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Reduced Aquaporin 4 Expression in the Muscle Plasma Membrane of Patients With Duchenne Muscular Dystrophy
Yoshihiro Wakayama, MD, PhD;
Takahiro Jimi, MD;
Masahiko Inoue, MD;
Hiroko Kojima, BS;
Makoto Murahashi, MD;
Toshiyuki Kumagai, MD;
Sumimasa Yamashita, MD;
Hajime Hara, MD;
Seiji Shibuya, MD
Arch Neurol. 2002;59:431-437.
ABSTRACT
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Background In Duchenne muscular dystrophy (DMD), previous freeze-fracture electron
microscopic studies demonstrated that muscle plasma membrane contained markedly
decreased numbers of orthogonal arrays. Recent investigations showed that
orthogonal arrays were composed of aquaporin 4 (AQP4) molecules, a member
of the water channel protein family.
Objectives To study whether the immunostainability of anti-AQP4 antibody is reduced
in muscles of patients with DMD and whether, if it is reduced, the problem
is at the genomic DNA, messenger RNA (mRNA), or posttranscriptional level.
Patients and Methods We analyzed the muscle and blood samples from 6 boys with DMD, 6 normal
control subjects, and 12 patients with neuromuscular diseases at the protein,
genomic DNA, and mRNA levels. At the protein level, immunohistochemical staining
and immunoblot analysis were performed. At the genomic DNA and mRNA levels,
the polymerase chain reaction and reverse transcription polymerase chain reaction,
respectively, were used to screen for mutations in the AQP4 gene.
Results At the protein level, immunohistochemical staining of our originally
generated rabbit anti-AQP4 antibody in DMD muscles was markedly reduced. Most
of the DMD myofibers showed negative staining with sporadic partially positive
fibers at their myofiber surface, whereas the control muscles displayed continuous
myofiber surface staining. Immunoblot analysis showed that the content of
AQP4 in DMD muscles was remarkably decreased. Amplification of leukocyte genomic
DNA by polymerase chain reaction showed that the patients with DMD had genomic
DNA of the AQP4 molecule. Quantitative reverse transcription polymerase chain
reaction demonstrated that DMD skeletal muscles contained markedly decreased
AQP4 mRNA compared with controls.
Conclusion The reduction in AQP4 in DMD muscles results from decreased levels of
AQP4 mRNA in DMD myofibers.
INTRODUCTION
IN DUCHENNE muscular dystrophy (DMD), previous freeze-fracture electron
microscopic studies demonstrated that muscle plasma membrane contained markedly
decreased numbers of orthogonal array (OA) particles with decreased numbers
of subunit particles in the cytoplasmic half of the frozen cleaved plasma
membrane.1-2 Our group reported
a similar finding in the dystrophin-deficient X chromosome–linked muscular
dystrophy (mdx) mice.3
Recent investigations showed that the specific cellular sites of mercurial
insensitive water channel (MIWC)4-5
expression in astrocytes, trachea, sarcolemma, gastric parietal cells, and
kidney principal cells correspond exactly to sites where OA particles have
been visualized by freeze-fracture electron microscopy, suggesting that MIWC
may be the OA protein.6 Yang et al7 tested the hypothesis that MIWC protein forms OA particles.
They conducted the transfection of the coding sequence of rat MIWC molecule
into Chinese hamster ovary cells under a cytomegalovirus promoter. Immunostaining
of clonal cell populations showed MIWC expression at the plasma membrane,
and a single band of 31 kd was detected on immunoblot analysis. Freeze-fracture
electron microscopy disclosed the distinct OA particles on the plasma membrane
P face and the corresponding OA pits on the plasma membrane E face of the
MIWC-expressing cells, whereas the OA particles were not noted in the empty
vector-transfected cells.7 In addition, by
using fracture label electron microscopic technique, aquaporin 4 (AQP4) has
been demonstrated to be a major protein of OA particles in astrocyte8 and skeletal muscle plasma membrane.9
On the basis of these observations, OA particles have been proved to consist
of MIWC (AQP4) molecules.
Previously, our group generated rabbit anti-AQP4 antibody and demonstrated
markedly reduced immunostaining in mdx skeletal muscle.10
The aim of these studies was to determine whether the immunostainability of
anti-AQP4 antibody is reduced in muscles of patients with DMD and whether,
if it is reduced, the problem is at the genomic DNA, messenger RNA (mRNA),
or posttranscriptional level.
MATERIALS AND METHODS
MUSCLE AND BLOOD SAMPLES
Biopsy specimens of quadriceps femoris muscles were obtained with the
patient under local anesthesia from 6 boys with DMD whose ages at muscle biopsy
ranged from 1 year 6 months to 8 years 5 months. They had proximal muscle
weakness, atrophy in various degrees and calf pseudohypertrophy. All patients
with DMD had markedly high serum creatine kinase levels, ranging from 10 960
to 20 610 U/L at the time of muscle biopsy. The examination of leukocyte
genomic DNA from 2 of 6 patients with DMD demonstrated the dystrophin gene
deletion at exons 8, 12, 13, 17, and 19 in one boy and exons 49 through 52
in another boy. All muscles from patients with DMD showed negative dystrophin
immunostaining. For normal control specimens, 6 histochemically normal biopsy
specimens of quadriceps femoris muscles were obtained from patients who were
deemed to have myopathy but were free of neuromuscular disorders after histochemical
and immunologic examinations. Muscle specimens obtained by biopsy from 5 patients
with myotonic dystrophy, 4 patients with neurogenic muscle atrophy, 2 patients
with limb girdle muscular dystrophy, and 1 patient with myasthenia gravis
served as disease controls.
The leukocyte genomic DNA–coding AQP4 molecule was analyzed in
blood samples of 6 patients with DMD, 6 healthy control subjects, and 5 patients
with myotonic dystrophy, 4 patients with neurogenic atrophy, 2 patients with
limb girdle muscular dystrophy, and 1 patient with myasthenia gravis. In this
study, blood and muscle samples were taken from patients under informed consent.
PEPTIDE SYNTHESIS OF AQP4 AND ANTIBODY PRODUCTION
General procedures for peptide synthesis and antibody generation were
similar to those described previously.11 Briefly,
the peptide (C-EKKGKDSSGEVLSSV) of the C-terminal end of the cytoplasmic domain
in the rat AQP4 molecule4, 11 was
synthesized and extra cysteine was added to the N-terminus of this peptide.
Bovine thyroglobulin was added at an extra cysteine residue. The antibody
against this peptide was generated in rabbits. Solid-phase enzyme-linked immunosorbent
assay was used to determine the rabbit polyclonal antibody titer, which was
x204 800.
IMMUNOBLOT ANALYSIS OF ANTIBODY
Immunoblot analysis of antiserum against AQP4 in the histochemically
normal specimens of human quadriceps femoris muscles and those of patients
with DMD was performed with a previously described method12
with minor modifications. Sodium dodecyl sulfate polyacrylamide gel electrophoresis
was performed with a 12.5% homogeneous gel for AQP4, and 3% to 10% gradient
gel for dystrophin and ß-spectrin. The protein was transferred from gel
to a clear blot P membrane sheet (ATTO, Tokyo, Japan) by horizontal electrophoresis
at 108 mA for 90 minutes at room temperature. Immunostainings were done with
rabbit anti-AQP4 and anti–ß-spectrin13
antisera diluted 1:500 and 1:200, respectively, and with mouse monoclonal
anti–dystrophin (Dys 1) antibody (Novocastra Laboratories Ltd, Newcastle
upon Tyne, England) diluted 1:500.
IMMUNOHISTOCHEMISTRY
The muscle biopsy specimens were immediately frozen in isopentane cooled
with liquid nitrogen. Frozen, 6-µm-thick cross sections of the muscles
were placed on coverslips and incubated with primary antiserum, diluted 1:200
for rabbit anti-AQP4. Serial cross sections of muscles were also incubated
with primary monoclonal anti–Dys 1 antibody (Novocastra) diluted 1:30
and rabbit anti–ß-spectrin antiserum13
diluted 1:200. Indirect immunofluorescent staining was performed according
to the method previously described.14
POLYMERASE CHAIN REACTION
Polymerase chain reaction (PCR) was performed on genomic DNA extracted
from DMD, control, and disease control leukocytes with a kit (TaKaRa Ex Taq
[code RR001A]; TaKaRa Co, Kyoto, Japan) according to the manufacturer's protocol.
The oligonucleotide primers were designed from human AQP4 sequence5: sense strand, 5'-CTCAGCATTGCAACCATG-3',
and antisense strand, 5'-GGATTCCTGCTCCAATGA-3'. The PCR amplification
was performed by denaturing the genomic DNA at 94°C for 5 minutes and
conducted by 30 cycles of 30 seconds at 94°C and 52°C and 1 minute
at 72°C. The reaction mixture contained 1x PCR buffer, 2mM magnesium
chloride, 0.8mM of deoxyribonucleoside 5'-triphosphate (dNTP), 0.8µM
each of primer pair, and 0.1-U/µL Ex Taq polymerase. The PCR products
were observed by means of 2% agarose gel electrophoresis.
QUANTITATIVE REVERSE TRANSCRIPTION PCR
Total RNA was extracted from approximately 30 mg of each DMD, control,
or disease control muscle sample with an acid phenol extraction reagent (TRIzol
[code 15596-026]; Gibco BRL, Rockville, Md). The concentration of AQP4 mRNA
was estimated by quantitative reverse transcription PCR (RT-PCR). The oligonucleotide
primers were designed from human AQP4 sequence5:
sense strand (AQP4F), 5'-GGTGCTCATCTCCCTTTGCTTT-3'; antisense
strand (AQP4R), 5'-GTCTTTCCCCTTCTTCTCCTCTC-3'. The synthetic DNA
competitor was made by PCR from synthetic DNA templates provided in the kit
(Competitive DNA Construction Kit [code RR017]; TaKaRa Co), which included
both the upper (AQP4F) and lower (AQP4R) sequences specific for human AQP4.
We used a kit (Competitive RNA Transcription Kit [code 6125]; TaKaRa Co) to
make the synthetic RNA competitor from the synthetic DNA template by transcription
with SP6 RNA polymerase. The concentration of purified AQP4 RNA competitor
was calculated as a copy number per microgram of total RNA. Serial dilutions
of synthetic AQP4 RNA competitor were prepared. With 50 ng of total RNA of
each sample and serial dilutions of synthetic AQP4 RNA competitor, several
rounds of competitive RT-PCR for each sample were carried out according to
the kit instruction (mRNA Selective PCR Kit [code RR025A]; TaKaRa Co). Both
the sample and the competitor RNA were reverse transcribed for 30 minutes
at 50°C followed by 25 cycles of PCR (30 seconds each at 85°C and
60°C and 1 minute at 72°C) with the reaction mixture containing 1x
PCR buffer, 0.4µM each of primer pair, 5mM magnesium chloride, 1mM each
of dNTP analogue mixture, 0.8-U/µL ribonuclease inhibitor, 0.1-U/µL
reverse transcriptase, and 0.1-U/µL Taq polymerase. The competitive
RT-PCR products were separated by electrophoresis, and the concentration of
AQP4 mRNA in the sample was estimated by image analysis with an image processing
system (NIH image 1.61; National Institutes of Health, Bethesda, Md). To compare
the concentration of AQP4 mRNA with that of a housekeeping gene, a parallel
assay of ß-actin was performed for each sample by using human ß-actin
Competitive PCR Set ([code 6607] TaKaRa Co).
RESULTS
IMMUNOBLOT ANALYSIS OF ANTIBODY
Immunoblot analysis showed that the antibodies against AQP4, dystrophin,
and ß-spectrin reacted with 31-, 427-, and 270-kd protein extracts, respectively,
of normal and disease control human quadriceps femoris muscles (Figure 1). The reactions for AQP4 and ß-spectrin were markedly
and slightly decreased, respectively, in DMD muscle extracts (Figure 1A, C), whereas the reaction for dystrophin was absent in
DMD muscle extract (Figure 1B).
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Figure 1. Immunoblot analysis of aquaporin
4 (AQP4) (A), dystrophin (B), and ß-spectrin (C) expression in extracts
of histochemically normal human quadriceps femoris muscles (lane 1) and muscles
of patients with Duchenne muscular dystrophy (DMD) (lane 2). Electrophoresis
and blotting were performed as described in the "Materials and Methods" section.
In normal human muscle extracts, the bands of AQP4, dystrophin, and ß-spectrin
were observed at 31, 427, and 270 kd, respectively (A, B, and C, respectively).
The reactions for AQP4 and ß-spectrin were markedly and mildly decreased
in DMD muscle extracts, respectively (A and C, respectively), whereas the
reaction for dystrophin was absent in DMD muscle extract (B). Numbers to the
left in each figure indicate molecular masses of standards.
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IMMUNOHISTOCHEMISTRY
The immunoreaction for AQP4, dystrophin, and ß-spectrin with normal
and disease control human quadriceps femoris muscles was noted at the myofiber
surface (Figure 2A-C). The stainability
of the anti-AQP4 antibody in DMD muscle was markedly decreased and the pattern
of staining of AQP4 in the DMD muscles was strikingly patchy, with some large
fibers having a normal amount and almost all the small fibers being negative
(Figure 2D). The immunoreaction
with anti-dystrophin antibody at the DMD myofiber surface was negative (Figure 2E), whereas that with anti–ß-spectrin
antibody appeared to be normal in this study (Figure 2F). The myofibers with partial positive staining for anti-AQP4
antibody at their surface membranes showed negative staining with anti-dystrophin
antibody (Figure 2D-E).
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Figure 2. A through C, Immunohistochemical
staining of normal muscle with anti–aquaporin 4 (anti-AQP4) (A), anti-dystrophin
(B), and anti–ß-spectrin (C) antibodies. The cell surface of each
myofiber showed a thin layer of immunofluorescence with these antibodies.
No immunostaining of intracellular structures was noted. D through F, In muscle
samples from patients with Duchenne muscular dystrophy (DMD), immunostaining
for AQP4 was markedly reduced and the pattern of staining of AQP4 in the DMD
muscles was strikingly patchy, with some large fibers having normal amounts
and almost all the small fibers being negative (D). The immunoreaction with
anti-dystrophin antibody at the DMD myofiber surface was negative (E), whereas
that with anti–ß-spectrin antibody showed positive staining (F).
The myofibers with partial positive staining for anti-AQP4 antibody at their
surface membranes (D) showed negative staining with anti-dystrophin antibody
(E) (original magnification x330).
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PCR AMPLIFICATION OF GENOMIC DNA
The PCR amplification of leukocyte genomic DNA showed that all patients
with DMD and all healthy control subjects and disease control patients had
genomic DNA of human AQP4 as a single band (Figure 3).
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Figure 3. The polymerase chain reaction
(PCR) products obtained with primers for human aquaporin 4 as described in
the "Materials and Methods" section. The 164–base pair (bp) PCR product
was observed with template DNA from a boy with Duchenne muscular dystrophy
(lane 1), a control subject (lane 2), and a patient with myotonic dystrophy
(lane 3). Lane M is  x174 HaeIII digest
marker.
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QUANTITATIVE RT-PCR STUDY
The gel electrophoresis of competitive RT-PCR product of AQP4 in patients
with DMD (Figure 4A), healthy control
subjects (Figure 4B), and disease
control patients contained 2 bands of 733 and 568 base pairs (bp), which corresponded
to the RT-PCR products of total RNA samples extracted from muscle biopsy specimens
and AQP4 RNA competitor, respectively. The markedly decreased 733-bp RT-PCR
products of AQP4 were observed with 50 ng of total RNA from 6 patients with
DMD as compared with those of healthy subjects and disease control patients.
The gel electrophoresis of competitive RT-PCR products of ß-actin in
patients with DMD (Figure 4C), healthy
subjects (Figure 4D), and disease
control patients included 2 bands of 275 and 365 bp, which corresponded to
the RT-PCR products of total RNA samples extracted from muscle biopsy specimens
and ß-actin RNA competitor, respectively. The 275-bp RT-PCR products
of ß-actin appeared to be normal in 3 of 6 patients with DMD as compared
with those of healthy subjects and disease control patients.
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Figure 4. The competitive reverse transcription
polymerase chain reaction (RT-PCR) products obtained with primers for human
aquaporin 4 (AQP4) as described in the "Materials and Methods" section. The
gel electrophoresis of competitive RT-PCR products of AQP4 in patients with
Duchenne muscular dystrophy (DMD) (A) and healthy subjects (B) contained 2
bands of 733 and 568 base pairs (bp), which corresponded to the RT-PCR products
of total RNA samples extracted from muscles studied by biopsy and AQP4 RNA
competitor, respectively. The markedly decreased 733-bp RT-PCR products of
AQP4 were observed with 50 ng of total RNA in each boy with DMD (each lane)
of 6 patients with DMD as compared with those of healthy control subjects.
The gel electrophoresis of competitive RT-PCR products of ß-actin in
patients with DMD (C) and healthy subjects (D) included 2 bands of 275 and
365 bp, which corresponded to the RT-PCR products of total RNA samples extracted
from muscles studied by biopsy and ß-actin RNA competitor, respectively.
The 275-bp RT-PCR products of ß-actin appeared to be normal in 3 patients
with DMD (lanes 1-3) of 6 patients with DMD as compared with those of healthy
control subjects. Lane M is x174 HaeIII
digest marker. Each of lanes 1 through 6 represents the result of a boy with
DMD (A, C) or a healthy control subject (B, D).
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The group mean concentration of AQP4 mRNA in DMD muscles by quantitative
RT-PCR was 82 million molecules per microgram of total RNA, whereas that in
normal and disease control muscles was 3962 and 3404 million molecules per
microgram of total RNA, respectively. The group mean concentration of ß-actin
mRNA in DMD muscles by quantitative RT-PCR was 5458 million molecules per
microgram of total RNA, whereas that in normal and disease control muscles
was 16 236 and 18 066 million molecules per microgram of total RNA,
respectively (Figure 5). To compare
AQP4 mRNA of DMD muscles with that of normal control muscles, the ratio of
copy number of AQP4 mRNA to copy number of ß-actin mRNA was calculated
in patients with DMD, healthy subjects, and disease control patients. The
ratios were (26.1 ± 8.4) x 10 -3 (group mean
± SEM), (247.5 ± 65.4) x 10-3, and (261.7
± 71.2) x 10 -3, respectively. The ratio in
DMD muscles was statistically significantly lower than that in normal (P<.01) and disease control (P<.05)
muscles.
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Figure 5. Quantitative reverse transcription
polymerase chain reaction study of aquaporin 4 (AQP4) messenger RNA (mRNA).
In muscles from patients with Duchenne muscular dystrophy (DMD), AQP4 mRNA
level was markedly decreased. In contrast, AQP4 mRNA was expressed in normal
control and disease control muscles at the same level. The level of ß-actin
mRNA in muscles of patients with DMD was about one third that of normal control
and disease control muscles.
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COMMENT
Duchenne muscular dystrophy is an X-linked recessive muscle-wasting
disorder caused by the mutation of the dystrophin gene,15
which produces the membrane-associated cytoskeletal protein with 427-kd molecular
weight.16 Before the discovery of dystrophin
and its causative role in DMD, Schotland et al1, 17
and Wakayama et al2, 18 conducted
freeze-fracture electron microscopic studies of the muscle plasma membrane
in DMD in accordance with the membrane theory of DMD19
and membrane defects in DMD.20 They found marked
depletion of OA particles in the muscle plasma membrane P face and of OA pits
in the muscle plasma membrane E face of DMD,1-2,18
in addition to reduced individual intramembranous particles in this disease.
Recently, OA particles have been proved to consist of AQP4 molecule.7-9 At present, the relationship
between OA and dystrophin molecule is unknown. However, the indirect association
of AQP4 molecule with dystrophin was suggested via the modular protein-protein
interaction domain (PDZ domain)21 of  1-syntrophin,22-23 which is the 59-kd intracellular
dystrophin–associated protein22 and whose
expression is reduced in DMD24 and mdx mice.25 In fact, the immunoreactivity of rabbit anti-AQP4
antibody was markedly reduced in the sarcolemma and the vascular foot membrane
of brain astrocytes in 1-syntrophin null mice,23
and AQP4 mRNA was detected in soluble fractions but not in the membrane-associated
fraction of 1-syntrophin null mice.23
In our group's freeze-fracture studies of DMD muscle plasma membrane,2, 18 we noticed that most of the DMD myofiber
plasma membranes contained a markedly reduced density of OA particles; however,
some of the DMD myofibers contained the normal density of OA particles at
their plasma membranes. The results of the present immunohistochemical study
with anti-AQP4 antibody in DMD muscles coincided well with those of freeze-fracture
studies of DMD muscles, since the sporadic partially positive–staining
large fibers with anti-AQP4 antibody were noted in DMD muscles. We were interested
in whether the immunoreactivity of these AQP4-positive DMD myofibers was also
positive for the anti-dystrophin antibody. However, the serial cross sections
of DMD myofibers showed that the AQP4-positive fibers displayed the negative
dystrophin immunostaining.
In the next step, we investigated the mechanism of the depletion of
AQP4 molecule in DMD muscles at the genomic DNA and mRNA levels. As expected,
the PCR study disclosed that the genomic DNA of human AQP4 molecule was present
normally in DMD leukocytes and the decrease of AQP4 molecule in DMD muscles
was not due to the mutation of genomic DNA. In addition, the mRNA of human
AQP4 molecule was present in DMD muscles. The AQP4 staining pattern of DMD
muscles with some large fibers being positive and almost all small fibers
being negative suggested a secondary rather than a primary change. However,
quantitative RT-PCR showed that the amount of mRNA of AQP4 was markedly decreased
in DMD muscles. With regard to other membrane cytoskeletal proteins in DMD
muscles, the contents of dystroglycans and sarcoglycans have been reported
to be decreased.24, 26-28
The contents of mRNA for these proteins of DMD muscles were examined in ß-dystroglycan
and -sarcoglycan; they were described to be normal in dystroglycan26 and slightly decreased in -sarcoglycan,27 although the mRNA levels of other components of sarcoglycans
such as ß-,  -, and  -sarcoglycans have not been reported
so far.
This study clarified that the depletion of AQP4 molecule at the DMD
muscle plasma membrane was due to the decrease of mRNA of human AQP4 in DMD
muscles. The reduced content of AQP4 mRNA in DMD muscles implies either decreased
transcription or increased degradation of the message. The altered motor nerve
influence on the DMD muscles might have some relationship to decreased transcription.
In fact, the dystrophin protein of 116 kd (Dp116) is present in normal peripheral
nerve,29 but this dystrophin short form is
absent in DMD, resulting in the altered function of motor innervation in DMD
muscles. The abnormal innervation of DMD myofibers might be also brought about
by the proliferated connective tissue of DMD muscles. In addition, the freeze-fracture
study of the experimentally denervated regenerating myofibers showed no OA
particles at their muscle plasma membrane P face,30
and the mRNA of AQP4 molecule was absent in these muscles.31
Further investigations on the mechanism of the decrease of AQP4 mRNA in DMD
muscles will throw light onto the mechanism of the depletion of OA particles,
DMD muscle membrane lysis causing high serum levels of sarcoplasmic enzymes
and the formation of delta lesions of DMD myofibers, and finally the dysfunction
and degeneration of DMD muscles.
AUTHOR INFORMATION
Accepted for publication September 12, 2001.
Author contributions: Study
concept and design (Dr Wakayama); acquisition of
data (Drs Wakayama, Jimi, Inoue, Murahashi, Kumagai, Yamashita, Hara,
and Shibuya and Ms Kojima); analysis and interpretation
of data (Drs Wakayama and Jimi); drafting of the
manuscript (Drs Wakayama, Jimi, Inoue, Murahashi, Kumagai, Yamashita,
Hara, and Shibuya and Ms Kojima); critical revision of the
manuscript for important intellectual content (Drs Wakayama and Jimi); statistical expertise (Drs Wakayama and Jimi); obtaining funding (Dr Wakayama); administrative,
technical, or material support (Drs Wakayama, Jimi, Inoue, Murahashi,
Kumagai, Yamashita, Hara, and Shibuya and Ms Kojima); study
supervision (Dr Wakayama).
We thank Yoko Matsuzaki, MP, for her skillful technical assistance.
Corresponding author and reprints: Yoshihiro Wakayama, MD, PhD, Department
of Neurology, Showa University Fujigaoka Hospital, 1-30 Fujigaoka, Aoba-ku,
Yokohama 227-8501, Japan (e-mail: wakayama{at}showa-university-fujigaoka.gr.jp).
From the Department of Neurology, Showa University Fujigaoka Hospital,
Yokohama (Drs Wakayama, Jimi, Inoue, Murahashi, Hara, and Shibuya and Ms Kojima);
Department of Pediatric Neurology, Aichi Prefectural Colony, Kasugai (Dr Kumagai);
and Department of Neurology, Kanagawa Children's Medical Center, Yokohama
(Dr Yamashita), Japan.
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