Missense mutation of the sodium channel gene SCN2A causes Dravet syndrome.

Abstract

Mutations of the gene encoding the alpha2 subunit of the neuronal sodium channel, SCN2A, have been found in benign familial neonatal-infantile seizures (BFNIS). In Dravet syndrome, only one nonsense mutation of SCN2A was identified, while hundreds of mutations were found in the paralogue gene, SCN1A, which encodes the alpha1 subunit. This study examines whether SCN2A mutations are associated with Dravet syndrome. We screened for mutations of SCN1A, SCN2A and GABRG2 (the gene encoding gamma2 subunit of the GABA(A) receptor) in 59 patients with Dravet syndrome and found 29 SCN1A mutations and three missense SCN2A mutations. Among the three, one de novo SCN2A mutation (c.3935G>C: R1312T) identified in a patient was thought to affect an arginine residue in a voltage sensor of the channel and hence, to be pathogenic. This finding suggests that both nonsense mutations and missense SCN2A mutations cause Dravet syndrome.

Full text

Page 1

Rapid communication
Missense mutation of the sodium channel gene SCN2A
causes Dravet syndrome
Xiuyu Shia, Sawa Yasumotoa, Eiji Nakagawab, Tatsuya Fukasawac, Satoshi Uchiyad,
Shinichi Hirosea,*
aDepartment of Pediatrics, School of Medicine, Fukuoka University, Fukuoka, Japan
bDepartment of Pediatric Neurology, Musashi Hospital, National Center of Neurology and Psychiatry, Tokyo, Japan
cDepartment of Pediatrics, Nagoya Memorial Hospital, Nagoya, Japan
dDepartment of Pediatrics, Mito Saiseikai General Hospital, Mito, Japan
Received 16 July 2009; received in revised form 18 August 2009; accepted 26 August 2009
Abstract
Mutations of the gene encoding the a2 subunit of the neuronal sodium channel, SCN2A, have been found in benign familial
neonatal-infantile seizures (BFNIS). In Dravet syndrome, only one nonsense mutation of SCN2A was identified, while hundreds
of mutations were found in the paralogue gene, SCN1A, which encodes the a1 subunit. This study examines whether SCN2A muta-
tions are associated with Dravet syndrome. We screened for mutations of SCN1A, SCN2A and GABRG2 (the gene encoding c2
subunit of the GABAAreceptor) in 59 patients with Dravet syndrome and found 29 SCN1A mutations and three missense SCN2A
mutations. Among the three, one de novo SCN2A mutation (c.3935G>C: R1312T) identified in a patient was thought to affect an
arginine residue in a voltage sensor of the channel and hence, to be pathogenic. This finding suggests that both nonsense mutations
and missense SCN2A mutations cause Dravet syndrome.
? 2009 Elsevier B.V. All rights reserved.
Keywords: Dravet syndrome; SCN1A; SCN2A; GABRG2; Genetics
1. Introduction
Dravet syndrome, which includes severe myoclonic
epilepsy in infancy (SMEI) and its borderline phenotype
(SMEB), is a rare and malignant epilepsy syndrome,
which usually develops in the first year of life [1,2].
The majority of the genetic abnormalities underlying
Dravet syndrome have been found in SCN1A, though
a few mutations were found in GABRG2 [3]. To date,
more than 300 different mutations of SCN1A have been
reported [4]. Moreover, microchromosomal deletions
involving SCN1A have been reported as a cause of Dra-
vet syndrome [5]. Still, more than 20% patients with
Dravet syndrome are free from such genetic abnormali-
ties [6]. Further genetic studies are needed to determine
other causative or associative genetic abnormalities in
Dravet syndrome (i.e., SCN2A).
Both SCN1A and SCN2A are clustered within 600 kb
on human chromosome 2q24. The first SCN2A muta-
tion was reported in a patient with febrile and afebrile
seizures similar to those of generalized epilepsy with feb-
rile seizures plus (GEFS+) [7]. Subsequent studies have
identified eight different mutations of SCN2A in benign
familial neonatal-infantile seizures (BFNIS) [8]. Until
now, all SCN2A mutations identified in BFNIS were
missense mutations inherited from a single parent; each
0387-7604/$ - see front matter ? 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.braindev.2009.08.009
*Corresponding author. Address: Department of Pediatrics, School
of Medicine, Fukuoka University, 45-1, 7-chome Nanakuma, Jonan-
ku Fukuoka 814-0180, Japan. Tel.: +81 92 801 1011x3390, +81 92 862
1290; fax: +81 92 862 6955.
E-mail address: hirose@fukuoka-u.ac.jp (S. Hirose).
www.elsevier.com/locate/braindev
Brain & Development 31 (2009) 758–762

Page 2

demonstrated impairment of the function of the sodium
channel bearing a2 subunit or NaV1.2. Only one non-
sense mutation of SCN2A, however, has been reported
in patients with Dravet syndrome [9]. The aim of this
study is to examine whether SCN2A mutations are asso-
ciated with Dravet syndrome.
2. Materials and methods
2.1. Patients
This study included 59 Japanese patients who had
been diagnosed with Dravet syndrome (SMEI n = 33
and SMEB n = 26) at departments of child neurology
in various regional tertiary hospitals. The diagnoses of
SMEI and SMEB were made by the method described
previously [10]. We also recruited 96 healthy volunteers
as the control group. Each participant or the parent/
guardian signed an informed consent form approved
by the Ethics Review Committee of Fukuoka University
or similar committees of the participating institutions.
2.2. Genetic analysis
Genomic DNAs were prepared from ethylenedi-
aminetetraaceticacid (EDTA)-treated
samples using QIAamp DNA Blood kit (Qiagen, Hil-
den, Germany). Screening for genetic abnormalities of
SCN1A, SCN2A and GABRG2 was performed using
direct sequencing methods and multiple ligand polymer-
ase amplification (MLPA). Details of the PCR condi-
tions and the primers used are available upon request.
Reference sequences of messenger RNA (mRNA) were
based on information available from RefSeq (accession
numbers): Human SCN1A NM 006920; Human SCN2A
NM 021007; Human GABRG2, NM 198904.
whole-blood
3. Results
A total of 29 SCN1A mutations were found; none
were found in GABRG2. Three novel missense muta-
tions of SCN2A were identified in 3 (Table 1, 5.1%) of
59 patients but were not found in the 96 healthy
volunteers.
Patient A with SMEI had a missense mutation of
SCN2A (c.964G>A: D322N, exon 6, Fig. 1) and a splice
site mutation of SCN1A (IVS4+1G>A), the mother had
same mutation of SCN2A. Patient B with SMEB had
missense mutations of SCN2A (c.982T>G: F328V, exon
7, Fig. 1) and SCN1A (c.4507G>A: E1503K), the
mother had same mutation of SCN2A. Patient C with
SMEI had a de novo missense mutation of SCN2A
(c.3935G>C: R1312T, exon 20, Fig. 1) without any
SCN1A mutations, parents had no such mutation.
These three SCN2A mutations were all novel and
were considered to affect highly conserved amino acids
in many species (Fig. 1). Mutations of exon 6 (patient
A) and exon 7 (patient B) located in the loop between
the fifth and sixth transmembrane segment of domain
I (Fig. 2). Mutation of exon 20 (patient C) located in
the fourth transmembrane segment of domain III
(Fig. 2).
4. Discussion
This study reports three novel missense mutations of
SCN2A in three Dravet syndrome patients who have no
neonatal seizures. All three mutations affect highly con-
served amino acids in many species. Two SCN2A muta-
tions, c.964G>A: D322N and c.982T>G: F328V,
however, coexisted with de novo SCN1A mutations,
IVS4+1G>A and c.4507G>A: E1503K in patients with
SMEI and SMEB, respectively. These mutations were
also inherited from one of their parents and accordingly
were not likely to be pathogenic. In contrast, patient C
with SMEI had a de novo missense mutation of SCN2A
(c.3935G>C: R1312T) without any SCN1A mutations.
Arginine at the 1312 position is a crucial charged amino
acid in the fourth transmembrane segment of domain
III, which functions as a voltage sensor of the NaV1.2
channel. This mutation in such important position
may hamper the function of the channel and contribute
to the case’s pathogenesis of Dravet syndrome.
Severe myoclonic epilepsy in infancy (SMEI) was first
described in 1982, recently the term “Dravet syndrome”
has been proposed [2,11]. Genetic abnormalities, espe-
cially sodium channels (SCN1A) are a major cause of
Dravet syndrome, as voltage-gated sodium channels
are responsible for the initiation and propagation of
action potentials in excitable tissues. In this study 29
SCN1A mutations are found in 59 patients with Dravet
syndrome, confirming that genetic factors play an
important role in the etiology of Dravet syndrome. Fur-
thermore, novel SCN2A mutations expand the sodium
channel mutation spectrum and suggest that missense
mutation of SCN2A is also a cause of Dravet syndrome.
Table 1
Clinical and genetic characteristics of the patients with SCN2A mutations.
PtDiagnosis Sex
SCN2A mutation
SCN1A mutationAge at onset Myoclonic seizuresAtypical absence Parents
A
B
C
SMEI
SMEB
SMEI
F
F
F
D322N (exon 6)
F328V (exon 7)
R1312T (exon 20)
IVS4+1 G>A7
E1503K
No
7 M
8 M
11 M
+
?
+
–
–
–
Mother (D322N)
Mother (F328V)
No
X. Shi et al./Brain & Development 31 (2009) 758–762
759

Page 3

SCN2A encoding NaV1.2, a voltage-gated sodium
channel pore-forming an a subunit, expresses abun-
dantly in the adult central nervous system. Early in
development, NaV1.2 is highly expressed in the regions
destined to become Ranvier nodes, and is developmen-
tally replaced by NaV1.6 in adult rat brains. NaV1.2 is
also enriched at axon initial segments (AIS) in develop-
ing neurons. Clusters of NaV1.2 at nodes and at the AIS
contribute to determining axonal firing frequency and
action potential propagation [12]. Abnormal NaV1.2
function may disrupt this channel’s physiological role
in controlling excitability.
The first mutation of SCN2A was reported in a
patient with febrile and afebrile seizures similar to those
of GEFS+, biophysical analysis revealed that the
NaV1.2 mutant channel inactivated more slowly, sug-
gesting an increase of sodium ion influx. Subsequent
mutations were reported mostly in BFNIS. To date
eight SCN2A mutations have been reported in BFNIS
[8]. Only one nonsense mutation has been identified in
a patient with intractable epilepsy and mental decline.
Mutations in NaV1.2 have been supposed less frequently
associated with severe forms of epilepsy. In this study,
however, three missense mutations of SCN2A were
found in patients with severe forms of epilepsy (SMEI
or SMEB) and none of our patients had neonatal sei-
zures. In addition, we found no GABRG2 mutation.
Our study indicates that mutations in NaV1.2 are
Fig. 1. Pedigree of the patients with SCN2A mutation and alignment of affected amino acid. Filled symbols represent affected individuals. N/A
means not available. Arrows indicate where mutations occur. Rectangular boxes represent the corresponding amino acids to the amino acids where
the mutations occur, that are highly conserved throughout different species.
760
X. Shi et al./Brain & Development 31 (2009) 758–762

Page 4

involved not only in benign but also severe forms of
epilepsy.
Recently, Misra [13] examined the functional charac-
terization of three SCN2A mutations associated with
BFNIS. They observed that cells expressing mutant
channels exhibited lower peak current levels than WT.
Furthermore, all three mutants exhibited between 47%
and 79% lower cell surface expression of channel protein
compared to WT in tsA201 cells, and the relative mag-
nitude of the reduction was concordant with mean
whole-cell peak current levels. This finding suggests that
reduced sodium channel density may be an important
factor in the pathogenesis of BFNIS.
In another study [14] Kile investigated the cellular
network activity of the hippocampus in Q54 transgenic
mice displaying spontaneous seizures as a result of a
gain-of-function mutation of the SCN2A sodium chan-
nel gene. Increased spontaneous extracellular activity
was found in both CA1 and CA3 regions of Q54 hippo-
campal slices. Q54 slices demonstrated significantly
greater spontaneous and afterdischarge activity than
did WT slices; they also showed a greater population
spike amplitude and duration following tetanic stimulus.
All of these findings highlight the importance of NaV1.2
function in the development of these seizures.
We identified three novel missense mutations of
SCN2A in patients with Dravet syndrome. Among
them,one solely
de
(c.3935G>C: R1312T) identified in a patient with SMEI
was thought to be pathogenic. To our knowledge, there
have been no previous reports of missense mutation of
SCN2A causing Dravet syndrome. These results would
pave the way toward an understanding of NaV1.2 chan-
nel dysfunctions and their involvement in the molecular
etiology of epilepsy.
novo SCN2A
mutation
Acknowledgment
We are indebted to all members of the family for their
helpful cooperation in this study. We thank Ms. Takako
Umemoto and Hideko Takeda for formatting and typ-
ing the manuscript and Ms. Minako Yonetani and
Akiyo Hamachi for the technical assistance. This study
was supported in part by Grants-in-Aid for Scientific
Research (S) 16109006, (A) 18209035 and 21249062,
Exploratory Research 1659272, and “High-Tech Re-
search Center” Project for Private Universities-matching
fund subsidy from the Ministry of Education, Culture,
Sports, Science and Technology, 2006–2010 “The Re-
search Center for the Molecular Pathomechanisms of
Epilepsy, FukuokaUniversity”,
(19A-6) and (21B-5) and (21210301) from the Ministry
of Health, Labor and Welfare and the Central Research
Institute of Fukuoka University.
Research Grants
References
[1] Proposal for revised classification of epilepsies and epileptic
syndromes. Commission on Classification and Terminology of the
International League Against Epilepsy. Epilepsia 1989;30:389–99.
[2] Engel Jr J. A proposed diagnostic scheme for people with epileptic
seizures and with epilepsy: report of the ILAE Task Force on
Classification and Terminology. Epilepsia 2001;42:796–803.
[3] Harkin LA, Bowser DN, Dibbens LM, Singh R, Phillips F,
Wallace RH, et al. Truncation of the GABAA-receptor c2 subunit
in a family with generalized epilepsy with febrile seizures plus. Am
J Hum Genet 2002;70:530–6.
[4] LossinC.AcatalogofSCN1Avariants.BrainDev2009;31:114–30.
[5] WangJW,KurahashiH,IshiiA,KojimaT,OhfuM,InoueT,etal.
MicrochromosomaldeletionsinvolvingSCN1Aandadjacentgenes
inseveremyoclonicepilepsyininfancy.Epilepsia2008;49:1528–34.
[6] Depienne C, Trouillard O, Saint-Martin C, Gourfinkel-An I,
Bouteiller D, Carpentier W, et al. Spectrum of SCN1A gene
Fig. 2. Schematic representation of the missense mutations identified in this study on the NaV1.2 protein. Neuronal voltage-gated Na+channels are
composed of three subunits; an a subunit and two auxiliary b subunits, b1 and b2. The a subunit is a large pore-forming, bell-shaped molecule whose
expression alone is sufficient to function as a Na+channel. The a subunit has four homologous domains (I–IV), each comprising six membrane-
spanning segments. The fourth segment of each domain functions as a voltage sensor. The ion pore is formed between the fifth and sixth segments.
Round spots represent mutation positions which located in the loop between the fifth and sixth transmembrane segment of domain I and the fourth
transmembrane segment of domain III, respectively.
X. Shi et al./Brain & Development 31 (2009) 758–762
761

Page 5

mutations associated with Dravet syndrome: analysis of 333
patients. J Med Genet 2009;46:183–91.
[7] Sugawara T, Tsurubuchi Y, Agarwala KL, Ito M, Fukuma G,
Mazaki-Miyazaki E, et al. A missense mutation of the Na+
channel aII subunit gene Nav1.2 in a patient with febrile and
afebrile seizures causes channel dysfunction. Proc Natl Acad Sci
USA 2001;98:6384–9.
[8] Herlenius E, Heron SE, Grinton BE, Keay D, Scheffer IE, Mulley
JC, et al. SCN2A mutations and benign familial neonatal-infantile
seizures: the phenotypic spectrum. Epilepsia 2007;48:1138–42.
[9] 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.
[10] Fukuma G, Oguni H, Shirasaka Y, Watanabe K, Miyajima T,
Yasumoto S, et al. Mutations of neuronal voltage-gated Na+
channel a1 subunit gene SCN1A in core severe myoclonic epilepsy
in infancy (SMEI) and in borderline SMEI (SMEB). Epilepsia
2004;45:140–8.
[11] Dravet C, Bureau M, Guerrini R, Giraud N, Roger J. Severe
myoclonic epilepsy in infants. Epileptic syndromes in infancy. 2nd
ed. London: John Libbey; 1992. p. 14–88.
[12] Boiko T, Van Wart A, Caldwell JH, Levinson SR, Trimmer JS,
Matthews G. Functional specialization of the axon initial segment
byisoform-specific sodium
2003;23:2306–13.
[13] Misra SN, Kahlig KM, George Jr AL. Impaired NaV1.2 function
and reduced cell surface expression in benign familial neonatal-
infantile seizures. Epilepsia 2008;49:1535–45.
[14] Kile KB, Tian N, Durand DM. Scn2a sodium channel mutation
results in hyperexcitability in the hippocampus in vitro. Epilepsia
2008;49:488–99.
channeltargeting.J Neurosci
762
X. Shi et al./Brain & Development 31 (2009) 758–762