respectively. PCR products were size-resolved and bands
were excised and purified (Qiaquick Gel Extraction, Qiagen) for
sequence confirmation (Genewiz).
In situ hybridization of brain Clcn1 mRNA. An in situ
hybridization (ISH) probe template targeting mouse Clcn1 exon
23 was produced by 2 rounds of PCR amplification (table e-3).
Initial PCR templates were generated by PCR (Hotstar, Qiagen) of
30-second extension time). The single amplicon was excised and
purified (Qiagen Qiaquick Gel Extraction) for sequence confirma-
tion and used in a 100-mL large-scale PCR reaction to produce
.1 mg probe template. In vitro transcription reactions were per-
formed on purified templates (QiaQuick PCR Purification, Qia-
gen) using the 103 DIG RNA Labeling Mix (Roche, Branchburg,
NJ) to generate the antisense detection probe (SP6 promoter) or
control sense probe (T7 promoter) ISH was performed by the
IDDRC RNA In Situ Hybridization Core.17Fresh-frozen wild-
type mouse brains were sagittally sectioned at 25 mm, and incu-
bated with labeled riboprobe for 5.5 hours at 63.5°C followed by a
series of desalinizing washes. Colorimetric detection of probe
binding used horseradish peroxidase (HRP)–conjugated anti-
digoxigenin antibody followed by treatment with a neutravidin-
alkaline phosphatase and nitroblue tetrazolum resulting in a blue
precipitate. Slides were washed, postfixed with 4% paraformalde-
hyde, and coverslipped. Sections were scanned at 103 magnifica-
tion in a series of overlapping tiles per section, then compiled into a
Clcn1 transcripts based on relative expressionlevels,whilecells with
no detectable precipitate remain uncolored.
Western blot detection of ClC-1 protein. Tissues were
homogenized (Tissue Tearor) in ice-cold RIPA buffer containing
protease/phosphatase inhibitors (Santa Cruz Biotechnology, Santa
sample was separated on 8% Tris–HEPES sodium dodecyl sulfate
polyacrylamide gel electrophoresis gels, analyzed by Western blot
(1:200 in vehicle, Millipore, Billerica, MA) and HRP-tagged goat
antirabbit F(ab’)2 secondary antibody (1:500 dilution in vehicle,
Molecular Probes, Eugene, OR), and detected with a chemilumines-
cent substrate (SuperSignal, Pierce Chemical, Rockford, IL).
RESULTS CLCN gene variant enrichment in epilepsy.
We newly evaluated the personal ion channel variant
profiles in 152 individuals with sporadic IE and 139
neurologically asymptomatic controls.16Among the
enrichment in known and novel nonsynonymous single
nucleotide polymorphisms (nsSNPs) in the voltage-
gated chloride channel gene family (CLCN1-7). When
examined cumulatively, 96.7% of individuals with IE
had at least one missense variant in the CLCN genes
compared with 28.2% of controls. The total number of
CLCN missense variants observed in a personal profile
(“channotype”) was independent of the number of
nsSNPs identified in the individual regardless of disease
status (figure 1A). While not detected in CLCN3
(NM_001243372) or CLCN4 (NM_001830), we
identified missense variants in all other CLCN genes
in individuals with IE (figure 1B). Notably, 3 times as
Neurology 80March 19, 2013 1079
many cases had nsSNPs in CLCN1 (NM_000083)
and CLCN2 (NM_004366) vs controls. Of these
nsSNPs, only 3 in CLCN1 (rs10282312, rs41276054,
rs13438232) and2in CLCN2 (rs9820367,rs2228292)
were previously reported in the database of single nucle-
otide polymorphisms (dbSNP). Interestingly, with the
in a single control individual, the novel variants in these
known channelopathy genes were found only in those
with IE (9/9 CLCN1; 9/10 CLCN2). This trend was
also observed in CLCN5-7 (accessions NM_000084,
NM_001286, NM_001287, respectively), where 8/11
missense variants were novel to our study and 7/8 of
these were identified solely in the IE population. We
performed an in silico analysis of each singular nsSNP
using SIFT19and Polyphen220algorithms to assess the
projected functional impact of the amino acid substitu-
tion and found that 70.4% of patient genomes had one
or more predicted deleterious mutations in the CLCN
gene family as compared with 18.4% of controls. We
also observed a greater frequency of compound (.1)
missense variants within a single gene in the IE cohort
where 7.9% of individuals with IE had 2 or more
nsSNPs in the CLCN1 gene compared with ,1%
(1/139) in controls. This disparity in variant burden
and enrichment of predicted damaging mutations in
IE cases suggests that, if expressed within appropriate
brain networks, CLCN genes could be considered as
preeminent candidate genes for epilepsy.
Channotype analysis of a proband with a novel de novo
truncation in CLCN1.Weidentifiedasolitarynovelnon-
sense mutation in the CLCN1 channel gene in one
proband (1/291). This heterozygous nsSNP encodes a
novel premature stop codon (R976X), resulting in
truncation of the distal C-terminus of ClC-1 protein
by 12 residues (figure 2A). This mutation has not
been described in dbSNP or the 1000 Genomes
Project. Since CLCN1 mutations produce hyperex-
citable muscle phenotypes, we reasoned that, if de
novo in the proband, the novel truncating mutation
would identify CLCN1 as a priority candidate gene
for epilepsy in this individual. A PCR fragment con-
taining CLCN1 exon 23 was generated for both
maternal and paternal alleles (figure 2B). Sequence
analysis showed that the C to T transition was not
Figure 1Genetic variation in CLCN genes among individuals with sporadic idiopathic epilepsy compared with neurologically normal controls
(A) Parallel exomic sequencing revealsthat the total number ofindividuals with structural variants (nsSNPs) inCLCN genes is greaterin an idiopathic epilepsy
(IE) population compared with controls. Scattergrams of all individuals within each cohort show the total number of CLCN nsSNPs per individual plotted
against number of single nucleotide polymorphisms (SNPs) (top panel) or nsSNPs contained in their channotype (bottom panel). The number of nsSNPs in the
CLCN genes isindependent of total SNP and nsSNP load inan individualchannotype. The proband with the de novo truncation is identified as a black triangle.
(B) Histograms show mutation burden of CLCN1–7. Individuals with IE have 3 times as many nsSNPs in CLCN1 and CLCN2, including a small number of
individuals with 2 or more nonsynonymous variants within the same channel gene. No nsSNPs were identified in the CLCN3 or CLCN4 genes.
1080Neurology 80 March 19, 2013
present in either parent (figure 2C), confirming it as
a spontaneous de novo mutation and therefore likely
to contribute to the phenotype. The proband is also
heterozygous for a known CLCN1 single nucleotide
polymorphism (rs13438232, P727L)21found in
both patients and controls.16
We further examined the proband channotype and
failed to find other compelling candidate variants
among the 15 homozygous nsSNPs and 30 heterozy-
gous nsSNPs in other ion channel genes. The only
other novel variant identified in the proband was in
CLCNKA (A287V), a subunit expressed primarily in
kidney, which was found in both patient and control
populations within our study.16The remaining
nsSNPs in the proband’s channotype are considered
common polymorphisms and have all been previously
described in dbSNP. The only variant among known
ion channel screen of this individual was a single pol-
ymorphic nsSNP (rs891398) encoding a T125A mis-
sense variant in CHRNA2, a known gene for a
nocturnal frontal lobe epilepsy syndrome.22This vari-
ant isconsideredbenignby bothPolyphen2 and SIFT,
which is consistent with its polymorphic prevalence in
the human population, and is unlikely to be a cause of
the generalized absence epilepsy phenotype in this
Clinical history and comorbidities in the de novo R976X
proband. We explored the clinical history of the R976X
proband and noted the neurologic phenotype of general-
ized pharmacoresistant epilepsy with mixed seizure types
as well as mild myotonic features. The female proband is
the only child of healthy, unrelated Italian parents. Fol-
occurred during a febrile illness at 11 months of age. An
EEG showed generalized spike-wave discharges, and val-
proate treatment was started. A second seizure appeared
at 3 years, ethosuximide (ESM) was added, and the
patient remained seizure-free. During a medication taper
at age 14, generalized tonic-clonic seizures (GTC) ap-
peared and persisted despite various drug combinations,
including phenobarbital (PB) and lamotrigine (LTG).
ical absences, lasting about 10–12 seconds and accompa-
nied by 3-Hz generalized spike-wave complexes (figure
2D), occurring several times daily. Background EEG
activity remained normal. On occasion it was noticed
that an absence seizure would evolve into a GTC seizure.
Reintroduction of ESM and PB withdrawal were fol-
lowed by full control of spontaneous absence seizures
since age 21. The patient is right-handed. At age 9, a
clinically significant dysfacility with handwriting raised a
provisional diagnosis of myotonic writer’s cramp.
Figure 2 Detection of a heterozygous de novo nonsense mutation in CLCN1 in a proband with childhood-onset idiopathic generalized epilepsy
including cortical spike-wave absence seizures and a history of writer’s cramp
(A) Schematic diagram of a single a subunit of the ClC-1 channel protein showing the location of the novel C-terminal truncation mutation (R976X) identified
in a single proband inthe studycohort. (B)PCRamplification ofthe finalcoding exon (exon 23)of CLCN1 inthe trioyielded a 550 bp product (arrow) used as a
template in a Sanger sequencing reaction. (C) Sequence chromatograms for the trio compared to the NCBI reference gene (NM_000083) indicating the
heterozygous base pair substitution encoding a premature stop codon in the proband. Neither parent has the mutation, indicating that it is de novo, resulting
from a spontaneous C to T transition inthe proband.(D) A multi-lead EEG recording from the proband during a typical absence seizure 20 seconds in duration
exhibiting 3-Hz generalized spike-and-wave complexes.
Neurology 80 March 19, 20131081
EEGs obtained over the last several years continue to
clinical seizures. Current treatment includes LTG, ESM,
followedbyreappearance of absence seizures.AnMRIof
the brain was normal. Cognitive testing with the Wechs-
ler Adult Intelligence Scale and the Raven Progressive
Matrices at 24 years of age demonstrated normal verbal
scores and borderline performance scores. Handwriting
that was once considered dysgraphic appeared normal.
At age 26, her neurologic examination remains unre-
electrocorticography, blink reflex, motor and sensory
normal limits with no evidence of myotonic discharge.
CLCN1 mRNA and protein expression in the brain. We
hypothesized that if CLCN1 is indeed expressed in
brain, then loss-of-function mutations that reduce
chloride conductance could contribute to enhanced
network excitability and increased susceptibility to
seizures or other neurologic phenotypes. We used
reverse transcriptase PCR to detect mRNA in human
brain, and found that CLCN1 mRNA transcripts
were expressed in all sampled regions, including cer-
ebellum, hippocampus, spinal cord, occipital, parie-
tal, and frontal lobes, and heart (figure 3A). A scan
of human tissue revealed expression of the remain-
ing voltage-gated chloride channel family members
CLCN2–7 in the same regions (figure 3C). Inter-
estingly, the primers targeting the N-terminus of
CLCN2 detected a second 330-bp band in the spinal
cord that was most visible in human hippocampus
and neocortical regions (figure 3C) corresponding
to a known CLCN2 isoform lacking the 132 bp
encoded by exon 3.23Similarly, we found Clcn1 mRNA
in the brain and heart of developing and adult mice
(figure 3B). Cellular localization of Clcn1 expression
was performed by in situ hybridization in wild-type
mouse brain. Neuronal expression level was highest in
pyramidal and dentate granule cells of the hippocampal
formation, cerebellar Purkinje cell layer, and in scattered
brainstem nuclei. Medium levels of expression were evi-
dent in all laminae of frontal neocortex and thalamic
relay nuclei. Lower levels were also diffusely present in
other brain regions (figure 3D).
Using Western immunoblot analysis with a specific
band with apparent molecular weight of ;170 kDa in
membrane fractions of human brain and heart tissue
(figure 3E). Preincubation with the antigenic peptide
abolished detection, confirming specific epitope interac-
tion. We also detected a single immunoreactive band
of ;150 kDa in protein lysates of whole mouse brain
and, as a positive control, in murine skeletal muscle
(figure 3F). Peptide-preincubation abolished detection
in both tissues. The difference in apparent molecular
weight between murine and human ClC-1 subunits
likely reflects species differences in post-translational
modification, or presence of dissociation-resistant auxil-
DISCUSSION Directed by the results of a large-scale
exome screen for ion channel gene variants in epilepsy,
we show for the first time that the voltage-gated chloride
channel protein ClC-1 is expressed in human and
murine brain, and that de novo mutation of this
gene makes it a strong candidate for contribution to
human idiopathic generalized epilepsy with mild focal
myotonic/dystonic features in our proband. We postu-
mutations could contribute to hyperexcitability by com-
promising the chloride channel’s contribution to the
total membrane conductance and resting membrane
potential, a mechanism that is well-established in my-
ocytes.1Regional expression was identified in cortical,
hippocampal,and thalamic regionswhere loss of chlo-
ride currents could contribute to the mixed seizure
phenotypes observed in the patient. The presence of
Clcn1 in other subcortical structures, including the
basal ganglia, subthalamus, and cerebellar Purkinje cell
layer, raises the possibility of motor outflow deficits
potentially contributing to the proband’s mild myo-
tonic/dystonic motor features, and, intriguingly, to the
dystonia phenotype of other CLCN1-linked individu-
als with what has previously been regarded as a purely
muscular movement disorder.
The distal location of the proband’s CLCN1
C-terminal truncation is relevant to ClC-1 function
in the membrane, and nearby mutations in the 117
amino acids downstream of the second cystathionine
b-synthase (CBS2) domain have been identified in
myotonia patients with varying loss-of-function effects
on expression levels, macroscopic conductance, and
voltage dependence.24,25A known distal C-terminal
myotonia mutation at residue 946 in human ClC-1 re-
duces macroscopic conductance and subunit protein
truncations made further upstream result in variable
current densities as critical residues in CBS2 are
removed.26Likewise, different ClC-1 C-terminal var-
iants in human myotonia result in a wide spectrum of
clinical severity with variable penetrance. For example,
the distal C-terminal truncation variant R894X in ClC-
1 causes recessive myotonia congenita in some families
and dominant myotonia in others.25,27Possible explan-
ations for this spectrum include allelic variation within
the remainder of the gene, reduced penetrance of dom-
inant-negative mutations, incomplete dominance, and
founder effect.27–29Indeed, the dose dependency of
ClC-1 mutations on clinical phenotype has been
described, and accumulation of deleterious mutations
1082Neurology 80 March 19, 2013
Figure 3 The ClC-1 “skeletal” chloride channel is expressed in human and murine nervous system
(A) Reverse transcriptase PCR (RT-PCR) amplification using exon spanning primers detected the expected 330-bp product in all regions of the human brain
as well as heart. (B) Similar amplification of a 400-bp product revealed that Clcn1 is expressed in the brain, heart, and skeletal muscle of the newborn,
neonatal, and adult mouse. The positive and negative PCR controls targeted a 770-bp amplicon of mouse Tuba-1 (b-actin). (C) PCR amplification of cDNA
from regionally dissected human pathology samples. All other members of the CLCN chloride channel family (CLCN2–7) were detected in all regions of the
brain as well as in human heart. A known alternatively spliced isoform of CLCN2 lacking the 132 bp encoded by exon 3 was detected (arrow) in addition to
the full-length N-terminal isoform. (D) In situ hybridization detection of Clcn1transcripts inthe mouse brain show distinct patterns of expression across brain
regions with low levels of expression (blue) throughout. Expression level is highest (red cells) in the hippocampus where transcripts were detected in neurons
of the pyramidal layer and dentate granule cell layer. Notable expression was also observed in the cerebellar Purkinje cell layer. Moderate expression (yellow)
levels are observed in the frontal neocortex and thalamic relay nuclei. High-resolution image provided as figure e-1. (E) An immunoreactive band correspond-
ing to the expected molecular weight of the ClC-1 a subunit protein was detected in protein lysate from human pathologic samples of human brain and heart
using Western immunoblotting using a rabbit polyclonal ClC-1–specific antibody. Preincubation of the antibody with the peptide against which the antibody
was raised abolishes detection of the bands in both brain and heart protein lysates. (F) An immunoreactive band corresponding to ClC-1 channel subunit
protein was detected in 200 mg of murine brain lysate. The apparent molecular weight of this immunoreactive band in brain was the same as that detected in
40 mg of murine skeletal muscle lysate. Preincubation of the antibody with the peptide against which the antibody was raised abolishes detection of the
bands in both brain and skeletal muscle protein lysates.
Neurology 80 March 19, 20131083
or allelic variation that favors increased variant mRNA
level correlates with a more severe pathology.27,30–32
Heteromeric subunit complexity may also account
for the uncommon co-occurrence of clinical epilepsy in
human myotonia. There is widespread overlap of
CLCN1–3 regional expression in brain networks.
Although skeletal muscle ClC-1 channels are homo-
dimers, ClC-1/ClC-2 heterodimers with unique bio-
physical properties can be formed.33While the
existence of native ClC-1/ClC-2 heterodimers has yet
to be explored in neurons, these chimeric channels could
and cell type, contribute to differences in phenotype
severity. Indeed, coexpression of a mutant ClC-1 found
in dominant human myotonia congenita (P480L) sup-
presses ClC-2 current in a dominant-negative fashion.33
Our study illustrates how genetic screening for de
novo truncations in voltage-gated ion channels can
uncover novel candidates for pathogenic phenotypes
occult comorbid neurologic syndromes. In addition, the
possibility of a central origin of the dystonic features of
CLCN1-linked clinical phenotypes may be interesting
nel throughout central motor control networks. Indi-
viduals with IE display a wide range of clinical
comorbidities, many of which are secondary to func-
tionally deficient ion channels in tissues outside the
CNS.34–36Elucidating the molecular determinants of
such comorbidities may enhance their clinical detec-
tion and provide a more efficient therapeutic target in
the treatment of multiple disorders caused by a single
ion channel gene defect. These results are the first to
absence epilepsy and to localize CLCN1 message and
ClC-1 protein to relevant regions in the brain. Our
findings identify the need for further exploration of
the role of CLCN-family channels in neuronal excita-
bility disorders and confirm the heuristic value of per-
sonalized genomic profiling in epilepsy.
Tim T. Chen: manuscript preparation, study design, molecular/biochemical
experiments. Tara L. Klassen: manuscript preparation, study design,
molecular/biochemical experiments. Alica M. Goldman: patient recruitment
and biochemical experiments. Carla Marini: patient recruitment and clinical
phenotyping. Renzo Guerrini: patient recruitment and clinical phenotyp-
ing. Jeffrey L. Noebels: manuscript preparation, study design.
Supported by the National Institute of Neurological Disorders and Stroke
NS 049130 and NS 29709 (J.L.N.), an Epilepsy Foundation Postdoc-
toral Fellowship (T.L.K.), the BCM IDDRC (5P30HD024064) and
the Blue Bird Circle Foundation.
The authors report no disclosures relevant to the manuscript. Go to
Neurology.org for full disclosures.
Received June 15, 2012. Accepted in final form September 21, 2012.
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