Molecular correlates of age-dependent seizures in an inherited neonatal-infantile epilepsy.

Abstract

Many idiopathic epilepsy syndromes have a characteristic age dependence, the underlying molecular mechanisms of which are largely unknown. Here we propose a mechanism that can explain that epileptic spells in benign familial neonatal-infantile seizures occur almost exclusively during the first days to months of life. Benign familial neonatal-infantile seizures are caused by mutations in the gene SCN2A encoding the voltage-gated Na(+) channel Na(V)1.2. We identified two novel SCN2A mutations causing benign familial neonatal-infantile seizures and analysed the functional consequences of these mutations in a neonatal and an adult splice variant of the human Na(+) channel Na(V)1.2 expressed heterologously in tsA201 cells together with beta1 and beta2 subunits. We found significant gating changes leading to a gain-of-function, such as an increased persistent Na(+) current, accelerated recovery from fast inactivation or altered voltage-dependence of steady-state activation. Those were restricted to the neonatal splice variant for one mutation, but more pronounced for the adult form for the other, suggesting that a differential developmental splicing does not provide a general explanation for seizure remission. We therefore analysed the developmental expression of Na(V)1.2 and of another voltage-gated Na(+) channel, Na(V)1.6, using immunohistochemistry and real-time reverse transcription-polymerase chain reaction in mouse brain slices. We found that Na(V)1.2 channels are expressed early in development at axon initial segments of principal neurons in the hippocampus and cortex, but their expression is diminished and they are gradually replaced as the dominant channel type by Na(V)1.6 during maturation. This finding provides a plausible explanation for the transient expression of seizures that occur due to a gain-of-function of mutant Na(V)1.2 channels.

Full text

Page 1

BRAIN
A JOURNAL OF NEUROLOGY
Molecular correlates of age-dependent seizures
in an inherited neonatal-infantile epilepsy
Yunxiang Liao,1,2,?Liesbet Deprez,3,?Snezana Maljevic,1,2,?Julika Pitsch,4Lieve Claes,3
Dimitrina Hristova,5Albena Jordanova,3,6Sirpa Ala-Mello,7,8Astrid Bellan-Koch,1
Dragica Blazevic,1Simone Schubert,1Evan A. Thomas,9Steven Petrou,9,10Albert J. Becker,4
Peter De Jonghe3,11and Holger Lerche1,2
1 Neurological Clinic and Institute of Applied Physiology, University of Ulm, Ulm, 89081, Germany
2 Department of Neurology and Epileptology, Hertie-Institute for Clinical Brain Research, University Hospital Tu ¨bingen, Tu ¨bingen, 72076, Germany
3 Neurogenetics Group, VIB Department of Molecular Genetics, University of Antwerp, Antwerpen, 2610, Belgium
4 Department of Neuropathology, University of Bonn Medical Center, Bonn, 53105, Germany
5 Tokuda Hospital Sofia, Sofia, 1407, Bulgaria
6 Department of Chemistry and Biochemistry, Medical University-Sofia, Sofia, 1407, Bulgaria
7 Department of Clinical Genetics, Helsinki University Central Hospital, Helsinki, 00290, Finland
8 Rinnekoti Foundation, Rinnekodintie 10, Espoo, 02980, Finland
9 Florey Neurosciences Institute, Melbourne, Parkville 3010, Australia
10 The Centre for Neuroscience, The University of Melbourne, Melbourne, Victoria 3010, Australia
11 Division of Neurology, University Hospital Antwerp, Antwerpen, Belgium
*These authors contributed equally to this work.
Correspondence to: Professor Dr Holger Lerche,
Department of Neurology and Epileptology,
Hertie-Institute for Clinical Brain Research,
University Hospital Tu ¨bingen,
D-72076 Tu ¨bingen, Germany
E-mail: holger.lerche@uni-tuebingen.de
Many idiopathic epilepsy syndromes have a characteristic age dependence, the underlying molecular mechanisms of which are
largely unknown. Here we propose a mechanism that can explain that epileptic spells in benign familial neonatal-infantile
seizures occur almost exclusively during the first days to months of life. Benign familial neonatal-infantile seizures are caused
by mutations in the gene SCN2A encoding the voltage-gated Na+channel NaV1.2. We identified two novel SCN2A mutations
causing benign familial neonatal-infantile seizures and analysed the functional consequences of these mutations in a neonatal
and an adult splice variant of the human Na+channel NaV1.2 expressed heterologously in tsA201 cells together with beta1 and
beta2 subunits. We found significant gating changes leading to a gain-of-function, such as an increased persistent Na+current,
accelerated recovery from fast inactivation or altered voltage-dependence of steady-state activation. Those were restricted to the
neonatal splice variant for one mutation, but more pronounced for the adult form for the other, suggesting that a differential
developmental splicing does not provide a general explanation for seizure remission. We therefore analysed the developmental
expression of NaV1.2 and of another voltage-gated Na+channel, NaV1.6, using immunohistochemistry and real-time reverse
transcription–polymerase chain reaction in mouse brain slices. We found that NaV1.2 channels are expressed early in develop-
ment at axon initial segments of principal neurons in the hippocampus and cortex, but their expression is diminished and they
doi:10.1093/brain/awq057Brain 2010: 133; 1403–1414 |
1403
Received April 30, 2009. Revised February 8, 2010. Accepted February 15, 2010. Advance Access publication April 5, 2010
? The Author (2010). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved.
For Permissions, please email: journals.permissions@oxfordjournals.org
at University of Washington on June 11, 2010
http://brain.oxfordjournals.org
Downloaded from

Page 2

are gradually replaced as the dominant channel type by NaV1.6 during maturation. This finding provides a plausible explanation
for the transient expression of seizures that occur due to a gain-of-function of mutant NaV1.2 channels.
Keywords: epilepsy; seizure; sodium channel; development; axon
Abbreviations: EGFP =enhanced green fluorescent protein; IRES =internal ribosomal entry site; mRNA =messenger RNA
Introduction
Idiopathic epilepsies comprise a group of clinically well-defined
syndromes. They have a primary genetic background, usually no
structural brain abnormalities and most of them have a benign
course without additional neurological symptoms. Therefore,
they constitute interesting models to study the primary patho-
physiological mechanisms of epilepsy. A hallmark of many of
these syndromes is a syndrome-specific age dependence with a
developmental pattern of seizure onset and remission, the mech-
anisms of which are poorly understood. One group of seizure
syndromes with a clear age dependence occurring in neonates
and infants can be divided in three subforms: benign familial neo-
natal seizures, benign familial neonatal-infantile seizures (BFNIS)
and benign familial infantile seizures. They are characterized by
clusters of partial or secondarily generalized seizures with onset
either in the first days of life (benign familial neonatal seizures),
between 3 and 12 months of age (benign familial infantile seizure)
or more variably between the first days or months after birth
(BFNIS) (Berkovic et al., 2004; Specchio et al., 2006). For two
of these syndromes, genetic defects have already been identified.
Whereasbenign familialneonatal
loss-of-functionmutationsin
KCNQ3 encoding the voltage-gated K+channels KV7.2 and
KV7.3 (Maljevic et al., 2008), mutations in the gene SCN2A
encoding the voltage-gated Na+channel NaV1.2 have been de-
tected in BFNIS (Heron et al., 2002; Berkovic et al., 2004). NaV1.2
is one of four voltage-gated Na+channel alpha-subunits expressed
in the mammalian brain (Vacher et al., 2008), which are respon-
sible for the initiation and conduction of action potentials. The first
two studies investigating the functional consequences of a few
SCN2A mutations using the rat or human isoforms of the channel
predicted subtle gain-of-function mechanisms and an increase in
neuronal firing (Scalmani et al., 2006; Xu et al., 2007). Another
study suggested a loss-of-function by decreased surface expres-
sion for some mutations (Misra et al., 2008).
Since the mechanisms underlying the striking age dependence
of these syndromes are elusive, we set out to investigate both the
mechanisms of seizure generation and seizure remission in BFNIS.
New mutations were identified in BFNIS families and functionally
studied with the patch clamp technique using two different splice
variants of the human NaV1.2 channel expressed either early (neo-
natal) or late (adult) in development (Kasai et al., 2001). While a
previous study already suggested for one BFNIS mutation that
alternative splicing by itself does not explain a predominant seizure
generation in neonates and infants (Xu et al., 2007), a differential
developmental expression of distinct Na+channel subunits could
provide anintriguingexplanation
seizures
two
arecaused
KCNQ2
by
thegenesand
foranage-dependent
occurrence of seizures. We investigated this hypothesis for
BFNIS, studying the developmental expression of two voltage-
gated Na+channels, NaV1.2 and NaV1.6 using immunohistochem-
istry in mouse brain slices. The expression of these two channels
had been shown previously to be developmentally regulated in
retinalganglioncells(Boiko
et al., 2001).
etal.,2001,2003;Kaplan
Materials and methods
Patients and pedigrees
In this study, eight unrelated patients with a benign form of epilepsy
starting in the first year of life were recruited. The two families in
which mutations have been detected are described here in more
detail. All patients and relatives or their legal representatives gave
written informed consent to participate in this study. Ethical approval
was obtained from the responsible local authorities.
In Family 1, of Bulgarian origin, the index patient (Fig. 1A, III.1)
experienced three seizures in 1day around the age of 4 months.
The seizures were bilateral tonic–clonic and associated with sweating
and eye deviation to the left. Acute treatment with diazepam was
administered. Eleven days later, seizures recurred in a cluster of 11
episodes over a period of 24h with the duration of about 1min
each. Therapy with valproate was started, and the patient remained
seizure-free. At the age of 2 years, anti-epileptic treatment was dis-
continued without a relapse (the patient is now 8 years old). The
interictal EEG was normal.
The proband’s brother (Fig. 1A, III.2), now aged 6 years, experi-
enced three bilateral clonic seizures at Day 6 of life. At the age of 10
weeks, he had recurring clusters of ?15 seizures in 24h for ?2 weeks.
Seizures lasted ?1min and were associated with eye deviation to the
right, sweating and blushing. He was also treated with valproate.
Interictal EEG recordings were normal. Seizures disappeared after the
age of 3 months and pharmacotherapy was stopped at the age of
2 years without further seizures.
Both parents experienced no epileptic seizures. The information
about the childhood of the maternal grandfather (Fig. 1A, I.1) was
based on history. He was reported to have experienced multiple seiz-
ures ‘as a baby’, characterized by clonic jerking of both arms and legs.
Several relatives of the maternal grandfather also had seizures early in
life, but no DNA samples were available for genetic analysis. They all
were reported to have a normal mental status and no seizures later
in life.
In Family 2 from Finland, the index patient (Fig. 1A, II.1), now aged
7 years, experienced his first seizure 24h after birth, characterized by
unilateral clonic jerks. Later on, other seizure types occurred including
bilateral clonic seizures, apnoeas and atypical absence seizures.
Seizures were difficult to control but finally responded well to a com-
bination of phenobarbitone and phenytoin. Interictal EEG recordings
1404 |
Brain 2010: 133; 1403–1414Y. Liao et al.
at University of Washington on June 11, 2010
http://brain.oxfordjournals.org
Downloaded from

Page 3

showed multi-spike bursts and irritative activity without focal findings.
Brain MRI was unremarkable. Seizures disappeared at the age of
3 months. At the age of 10 months, the interictal EEG was normal
and anti-epileptic treatment was discontinued. The further develop-
ment of the child was normal. None of the parents or close relatives
of the patient had a history of seizures.
Genetic analysis
DNA was extracted from peripheral blood of the patients and their
family members. Patients had different ethnic backgrounds and for
each background, DNA samples of 200 randomly selected control in-
dividuals were available. The 27 exons of SCN2A were amplified using
the polymerase chain reaction with flanking intronic primers designed
with the software tool SNP box (Weckx et al., 2005) (primers available
upon request). The primers for exon 6 were designed in order to
amplify both alternative exons 6A and 6N (from the adult and
neonatal splice variant, respectively) (Kasai et al., 2001). The resulting
polymerase chain reaction fragments were sequenced with the BigDye
Terminator v3.1 Cycle Sequencing kit from Perkin-Elmer Applied
Biosystems (Foster City, CA). Sequences were analysed on an
ABI3730 automated sequencer using Sequencing Analysis 5.0 software
(Applied Biosystems, Foster City, CA). Pyrosequencing with the
PSQTM96 System (Pyrosequencing AB, Uppsala, Sweden) was used
to confirm the presence of the mutations in patients and their absence
from control individuals. Mutations were numbered according to the
published cDNA sequence (accession number NM_021007) with
nucleotide +1 corresponding to the A of the ATG translation initiation
codon and the nomenclature followed the MDI/HGVS Mutation
Nomenclature Recommendations (http://www.hgvs.org/mutnomen)
(den Dunnen and Antonarakis, 2001).
To test paternity, we genotyped 23 short tandem repeat markers
located on 11 different autosomal chromosomes. The markers were
amplified in two multiplex polymerase chain reactions.
Cloning and mutagenesis
The neonatal and adult splice variants of the human NaV1.2 channel
had been cloned before into the mammalian expression vector
pcDNA3.1 (Xu et al., 2007). Site-directed mutagenesis was performed
to engineer both mutations into both splice variants of human NaV1.2
using overlap polymerase chain reaction strategy (primers are available
upon request). All mutant cDNAs were fully resequenced before used
in experiments to confirm the introduced mutations and exclude any
additional sequence alterations. The human auxiliary subunits hb1and
hb2in the pCLH vector were kindly provided by GlaxoSmithKline. We
exchanged the hygromycin coding region in the vector with the
sequence coding for either enhanced green fluorescent protein
(EGFP)orCD8markergenes
pCLH-hb2-CD8, respectively. The presence of the internal ribosomal
toobtainpCLH-hb1-EGFPor
Figure 1 Pedigrees and genetic analysis. (A) Pedigrees of the two BFNIS families with a SCN2A mutation. Square = male; circle=female;
open symbols=unaffected individual; filled symbol=patient with BFNIS; +/M=individual carrying a heterozygous SCN2A mutation;
+/+= individual not carrying a SCN2A mutation. (B) Chromatograms of the SCN2A mutations in comparison with wild-type sequences.
(C) Predicted transmembrane topology of NaV1.2 showing the location of the mutations. Green diamond=published missense mutations
causing BFNIS or benign familial infantile seizure; blue star=published de novo mutations causing intractable epileptic encephalopathies
including infantile spasms and Dravet syndrome; yellow square=missense mutation associated with febrile and afebrile seizures; black
triangle=splice variant polymorphism, N209D; red circles=new missense mutations reported in this study.
Molecular correlates of age-dependent seizures Brain 2010: 133; 1403–1414 |
1405
at University of Washington on June 11, 2010
http://brain.oxfordjournals.org
Downloaded from

Page 4

entry site (IRES) site in the vector enabled parallel expression of the
auxiliary subunit and the marker protein in transfected cells, as has
been described previously by Lossin et al. (2002).
Transfection and expression in tsA201
cells
Human tsA201 cells were cultured at 37?C, with 5% CO2humidified
atmosphere andgrown in 50%
medium+50% F-12 HAM (Invitrogen, Carlsbad, CA)+10% (v/v)
foetal bovine serum. A standard calcium phosphate transfection
method was performed for transient expression of wild-type or
mutant Na+channel ?-subunits together with b1- and b2-subunits in
tsA201 cells. Approximately 6 mg of total DNA was transfected in a
molar ratio 1:1:1. Anti-CD8 antibody-coated microbeads (Dynabeads
M450, Dynal, Norway) suspended in phosphate buffered saline were
added to the cells and gently shaken. Only the cells positive for both
CD8 antigen and green fluorescent protein fluorescence were used for
electrophysiological measurements.
Dulbecco’s modified Eagle
Electrophysiology
Standard whole-cell recordings were performed using an Axopatch
200B amplifier, a Digidata 1320A digitizer and pCLAMP 8 data acqui-
sition software (Axon Instruments, Union City, CA, USA). Leakage and
capacitive currents were automatically subtracted using a pre-pulse
protocol (?P/4). Currents were filtered at 5kHz and digitized at
20kHz. All measurements were performed at room temperature of
21–23?C. Na+currents of 1.5–12 nA were recorded from the trans-
fected tsA201 cells, at least 10min after establishing the whole cell
configuration. Borosilicate glass pipettes were fire polished with a final
tip resistance of 1–1.3 MV when filled with internal recording solution
(see below). We carefully checked that the maximal voltage error due
to residual series resistance after up to 90% compensation was always
55mV. The pipette solution contained (in mM): 105 CsF, 35 NaCl,
10 EGTA, 10 (4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid
(HEPES) (pH 7.4). The bath solution contained (in mM) 150 NaCl,
2 KCl, 1.5 CaCl2, 1 MgCl2, 10 HEPES (pH 7.4).
Data recording and analysis
The following voltage clamp protocols were used for recordings. The
membrane was depolarized to various test potentials from a holding
potential of?140mV. A second-order exponential function was the
best fit to the time course of fast inactivation during the first 70ms
after onset of the depolarization, yielding two time constants.
The weight of the second slower time constant was relatively small
(55%). Only the fast time constant, named ?h, was therefore used for
data presentation in the ‘Results’ section. Persistent Na+currents
(ISS, for the ‘steady-state’ current) were determined at the end
of depolarizing pulses, lasting 70ms, to different test potentials
and are given relative to the initial peak current (IPEAK). Recovery
fromfastinactivation was recorded
of?140mV. Cells were depolarized to?20mV for 100ms to
inactivate all Na+channels and then repolarized to various recovery
potentials (?80,
first-order exponential function with an initial delay was the best fit
to the time course of recovery from inactivation. ?rec, is shown for data
evaluation.
fromholdingpotentials
?100 or?120mV) for increasing duration. A
The activation curve (conductance–voltage relationship) was derived
from the current–voltage relationship that was obtained by measuring
the peak current at various step depolarizations from the holding po-
tential of?140mV. The following Boltzmann function was fit to the
obtained data points:
g
gmaxVð Þ¼
1
f1 þ exp½ðV ? V1=2Þ=kV?g,
with g=I/(V?Vrev) being the conductance, I the recorded current
amplitude at test potential V, Vrev the Na+reversal potential, gmax
the maximal conductance, V1/2the voltage of half-maximal activation
and kVa slope factor. Steady-state inactivation was determined using
300ms conditioning pulses to various potentials followed by the test
pulse to?20mV at which the peak current reflected the percentage of
non-inactivated channels. A standard Boltzmann function was fit to
the inactivation curves:
I
ImaxVð Þ¼
1
f1 þ exp½ðV ? V1=2Þ=kV?g
with I being the recorded current amplitude at the conditioning po-
tential V, Imaxbeing the maximal current amplitude, V1/2the voltage
of half-maximal inactivation and kVa slope factor.
All data were analysed using a combination of pCLAMP, Microsoft
Excel and Origin (OriginLab Inc., Northampton, MA, USA) software.
For statistical evaluation, Student’s t-test was applied. All data are
shown as mean?SEM.
Preparation of brain slices and
immunohistochemistry
The use of animals and all experimental procedures were approved by
localauthorities(Regierungspraesidium
Germany). C57BL/6J mice at different postnatal days (P1, P3, P5,
P8, P10, P15, P20, P30, P40 and P90) were sacrificed by CO2inhal-
ation followed by decapitation. Brains were removed and frozen im-
mediately in liquid nitrogen vapour and stored at?80?C until
sectioning. Coronary sections (5 mm) were serially cut in a cryostat
and mounted on Superfrost microscope slides (Menzel GmbH,
Braunschweig, Germany). After drying at room temperature overnight,
brain slices were frozen and stored at?80?C. For immunohistochem-
istry, the cryostat sections were air-dried for 30min at room tempera-
ture and then fixed in pre-cooled acetone at?20?C for 10min.
Blocking was performed in 3% normal goat serum in tris buffered
saline containing 0.3% Triton-X. The polyclonal rabbit anti-AnkyrinG
antibody (Santa Cruz Biotechnology, Santa Cruz, CA) was diluted
1:1000 and combined with either anti-NaV1.2 (clone K69/3, 1:500
dilution) or anti-NaV1.6 (clone K87 A/10, 1:200 dilution), which
were both mouse monoclonal antibodies obtained from Neuromab
(Davis, CA). Sections were incubated with primary antibodies at 4?C
(NaV1.2) or room temperature (NaV1.6) overnight. After extensive
washing with Tris buffered saline, detection was performed using
fluorescently labelledsecondary
anti-mouse and Alexa 568 goat anti-rabbit (Invitrogen, Carlsbad,
CA) at room temperature for 1h. The slides were then stained with
40,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich, St Louis, MO) to
identify the nuclei. After the final washing step sections were air-dried
and covered with a mounting medium (Vecta Shield, Vector
Laboratories, Burlingame, CA). The stained sections were stored at
4?C prior to examination on an Axiovision2 plus Zeiss microscope
(Jena, Germany).
Tuebingen,Tuebingen,
antibodies–Alexa 488, goat
1406 |
Brain 2010: 133; 1403–1414 Y. Liao et al.
at University of Washington on June 11, 2010
http://brain.oxfordjournals.org
Downloaded from

Page 5

Freshly frozen hippocampal dissections were obtained from pharma-
coresistant epilepsy patients who underwent epilepsy surgery for seiz-
ure control at the Bonn University Medical Centre. All patients gave
written informed consent. Procedures were in accordance with the
Declaration of Helsinki and approved by the local ethics committee.
Cryosections, 12 mm thick, were fixed and stained using the same
protocol as for the mouse brain sections. The antibodies used were
(i) rabbit polyclonal anti-NaV1.2 (1:200; Alomone Labs, Jerusalem,
Israel) with compatible anti-Ankyrin G antibodies (mouse monoclonal
anti-AnkG, 1:1000, Calbiochem, Merck, Darmstadt, Germany) and (ii)
mouse monoclonal anti-Pan NaV
Germany) combined with rabbit polyclonal anti-AnkyrinG antibodies
(see above).
(1:1000, Sigma, Deisenhofen,
Real-time reverse transcription
polymerase chain reaction
CA1 and DG regions were microdissected from hippocampal slices
(400 mm) of C57BL/6 mice at two different time points of postnatal
development (P8 and P30). Messenger RNA (mRNA) was isolated
usingaDynabeadsmRNA Direct
Invitrogen, Carlsbad, CA, USA) and the following cDNA-synthesis
was performed using a High-Capacity cDNA Reverse Transcription
Kit (Applied Biosystems, California, USA) according to the manufac-
turer’s protocols. NaV1.2 and NaV1.6 transcript quantification was per-
formed by real-time reverse transcription–polymerase chain reaction
(PRISM 7700; PE Biosystems, California, USA). Primers for NaV1.2
and NaV1.6 subunits and for synaptophysin were designed with
Primer Express software (PE
(Supplementary Table 1). No significant homology of the amplicon
sequences with other previously characterized genes was found
searching GenBank databases by the BLASTN program. Relative
quantification of the starting mRNA copy numbers using multiple
replicates for each reaction was performed according to the ??Ct
method (Fink et al., 1998). The signal threshold was set within the
exponential phase of the reaction for determination of the threshold
cycle. Relative quantification started from 5–10ng of mRNA. Real-time
reverse transcription polymerase chain reaction was performed in a
6.25ml reaction volume containing 3.125ml of SYBR Green PCR
Master Mix (Invitrogen, Carlsbad, CA, USA), 0.1875ml of forward
and reverse primers (10 pmol/ml), 1.5ml of diethylpyrocarbonate–
H2O and cDNA dissolved in 1.25ml of diethylpyrocarbonate–H2O.
Reactions were performed in triplets. After preincubation for 10min
at 94?C, we performed 40 polymerase chain reaction cycles (20s at
94?C followed by 30s at 59?C and 40s at 72?C). The SYBR Green
fluorescence signal was measured in each cycle.
Statisticalsignificance wasanalysed
Outliers were rejected using the Grubb’s test. The values were con-
sidered significantly different at P50.05. Results are shown as
means?SEM.
Micro kit(DynalBiotech,
Biosystems,California,USA)
using Student’s
t-test.
Results
Genetics
We identified two heterozygous SCN2A mutations: c.754A4G
predicting M252V in Family 1 and c.781G4A predicting V261M
in Family 2 (Fig. 1A, B). The clinical phenotypes of the patients
carrying these mutations are described in detail in the ‘Materials
and methods’ section. The M252V mutation was present in the
two affected siblings, their affected maternal grandfather and in
their asymptomatic mother. The V261M was only detected in the
index case of Family 2. All affected individuals presented with a
phenotype compatible with BFNIS, i.e. with a neonatal or infantile
onsetof benignpartial-onset
with maturation. The unaffected parents of the index case of
Family 2 did not carry the mutation. Since non-paternity was
ruled out, this strongly suggests that the mutation arose de
novo. Both mutations were absent from 200 ethnically matched
control individuals. The affected amino acids are highly conserved
among the ?-subunits of mammalian brain sodium channels
(Supplementary Fig. 1). They are located very near to each
other in segment S5 of domain I (Fig. 1C).
seizuresremittingcompletely
Effects of the mutations on the gating
of the neonatal and adult splice variants
of NaV1.2 channels
There are two common splice variants that are conserved among
neuronal voltage-gated Na+channels, a neonatal and an adult
form. The difference is determined by an amino acid substitution
at position 209, asparagine by aspartic acid, located in the S3–S4
extracellular loop of domain I. The mutations were introduced into
the cDNAs of the SCN2A gene in each of the two splice variants.
Wild-type or mutant plasmids together with those encoding
human b1- and b2-subunits were co-transfected into tsA201 cells
to study their gating properties. The co-expression of both
b-subunits was controlled by co-expression of the enhanced
green fluorescent protein or CD8 using bicystronic constructs
(Lossin et al., 2002).
Typical whole cell Na+currents for both splice variants of
wild-type and mutant channels were elicited by various depolariz-
ing voltagestepsfromaholding
(Fig. 2A–C). The presence of an increased persistent current com-
pared to the wild-type was observed for mutant M252V channels
when engineered into the neonatal splice variant (Fig. 2D and E;
Table 1). This non-inactivating current was quantified at the end
of a longer test depolarization (70ms) relative to the peak current
(ISS/IPEAK). It could be reversibly and completely blocked by the
applicationof20nMtetrodotoxin,
voltage-gated Na+channels (Fig. 2D, inset). Such an increase in
persistent current has been described for many different Na+chan-
nel disorders going along with a hyperexcitability including myo-
tonia, cardiac arrhythmia and epilepsy (George, 2005; Lerche
et al., 2005; Cannon, 2006). It increases the Na+influx and can
readily explain a membrane depolarization and increased firing in
neurons (Golomb et al., 2006; Vervaeke et al., 2006). In contrast
to these observations in the neonatal splice form, the persistent
current was only slightly and not significantly increased for this
mutation in the adult splice variant (Fig. 2D and E; Table 1).
The predicted increase in neuronal firing could thus disappear or
be diminished as soon as the neonatal splice variant is no longer
expressed during development (Kasai et al., 2001). All other
potentialof
?140mV
a specificblockerof
Molecular correlates of age-dependent seizuresBrain 2010: 133; 1403–1414 |
1407
at University of Washington on June 11, 2010
http://brain.oxfordjournals.org
Downloaded from

Page 6

investigated gating parameters of activation, fast and slow inacti-
vation, as well as the current density, were not significantly dif-
ferent between mutant M252V and wild-type channels for both
the neonatal and the adult splice forms, except a slight but sig-
nificant acceleration of recovery from slow inactivation for the
neonatal splice variant of M252V (Table 1; Supplementary Fig. 2
and Table 2). This latter finding may contribute to an increase in
neuronal excitability by a shortening of the refractory period after
high frequency firing, when slow inactivation may occur in native
neurons.
Typical whole-cell recordings from cells expressing mutant
V261M channels are displayed in Fig. 3A and B. In contrast to
the M252V mutant channel, a significantly increased persistent
Na+current was not observed in the background of the neonatal
but was seen in the adult splice variant, when compared to the
wild-type (Fig. 3C and D; Table 1). The slope of the activation
curve was significantly decreased for both splice variants (Fig. 3E;
Table 1) while the voltage of half-maximal activation was not
significantly changed. This finding predicts an increase in channel
availability at subthreshold voltages. Furthermore, we found a
subtle slowing of the fast inactivation time course for the adult
splice variant of V261M and a marked acceleration of recovery
from fast inactivation for both splice variants (Fig. 3F–H; Table 1).
In particular the latter finding predicts an increase in neuronal
firing via a shortening of the refractory period after an action
potential. Another finding for the V261M mutation was an accel-
eration of recovery from slow inactivation in the background of
theneonatal splicevariant, similarly
(Supplementary Fig. 2 and Table 2). The current density was not
significantly changed for the mutation (Table 1).
asseenforM252V
Incontrast totheM252Vmutation, however, the
gain-of-function gating changes for V261M were seen in both
the neonatal and the adult splice variants, with some changes
only seen in the adult, so that a developmentally regulated mod-
ifying effect of the polymorphism in the D1/S3-S4 loop cannot
provide a general explanation for seizure remission later in devel-
opment for BFNIS.
Comparison of neonatal and adult splice
variants of wild-type channels
It has been reported previously that there is a difference in the
gating properties between the neonatal and the adult human
splicevariantsof NaV1.2WT
b-subunits (Xu et al., 2007). The adult splice variant was predicted
to enhance neuronal excitability compared to the neonatal one by
a depolarizing shift in the voltage dependence of steady-state fast
inactivation, a slower time course of and a faster recovery from
fast inactivation, while a depolarizing shift of the steady-state ac-
tivation curve (which would decrease excitability) did not reach
statistical significance (Xu et al., 2007). We observed the same
gating changes between both splice variants when the NaV1.2
?-subunit was expressed alone, although these differences did
notreachstatisticalsignificance
Supplementary Table 3). However, when we co-expressed both
the b1- and b2-subunits, these differences could not be repro-
duced. Hence, the relatively small changes at the border of reso-
lution between the different splice variants may be influenced by
the co-expression of auxiliary subunits.
channelsexpressed without
(Supplementary Fig.3;
Figure 2 Representative wild-type and M252V whole-cell Na+currents and their electrophysiological properties. (A–C) Whole-cell
recordings of wild-type (WT) and M252V mutant channels from transfected tsA201 cells: (A) wild-type, neonatal (neo) splice variant, (B)
M252V in the neonatal splice variant, (C) M252V in the adult (ad) splice variant. Na+currents were elicited by step depolarizations ranging
from ?105 to +67.5mV from a holding potential of ?140mV. (D) Representative wild-type and M252V persistent, tetrodotoxin-sensitive
Na+currents. Current amplitudes were recorded at the end of a 70ms depolarization to 0mV and are normalized to the peak amplitude
(ISS/IPEAK). Upon application of 20 nM tetrodotoxin (TTX), the persistent current was completely abolished, as shown in the inset. (E)
Voltage dependence of the persistent current (ISS/IPEAK). (F) Voltage dependence of Na+channel steady-state activation and fast in-
activation. (G) Voltage dependence of the fast inactivation time constant, ?h. (H) Time course of recovery from fast inactivation recorded
at?100mV. (I) Voltage dependence of the time constant of recovery from fast inactivation, ?rec. Voltage clamp protocols are described in
the ‘Materials and methods’ section. Numbers of recorded cells and statistical analysis are provided in Table 1. All data are shown as
mean?SEM.
1408 |
Brain 2010: 133; 1403–1414 Y. Liao et al.
at University of Washington on June 11, 2010
http://brain.oxfordjournals.org
Downloaded from

Page 7

Differential developmental expression
of NaV1.2 and NaV1.6 channels at axon
initial segments of principal neurons
Since we did not find a clear explanation for the transient expres-
sion of the clinical phenotype during the neonatal-infantile period
in our electrophysiological experiments, we studied the expression
of NaV1.2 and NaV1.6 channels at axon initial segments, at which
voltage-gated Na+channels are highly concentrated to generate
action potentials.
We used unfixed mouse brains in different stages of postnatal
development (P1–P90) to study the differential expression of both
channels at the axon initial segments of pyramidal neurons in the
hippocampus and cortex. The specificity of both monoclonal anti-
bodies was tested by heterologous expression of the respective
subunits in tsA201 cells. We did not observe any cross reaction
between both channel subtypes in this system (Supplementary
Fig. 4). Furthermore, the antigen for the NaV1.2 antibody was
located in the C-terminal region of the channels, thus far away
from the polymorphism in the D1/S3-S4 loop that differs among
the neonatal and adult splice variants, so that both splice variants
are predicted to be stained with the same channel-specific
antibodies.
Both antibodies heavily stained the axon initial segments, as was
verified by co-staining with antibodies directed against AnkyrinG
(Fig. 4). Anti-NaV1.2 antibodies stained the axon initial segments
of pyramidal neurons in the CA1 region of the hippocampus, with
the highest intensity between P5 and P15 during development.
This staining was diminished at later stages of development, al-
though still visible at adult ages. In contrast, staining of the axon
initial segments with anti-NaV1.6 antibodies was not detectable
before P15 and then gradually increased to reach its highest
levels from P30 onwards (Fig. 4). The same pattern of differential
developmental staining was observed for the axon initial segments
of principal neurons in the cortex (Supplementary Fig. 5A).
Recently, Hu et al. (2009) published a study reporting a distinct
localization and function of NaV1.2 and NaV1.6 in the axon initial
segments of pyramidal neurons in the adult rat cortex. In some of
the cortical neurons we could observe a similar proximal staining
of the axon initial segments using the NaV1.2 antibody in adult
mouse brain sections (Supplementary Fig. 5B), indicating that the
diminishment of NaV1.2 may result from a redistribution of this
channel from the whole axon initial segments to the proximal part
during maturation.
The staining pattern within the dentate gyrus and CA3 region
revealed an additional aspect. A similar developmental staining
compared to CA1 and cortex with both anti-NaVantibodies was
observed at the axon initial segments of granule cells within the
dentate gyrus and pyramidal cells in the CA3 region. However,
heavy staining with anti-NaV1.2 antibodies of mossy fibres pro-
jecting from the dentate gyrus to CA3 increased with age and did
not disappear in the adult stage. There was no detectable staining
withanti-NaV1.6antibodies
(Supplementary Figs 6 and 7). A similar observation has been
made with unmyelinated fibre bundles of the optic nerve (Boiko
et al., 2001, 2003; Kaplan et al., 2001).
ofthese unmyelinatedfibres
Table 1 Main electrophysiological parameters for wild-type and both mutant channels
Steady-state activation
Steady-state inactivation
?hat 0mV (ms)
n
ISS/IPEAKat 0mV (%)
n
?recat ?100mV (ms)
n
Current density (A/F)
n
V1/2(mV)
k
N
V1/2(mV)
k
n
WT neo
?9?1.3
?4.8 ? 0.3
15
?75.5?1.1
5.1?0.1
11
0.24?0.01
11
0.8?0.0
8
4.8?0.4
10
?810?170
16
M252V neo
?42.3?1.2
?5.2?0.2
15
?75.9?1.1
5.3?0.2
12
0.25?0.01
13
1.8?0.1***
12
6.2?0.6
13
?540?160
21
V261M neo
?41.0?0.9
?6.4?0.5**
11
?77.8?0.6
5.2?0.2
9
0.25?0.02
10
0.9?0.2
6
3.4?0.2**
11
?600?180
11
WT ad
?43.3?1.4
?5.6?0.4
12
?77.9?1.4
4.5?0.1
9
0.24?0.01
10
0.8?0.1
7
6.6?0.7
9
?800?210
17
M252V ad
?42.3?1.8
?5.5?0.4
11
?77.8?1.7
4.8?0.2
10
0.26?0.01
10
1.1?0.2
9
6.1?0.8
8
?390?100
12
V261M ad
?41.8?1.3
?6.7?0.2*
10
?79.5?1.1
5.8?0.2***
10
0.33?0.03**
8
1.4?0.3*
7
4.6?0.3*
10
?320?40
13
Electrophysiological parameters as determined in whole cell patch clamp recordings of transfected tsA201 cells for activation and fast inactivation of wild-type (WT), M252V and V261M mutants in neonatal (neo) or adult (ad)
splice variants of NaV1.2 channels. See ‘Experimental procedures’ section for the voltage clamp protocols used. V1/2=voltage of half-maximal activation or inactivation; k=slope factor; n=number of recorded cells; ?h=time
constant of fast inactivation; ISS/IPEAK=persistent (steady-state) Na+current divided by the peak current; ?rec=time constant of recovery from fast inactivation. Mean?SEM values are shown. Significant differences between
mutant and wild-type channels are indicated as follows: *P50.05, **P50.01, ***P50.001.
Molecular correlates of age-dependent seizuresBrain 2010: 133; 1403–1414 |
1409
at University of Washington on June 11, 2010
http://brain.oxfordjournals.org
Downloaded from

Page 8

The observed immunohistochemical stainings suggest a transi-
ently higher expression of NaV1.2 in early stages of development,
and a gradual replacement by NaV1.6 channels as the predomin-
ant channel at the axon initial segments of principal neurons in
the hippocampus and cortex with further maturation. This may
concern mainly the distal axon initial segments, as Hu et al.
(2009) described a proximal localization of NaV1.2 in adult
rat cortical neurons that we also observed in some of our
stainings. These findings can nicely explain how gain-of-function
mutations in NaV1.2 led to an age-dependent expression of
epileptic seizures, regardless if these mutations affect more the
gating properties of neonatal or adult splice variants of this ion
channel.
To compare our findings in mice with human brain slices, we
tested several available anti-NaV antibodies and could stain the
axon initial segments in sections from unfixed, frozen hippocampal
tissue obtained from epilepsy surgery samples of adult individuals,
using a different anti-NaV1.2 and an anti-Pan NaVantibody, the
latter recognizing all NaV?-subunits (Supplementary Fig. 8). With
the Pan NaVantibody, we observed a very strong signal in the
axon initial segments, whereas NaV1.2 staining appeared weaker.
However, these data have to be interpreted with care, since we
had to use two different antibodies against AnkG for co-staining
(anti-mouse and anti-rabbit). We could not acquire sections from
neonates or young infants, so we were not able to compare the
expression at different time points. These two NaVantibodies were
also tested in mouse sections and revealed very similar expression
patterns compared to both our previous stainings with other NaV
antibodies in mice, as well as to the stainings of human samples
(data not shown).
Differential developmental
expression of Nav1.2 and Nav1.6 on
the mRNA level in the mouse
hippocampus
We used microdissected regions of the mouse hippocampus (CA1
and dentate gyrus) at two different time points of postnatal
development (P8 and P30) to confirm our immunohistochemical
results and analyse the relative gene expression of NaV1.2 and
NaV1.6 with a different method on the mRNA level. Similar to
the immunohistochemical studies, the results indicated that the
amount of NaV1.2 mRNA decreases significantly from P8 to P30
(relative reduction by 3.3-fold) in the hippocampal CA1 region,
whereas the NaV1.6 mRNA level largely increased by 5.5-fold.
In the dentate gyrus, the NaV1.2 mRNA amount also decreased
with maturation, but the difference did not reach statistical signifi-
cance, and for NaV1.6 the increase was less pronounced (2.3-fold)
than that in the CA1 region (Fig. 5).
Discussion
Our combined genetic, electrophysiological, immunohistochemical
and reverse transcription polymerase chain reaction studies provide
a comprehensive analysis of the molecular mechanisms that may
underlie the pathogenesis and the age dependence of an auto-
somal dominant idiopathic epileptic syndrome, BFNIS. The discus-
sion is divided into two parts dealing with the mechanisms of (i)
seizure generation and (ii) seizure remission.
Figure 3 Representative V261M whole-cell Na+currents and their electrophysiological properties compared to wild-type channels.
(A and B) Whole-cell recordings of V261M mutant channels from transfected tsA201 cells: (A) V261M in the neonatal (neo) splice variant,
(B) V261M in the adult (ad) splice variant. Na+currents were elicited by membrane depolarizations ranging?105 to+67.5mV from a
holding potential of?140mV. (C) Representative wild-type and V261M mutant persistent Na+currents normalized to the peak current
(ISS/IPEAK). (D) Voltage dependence of the persistent current (ISS/IPEAK). (E) Voltage dependence of Na+channel steady-state activation
and fast inactivation. (F) Voltage dependence of the fast inactivation time constant, ?h. (G) Time course of recovery from fast inactivation
recorded at?100mV. (H) Voltage dependence of the time constant of recovery from fast inactivation, ?rec. Voltage clamp protocols are
described in the ‘Materials and methods’ section. Numbers of recorded cells and statistical analysis are provided in Table 1. All data are
shown as mean?SEM.
1410 |
Brain 2010: 133; 1403–1414 Y. Liao et al.
at University of Washington on June 11, 2010
http://brain.oxfordjournals.org
Downloaded from

Page 9

Figure 4 Developmental expression of NaV1.2 and NaV1.6 channels at axon initial segments in principal neurons of mouse brains.
Immunohistochemical stainings of mouse brain slices in the CA1 region of the hippocampus in different stages of development (postnatal
days P1 until P90). The Na+channel ? subunits NaV1.2 and NaV1.6 are stained with specific monoclonal antibodies (red fluorescence).
Axon initial segments are co-stained with specific antibodies against Ankyrin G (green fluorescence); nuclei are stained with DAPI (blue
fluorescence). The overlay shows all three stainings together. The anti-NaV1.2 and anti-NaV1.6 immunofluorescence signals suggest that
NaV1.2 channels are expressed at the axon initial segments of CA1 pyramidal neurons early in development and that they are partially
replaced with increasing maturation by NaV1.6. Scale bar 100mm.
Molecular correlates of age-dependent seizures Brain 2010: 133; 1403–1414 |
1411
at University of Washington on June 11, 2010
http://brain.oxfordjournals.org
Downloaded from

Page 10

Mechanisms of seizure generation
Our genetic and electrophysiological investigations strongly sug-
gest that these mutations are the cause of the epilepsy in the
affected individuals. Although Family 1 is relatively small, mutation
c.754A4G predictingtheamino
co-segregated with the disease phenotype including one asymp-
tomatic mutation carrier, whereas mutation c.781G4A predicting
p.V261M clearly occurred de novo in the only affected individual
of Family 2. The clinical variability of disease onset (neonatal in
two and infantile in two of altogether four affected patients) and
the finding of the first de novo mutation in this syndrome indicate
that it is worthwhile to search for mutations in ‘sporadic’ patients
or families when the clinical picture is typical for either benign
familial neonatal seizures or BFNIS. Both mutations were not de-
tected in a large number of normal controls and are strongly
conserved both in evolution and among other voltage-gated Na+
channels suggesting their pathogenicity already based on genetic
circumstantial evidence.
acid exchangep.M252V
The electrophysiological investigations then clearly indicated
subtle but significant changes in channel gating, with several
gain-of-function mechanisms that have been previously described
in a similar way in other Na+channel diseases of skeletal muscle,
cardiac muscle or brain, including some mutations in NaV1.2 that
had been described to be associated with BFNIS in several or with
generalized epilepsy with febrile seizures plus in one family
(Sugawara et al., 2001; George, 2005; Lerche et al., 2005;
Cannon, 2006; Scalmani et al., 2006; Xu et al., 2007). Such
changes predict an increase in membrane excitability of the re-
spective cells. In our study, an increase of neuronal excitability by
the two mutations can be explained by (i) a membrane depolar-
ization due to an increase of the persistent Na+inward current
relative to the peak current or a slowing of fast inactivation; (ii) a
shortening of the refractory period after an action potential by an
acceleration of recovery from fast (and maybe slow) inactivation;
and (iii) by an increase of the availability of Na+channels at sub-
threshold voltages induced by a decreased slope of the activation
curve. We tested this hypothesis in a simple one-compartment
model based on the Hodgkin–Huxley theory. For both mutations,
an increase in neuronal firing was predicted by the model. For
M252V channels, which mainly increased the persistent current,
we obtained a longer duration of burst firing, whereas mainly the
altered slope of the activation curve led to a decrease of the action
potential threshold and increased the firing rate for V261M com-
pared to wild-type channels (Supplementary Fig. 9). Altogether,
these results can explain the occurrence of epileptic seizures in the
mutation carriers.
From a biophysical point of view, our results demonstrate the
importance of the S5 segment of domain D1 for fast inactivation
of the NaV1.2 channel, which had not been described so far for
any of the known voltage-gated Na+channels. The finding is of
interest, since domains D1 and D2 have been thought to be
mainly responsible for the activation process of the channel, at
least in the skeletal muscle Na+channel (Cha et al., 1999).
Another disease-causing mutation in the skeletal muscle Na+chan-
nel with similar functional consequences has been described pre-
viously in the S5 segment of domain D4 (Bendahhou et al., 1999).
Mechanisms of seizure remission
The mechanism of age dependence in BFNIS and other idiopathic
epilepsies with a defined onset, and of remission of seizures during
different phases of development had been unclear up to now. We
used two different approaches to investigate this open question
for BFNIS. When we first studied both mutations in the back-
ground of the known neonatal and adult splice variants of
human NaV1.2 channels, we did not obtain a general answer
applying to all mutations. The M252V mutant only revealed
gating changes (increased persistent Na+current) in the back-
ground of the neonatal splice variant, which could indeed be an
explanation for the remission of seizures at later stages of devel-
opment when the neonatal splice variant is no longer expressed.
However, this did not apply to the V261M mutant, for which
most of the observed gain-of-function changes (decreased slope
of the activation curve and acceleration of recovery from fast in-
activation) were observed in the background of both splice
Figure 5 Messenger RNA expression levels of NaV1.2 and
NaV1.6 at two developmental time points. A quantitative
analysis of the mRNA of NaV1.2 and NaV1.6 channels
(corresponding to the genes SCN2A and SCN8A) in the mouse
hippocampus at two different developmental stages was
performed using a real-time reverse transcription–polymerase
chain reaction approach. (A) In the CA1 region, a significant
3.3-fold reduction of the NaV1.2 (P50.05) mRNA expression
level was observed between P8 and P30 (values normalized to
P8 were: 1.0?0.2 at P8 and 0.3?0.1 at P30), whereas the
NaV1.6 mRNA expression level significantly increased (P50.05)
between the two time points (from 1.0?0.1 at P8 to 5.5?2.0
at P30). (B) In the dentate gyrus (DG), the mRNA of NaV1.2 was
also reduced from 1?0.3 at P8 to 0.3?0.1 at P30 (although
not significantly), and the mRNA of NaV1.6 showed a significant
upregulation (2.3-fold, P50.001, values normalized to
P8: 1.0?0.2 at P8 and 2.3?0.2 at P30).
1412 |
Brain 2010: 133; 1403–1414 Y. Liao et al.
at University of Washington on June 11, 2010
http://brain.oxfordjournals.org
Downloaded from

Page 11

variants, and two of the changes even occurred only for the adult
channel (increased persistent current and slowing of fast inactiva-
tion). The relative proximity of both mutations in D1/S5 and the
structural link via the D1/S4 voltage sensor to the extracellular
D1/S3–S4 loop, containing the splice mutation N209D, may ex-
plain the distinct effects observed in a differential splice back-
groundona molecularand
mutation has also been investigated in the background of both
splice variants (L1563V), and the results did not reveal an explan-
ation for the remission of seizures (Xu et al., 2007). Therefore, it is
likely that another more general mechanism is responsible for this
characteristic clinical observation.
In our second approach, we studied the developmental expres-
sion pattern of NaV1.2 channels in comparison to another
voltage-gated Na+channel, NaV1.6. Our results suggest that
both channels, NaV1.2 and NaV1.6, are highly expressed at the
axon initial segments of pyramidal cells in the hippocampus and
cortex but that their developmental expression patterns differ
markedly. Whereas NaV1.2 channels are expressed early in devel-
opment with a decrease of the staining intensity and a redistribu-
tion towards the proximal part of the axon initial segments at least
in some neurons during maturation, NaV1.6 channel expression
increases with development more or less replacing NaV1.2 chan-
nels, in particular at the distal axon initial segments. A similar
effect has been described previously in retinal ganglion cells
(Boiko et al., 2001, 2003; Kaplan et al., 2001), but here we
could show the differential developmental expression of specific
voltage-gated Na+channels at the axon initial segments of cortical
and hippocampal excitatory neurons on both protein and mRNA
level, and thereby provide a plausible and general explanation for
the transient expression of seizures caused by gain-of-function
NaV1.2 mutations in BFNIS. Our results thus support an intriguing
hypothesis for the age dependence of an idiopathic epilepsy
syndrome, which directly involves the mutated channels, here an
age-dependent expression matching with the period in which the
seizures occur. Of course, we need to assume that the expression
pattern of these two investigated Na+channels is transferable
from mouse to man. Examination of human brain slices presents
a challenge not only due to the unavailability of high quality sec-
tions from different developmental stages but also due to a limited
number of antibodies that can specifically stain NaVchannels in
human tissue. Hence, although the results we obtained with stain-
ings of human adult brain tissue may support our findings in mice,
they have to be interpreted with care. Furthermore, the available
data about a comparison between developmental stages of mouse
and man suggest that the observed pattern in the mouse would
match the neonatal and infantile period of man (Bayer et al.,
1993; Clancy et al., 2001).
An additional observation that also matches the results obtained
previously in retinal ganglion cells (Boiko et al., 2001, 2003;
Kaplan et al., 2001) was a developmental increase and persistence
of NaV1.2 channel expression in adult ages in unmyelinated mossy
fibres in which we found no evidence for an expression of NaV1.6.
We therefore hypothesize that hyperexcitability of mossy fibres
caused by gain-of-function NaV1.2 mutations does not induce epi-
leptic seizures in patients with BFNIS, but that the transient
expression in the axon initial segments of principal neurons is
biophysicallevel. Oneother
responsible for the clinical phenotype. In agreement with this hy-
pothesis, it has been described that mossy fibres mainly target
inhibitory neurons (Acsady et al., 1998), so that a gain-of-function
of Na+channels expressed along mossy fibre axons could predom-
inantly promote inhibition instead of triggering excitation. This
may also explain why a SCN2A mutation with a complete loss
of function, for which even a dominant-negative effect on the
wild-type channel has been described, is associated with a
severe epilepsy of infancy with a later onset, but not with neonatal
or infantile seizures (Kamiya et al., 2004). It is noteworthy, how-
ever, that three recently reported de novo missense mutations in
SCN2A, two of which have been shown to exert strong effects on
channel gating with either gain or loss of function, can cause in-
tractable epileptic encephalopathies (Ogiwara et al., 2009; Shi
et al., 2009), so that the pathophysiology of severe SCN2A-asso-
ciated syndromes still has to be elucidated. Knock-out mice of
SCN2A did not unravel the possible differential function of this
channel so far, since homozygous animals die early after birth
and heterozygous ones were reported to develop normally with-
out obvious epileptic seizures (Planells-Cases et al., 2000). Finally,
an increasing expression of NaV1.2 in unmyelinated axons (not
only mossy fibres) might explain why expression of this channel
has been found to increase steadily from neonatal to adult in
whole brain preparations (Gong et al., 1999), despite the observed
decrease of NaV1.2 expression in principal neurons as observed
here on both protein and local mRNA levels.
Conclusions
Our studies and previous investigations (Scalmani et al., 2006; Xu
et al., 2007) indicate that SCN2A (NaV1.2) mutations cause BFNIS
by a gain-of-function mechanism increasing the membrane excit-
ability at the axon initial segments, the site of action potential
generation, in principal neurons of the hippocampus and cortex.
This would fit with the clinical observation of partial onset seizures
with a variable seizure semiology. Only one study reported a
loss-of-function by a significantly decreased surface expression
of mutated NaV1.2 channels in tsA201 cells (Misra et al., 2008),
which was not observed in other studies. Our immunohistochem-
ical and real-time reverse transcription–polymerase chain reaction
investigations suggest a transiently higher expression of NaV1.2
channels at the axon initial segments of principal neurons during
developmentandareplacement
voltage-gated Na+channel, NaV1.6, upon maturation, in particular
in the distal part of the axon initial segments. This observation
provides a plausible explanation for the age dependence of
BFNIS. A differential regulation of the developmental expression
of ion channels, modifying or interacting proteins that alter neur-
onal excitability, might also apply to explain other age-dependent
epilepsy syndromes.
ofNaV1.2 by another
Acknowledgements
We thank all patients and relatives for participating in the study,
DrAnna-Elina LehesjokiandDrAnna-Kaisa Anttonen for
Molecular correlates of age-dependent seizuresBrain 2010: 133; 1403–1414 |
1413
at University of Washington on June 11, 2010
http://brain.oxfordjournals.org
Downloaded from

Page 12

screening Finnish control individuals, GlaxoSmithKline for provid-
ing the clones of hb1 and hb2 subunits, and Dr J. Trimmer for
providing antibodies before they became available commercially.
We acknowledge the contribution of the VIB Genetic Service
Facility to the genetic analyses.
Funding
German Research Foundation (DFG Le1030/10-1, /8-2; SFB TR3,
project C6), the National Genome Network of the Federal Ministry
for Education and Research (BMBF: NGFN2/01GS0478 and
NGFNplus/01GS08123, 01GS08122),
(Epicure: LSH 037315), the University of Ulm, the Fund for
Scientific Research Flanders (FWO-F), Methusalem excellence
grant of the Flemish Government and University of Antwerp,
and the National Health and Medical Research Council of
Australia program grant (NHMRC #400121).
theEuropeanUnion
Supplementary material
Supplementary material is available at Brain online.
References
Acsady L, Kamondi A, Sik A, Freund T, Buzsaki G. GABAergic cells are
the major postsynaptic targets of mossy fibers in the rat hippocampus.
J Neurosci 1998; 18: 3386–403.
Bayer SA, Altman J, Russo RJ, Zhang X. Timetables of neurogenesis in
the human brain based on experimentally determined patterns in the
rat. Neurotoxicology 1993; 14: 83–144.
Bendahhou S, Cummins TR, Tawil R, Waxman SG, Ptacek LJ. Activation
and inactivation of the voltage-gated sodium channel: role of segment
S5 revealed by a novel hyperkalaemic periodic paralysis mutation.
J Neurosci 1999; 19: 4762–71.
Berkovic SF, Heron SE, Giordano L, Marini C, Guerrini R, Kaplan RE,
et al. Benign familial neonatal-infantile seizures: characterization of a
new sodium channelopathy. Ann Neurol 2004; 55: 550–7.
Boiko T, Rasband MN, Levinson SR, Caldwell JH, Mandel G, Trimmer JS,
et al. Compact myelin dictates the differential targeting of two sodium
channel isoforms in the same axon. Neuron 2001; 30: 91–104.
Boiko T, Van Wart A, Caldwell JH, Levinson SR, Trimmer JS,
Matthews G. Functional specialization of the axon initial segment by
isoform-specific sodium channel targeting. J Neurosci 2003; 23:
2306–13.
Cannon SC. Pathomechanisms in channelopathies of skeletal muscle and
brain. Annu Rev Neurosci 2006; 29: 387–415.
Cha A, Ruben PC, George AL Jr, Fujimoto E, Bezanilla F. Voltage sensors
in domains III and IV, but not I and II, are immobilized by Na+channel
fast inactivation. Neuron 1999; 22: 73–87.
Clancy B, Darlington RB, Finlay BL. Translating developmental time
across mammalian species. Neuroscience 2001; 105: 7–17.
den Dunnen JT, Antonarakis SE. Nomenclature for the description of
human sequence variations. Hum Genet 2001; 109: 121–4.
Fink L, Seeger W, Ermert L, Hanze J, Stahl U, Grimminger F, et al. Real-
time quantitative RT–PCR after laser-assisted cell picking. Nat Med
1998; 4: 1329–33.
George AL Jr. Inherited disorders of voltage-gated sodium channels.
J Clin Invest 2005; 115: 1990–9.
Golomb D, Yue C, Yaari Y. Contribution of persistent Na+current and
M-type K+current to somatic bursting in CA1 pyramidal cells:
combined experimental and modeling study. J Neurophysiol 2006;
96: 1912–26.
Gong B, Rhodes KJ, Bekele-Arcuri Z, Trimmer JS. Type I and type II Na(+)
channel alpha-subunit polypeptides exhibit distinct spatial and tem-
poral patterning, and association with auxiliary subunits in rat brain.
J Comp Neurol 1999; 412: 342–52.
Heron SE, Crossland KM, Andermann E, Phillips HA, Hall AJ, Bleasel A,
et al. Sodium-channel defects in benign familial neonatal-infantile sei-
zures. Lancet 2002; 360: 851–2.
Hu W, Tian C, Li T, Yang M, Hou H, Shu Y. Distinct contributions of
Na(v)1.6 and Na(v)1.2 in action potential initiation and backpropaga-
tion. Nat Neurosci 2009; 12: 996–1002.
Kamiya K, Kaneda M, Sugawara T, Mazaki E, Okamura N, Montal M,
et al. A nonsense mutation of the sodium channel gene SCN2A in a
patient with intractable epilepsy and mental decline. J Neurosci 2004;
24: 2690–8.
Kaplan MR, Cho MH, Ullian EM, Isom LL, Levinson SR, Barres BA.
Differential control of clustering of the sodium channels Na(v)1.2
and Na(v)1.6 at developing CNS nodes of Ranvier. Neuron 2001;
30: 105–19.
Kasai N, Fukushima K, Ueki Y, Prasad S, Nosakowski J, Sugata K, et al.
Genomic structures of SCN2A and SCN3A—candidate genes for deaf-
ness at the DFNA16 locus. Gene 2001; 264: 113–22.
LercheH, WeberYG,Jurkat-Rott
channel defects in idiopathic epilepsies. Curr Pharm Des 2005; 11:
2737–52.
Lossin C, Wang DW, Rhodes TH, Vanoye CG, George AL Jr. Molecular
basis of an inherited epilepsy. Neuron 2002; 34: 877–84.
Maljevic S, Wuttke TV, Lerche H. Nervous system KV7 disorders:
breakdownofa subthreshold
1791–801.
Misra SN, Kahlig KM, George AL Jr. Impaired NaV1.2 function and
reduced cell surface expression in benign familial neonatal-infantile
seizures. Epilepsia 2008; 49: 1535–45.
Ogiwara I, Ito K, Sawaishi Y, Osaka H, Mazaki E, Inoue I, et al. De novo
mutations of voltage-gated sodium channel alphaII gene SCN2A in
intractable epilepsies. Neurology 2009; 73: 1046–53.
Planells-Cases R, Caprini M, Zhang J, Rockenstein EM, Rivera RR,
Murre C, et al. Neuronal death and perinatal lethality in voltage-
gated sodium channel alpha(II)-deficient mice. Biophys J 2000; 78:
2878–91.
Scalmani P, Rusconi R, Armatura E, Zara F, Avanzini G, Franceschetti S,
et al. Effects in neocortical neurons of mutations of the Na(v)1.2 Na+
channel causing benign familial neonatal-infantile seizures. J Neurosci
2006; 26: 10100–9.
Shi X, Yasumoto S, Nakagawa E, Fukasawa T, Uchiya S, Hirose S.
Missense mutation of the sodium channel gene SCN2A causes
Dravet syndrome. Brain Dev 2009; 31: 758–62.
Specchio N, Vigevano F. The spectrum of benign infantile seizures.
Epilepsy Res 2006; 70(Suppl 1): S156–67.
Sugawara T, Tsurubuchi Y, Agarwala KL, Ito M, Fukuma G, Mazaki-
Miyazaki E, et al. A missense mutation of the Na+ channel alpha II
subunit gene Na(v)1.2 in a patient with febrile and afebrile seizures
causes channel dysfunction. Proc Natl Acad Sci USA 2001; 98:
6384–9.
Vacher H, Mohapatra DP, Trimmer JS. Localization and targeting of
voltage-dependent ion channels in mammalian central neurons.
Physiol Rev 2008; 88: 1407–47.
Vervaeke K, Hu H, Graham LJ, Storm JF. Contrasting effects of the
persistent Na+current on neuronal excitability and spike timing.
Neuron 2006; 49: 257–70.
Weckx S, Del-Favero J, Rademakers R, Claes L, Cruts M, De Jonghe P,
et al. novoSNP, a novel computational tool for sequence variation
discovery. Genome Res 2005; 15: 436–42.
Xu R, Thomas EA, Jenkins M, Gazina EV, Chiu C, Heron SE, et al. A
childhood epilepsy mutation reveals a role for developmentally regu-
lated splicing of a sodium channel. Mol Cell Neurosci 2007; 35:
292–301.
K,Lehmann-Horn F.Ion
brake.J Physiol2008; 586:
1414 |
Brain 2010: 133; 1403–1414Y. Liao et al.
at University of Washington on June 11, 2010
http://brain.oxfordjournals.org
Downloaded from

Page 13

ISSN: 1524-4628
Copyright © 2010 American Heart Association. All rights reserved. Print ISSN: 0039-2499. Online
Stroke is published by the American Heart Association. 7272 Greenville Avenue, Dallas, TX 72514
DOI: 10.1161/STROKEAHA.110.583542
published online Jun 3, 2010;
Stroke
Vermeulen, Floris Groenendaal, Linda S. de Vries and Timo R. de Haan
H.L.M.(Irma) van Straaten, Bwee-Tien Poll-The, Gerda van Wezel-Meijler, Jeroen R.
Florieke J. Berfelo, Karina J. Kersbergen, C.H.(Heleen) van Ommen, Paul Govaert,

Neonatal Cerebral Sinovenous Thrombosis From Symptom to Outcome
http://stroke.ahajournals.org
located on the World Wide Web at:
The online version of this article, along with updated information and services, is
http://www.lww.com/reprints
Reprints: Information about reprints can be found online at

journalpermissions@lww.com
410-528-8550. E-mail:
Fax: Kluwer Health, 351 West Camden Street, Baltimore, MD 21202-2436. Phone: 410-528-4050.
Permissions: Permissions & Rights Desk, Lippincott Williams & Wilkins, a division of Wolters

http://stroke.ahajournals.org/subscriptions/
Subscriptions: Information about subscribing to Stroke is online at
at UNIV WASHINGTON on June 14, 2010 stroke.ahajournals.org Downloaded from

Page 14

Neonatal Cerebral Sinovenous Thrombosis From
Symptom to Outcome
Florieke J. Berfelo, MD; Karina J. Kersbergen, BM; C.H.(Heleen) van Ommen, MD, PhD;
Paul Govaert, MD, PhD; H.L.M.(Irma) van Straaten, MD, PhD; Bwee-Tien Poll-The, MD, PhD;
Gerda van Wezel-Meijler, MD, PhD; Jeroen R. Vermeulen, MD, PhD; Floris Groenendaal, MD, PhD;
Linda S. de Vries, MD, PhD; Timo R. de Haan, MD, PhD
Background and Purpose—Cerebral sinovenous thrombosis is a rare disease with severe neurological sequelae. The
aim of this retrospective multicenter study was to investigate the clinical course, possible risk factors, and outcome
of a cohort of neonatal patients with sinovenous thrombosis and, second, to estimate the incidence in The
Netherlands.
Methods—From January 1999 to March 2009, a review of all neonatal patients with sinovenous thrombosis from 6 tertiary
neonatal intensive care units was performed. Population characteristics, clinical presentation, (prothrombotic) risk
factors, neuroimaging, interventions, and neurodevelopment were evaluated. An estimated incidence was calculated
based on the Netherlands Perinatal Registry.
Results—Fifty-two neonates were included (39 boys) with a median gestational age of 39 weeks (range, 30 to 42 weeks;
5 preterm). An assisted or complicated delivery occurred in 32 of 52. Presenting symptoms developed at a median
postnatal age of 1.5 days (range, 0 to 28 days) and consisted mainly of seizures (29 of 52). All sinovenous thrombosis
cases were confirmed with MRI/MR venography. Multisinus thrombosis was most common followed by superior
sagittal sinus thrombosis. FII G20210A mutation was present in 2 of 18 tested neonates (11%). Anticoagulation therapy
(in 22 of 52) did not result in hemorrhagic complications. At follow-up (median age, 19 months; range, 3 to 72 months),
moderate to severe neurological sequelae were present in 38%. The mortality was 10 of 52 (19%). A variable, although
high yearly incidence of 1.4 to 12 per 100 000 term newborns was found.
Conclusions—Neonatal sinovenous thrombosis is a multifactorial disease. The estimated incidence in The Netherlands
seems higher than reported elsewhere. (Stroke. 2010;41:00-00.)
Key Words: neonatal stroke ? risk factors ? sinovenous thrombosis ? treatment
N
and improved neuroimaging techniques. Described inci-
dences of pediatric sinovenous thrombosis range from 0.35 to
0.67 per 100 000 children per year with a higher incidence in
neonates (2.6 to 2.69 per 100 000 newborns a year). An
important data source is the Canadian Pediatric Stroke Reg-
istry.1–3The reported incidence of neonatal SVT is probably
an underestimation; many patients may remain unidentified
due to nonspecific clinical presentation, high dependence
on clinician awareness, and use of appropriate neuroimag-
ing techniques.4Morbidity and mortality can, however, be
eonatal cerebral sinovenous thrombosis (SVT) is in-
creasingly diagnosed due to greater clinical awareness
significant and depend on extent and localization of
thrombosis and associated cerebral parenchymal le-
sions.1,4–6Adverse neurological sequelae consist of gen-
eral developmental delay, sensorimotor deficits, visual
impairments, and epilepsy.2,5–8
Most studies on pediatric SVT include children of all
ages1,3,5,8–12and only few, usually small case-series, focus
on neonates.13–16Therefore, the aim of our study was to gain
more knowledge regarding clinical presentation, recognition
of high-risk profiles, use of optimal neuroimaging techniques,
interventions, and outcome in a large group of exclusively
neonatal patients with SVT.
Received March 4, 2010; final revision received March 21, 2010; accepted March 30, 2010.
From the Department of Neurology (F.J.B.), Academic Medical Center, Amsterdam, The Netherlands; the Department of Neonatology (K.J.K., F.G.,
L.S.d.V.), Wilhelmina Children’s Hospital, University Medical Center, Utrecht, The Netherlands; the Department of Pediatric Haematology (C.H.v.O.),
Emma Children’s Hospital, Academic Medical Center, Amsterdam, The Netherlands; the Department of Neonatology (P.G.), Sophia Children’s Hospital,
Erasmus Academic Medical Center, Rotterdam, The Netherlands; the Department of Neonatology (H.L.M.v.S.), Isala Clinics, Zwolle, The Netherlands;
the Department of Pediatric Neurology (B.-T.P.-T.), Emma Children’s Hospital, Academic Medical Center, Amsterdam, The Netherlands; the Department
of Neonatology (G.v.W.-M.), Leiden University Medical Center, Leiden, The Netherlands; the Department of Pediatric Neurology (J.R.V.), Free
University Medical Center, Amsterdam, The Netherlands; and the Department of Neonatology (T.R.d.H.), Emma Children’s Hospital, Academic Medical
Center, Amsterdam, The Netherlands.
Correspondence to Timo R. de Haan, MD, PhD, Department of Neonatology, Room H3-147, Amsterdam Medical Center/Emma Children’s Hospital,
Meibergdreef 9, 1100 DD, Amsterdam, The Netherlands. E-mail t.r.dehaan@amc.uva.nl
© 2010 American Heart Association, Inc.
Stroke is available at http://stroke.ahajournals.org DOI: 10.1161/STROKEAHA.110.583542
1
at UNIV WASHINGTON on June 14, 2010 stroke.ahajournals.org Downloaded from

Page 15

Methods
A retrospective, multicenter review was performed in neonates
admitted to 6 of the 10 Level III neonatal intensive care units
covering the most densely populated areas of The Netherlands
between January 1999 and March 2009. Pediatric hematology,
radiology, and neonatology databases of all participating centers
were searched for patients with neonatal SVT. Only MRI and MR
venography (MRV)-proven SVT was included. Because this was a
retrospective study, data on suspected SVT cases were unreliable or
incomplete. To estimate yearly incidence of neonatal SVT, the number
of term deliveries (home- and hospital-based) in the regions of the
neonatal intensive care units concerned was requested from the Neth-
Figure 1. A, Axial T2-weighted spin echo (T2SE)
image showing predominantly left-sided punctate
white matter lesions. B, Time-of-flight MRV image
showing absent flow in the straight sinus.
Table 1.Population Characteristics of 52 Neonates With SVT
ParameterNo. (%) or Median (Range)
Gestational age, weeks
Birth weight, g
Apgar score 5 minutes
Apgar score 10 minutes
Male
Female
Caucasian
African
Umbilical cord pH (n?13)
Interval birth to symptoms, days
Interval symptoms to diagnosis, days
39.0 (30–42)
3030 (1403–4415)
8.0 (3–10)
9.0 (3–10)
39 (75%)
13 (25%)
49 (94%)
3 (6%)
7.13 (6.76–7.27; n?4, pH ?7.0)
1.5 (0–28)
4.0 (0–26)
No. (%)
Clinical symptoms
Generalized seizure
Focal seizure
Apnea
Asymptomatic (chance finding)
Agitated
Sepsis-like
Depressed consciousness
No data
Outcome
Died
Survived
Normal
Moderately abnormal
Severely abnormal
No follow-up data
Follow-up ?9 months
18 (34.6%)
11 (21.2%)
9 (17.3%)
7 (13.4%)
3 (5.8%)
2 (3.8%)
1 (2%)
1 (2%)
10 (19%)
42 (81%)
19/42 (45%)
12/42 (29%)
8/42 (19%)
3/42 (7%)
13/42 (36.1%)
Table 2.
Neonates With SVT
Clinical and Prothrombotic Risk Factors in 52
Risk Factors
Maternal
Pre-eclampsia/HELLP syndrome
Pregnancy induced- or pre-existing diabetes
Maternal shock
Maternal surgery
History of clotting disease*
Perinatal
Complicated delivery†
Perinatal asphyxia
Fetomaternal transfusion
Neonatal
Sepsis or meningitis
Dehydration
Congenital heart defect
ECMO
No. (%)
7 (13.4%)
3 (5.8%)
1 (1.9%)
1 (1.9%)
1 (1.9%)
31 (60%)
3 (5.8%)
1 (1.9%)
8 (15.4%)
1 (1.9%)
1 (1.9%)
1 (1.9%)
Prothrombotic Risk Factors
Antithrombin level
Patients Tested, Result,
Median Value, Range
29/52, normal, median: 69%, range
39–145%
32/52, normal, median: 37%, range
10–86%
32/52, normal, median: 56%, range
10–100%
41/52, present in 2/41 (4.9%)
18/52, present in 2/18 (11%)
23/52, present in 13/23 (56.5%)
Protein C level
Protein S level
FV G1691A mutation
FII G20210A mutation
MTHFR C677T- and A1298C
homo- or heterozygous mutations
Homocysteine level23/52, normal, median: 6.0 mg/L;
range 4–16.3 mg/L
16/52, normal, median: 21.5 mg/L;
range 0–322 mg/L
Lipoprotein a
*Deep venous thrombosis and pulmonary emboli.
†Ventouse, forceps delivery, or emergency cesarean section.
HELLP indicates hemolytic anemia, elevated liver enzymes, low platelet
count; ECMO, extracorporeal membrane oxygenation procedure; MTHFR,
methylene tetrahydrofolate reductase.
2Stroke
July 2010
at UNIV WASHINGTON on June 14, 2010 stroke.ahajournals.orgDownloaded from