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CAPN3 mutations in patients with idiopathic eosinophilic myositis.

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CAPN3 Mutations in Patients with
Idiopathic Eosinophilic Myositis
Martin Krahn, MD,1 Adolfo Lopez de Munain, MD, PhD,2 Nathalie Streichenberger, MD,3
Rafaëlle Bernard, MD,1 Christophe Pécheux, BSc,1 Hervé Testard, MD,4 José L. Pena-Segura, MD,5
Eugenia Yoldi, MD,6 Ana Cabello, MD,7 Norma B. Romero, MD, PhD,8 Juan J. Poza, MD, PhD,2
Sandrine Bouillot-Eimer, MD,9 Xavier Ferrer, MD,10 Maria Goicoechea, BSc,2 Federico Garcia-Bragado, MD,11
France Leturcq, PhD,12 J. Andoni Urtizberea, MD,13 and Nicolas Lévy, MD, PhD1,14
Objective: Eosinophilic myositis (EM) constitutes a rare pathological entity characterized by eosinophilic infiltration of skeletal
muscles, usually associated with parasite infections, systemic disorders, or the intake of drugs or L-tryptophan. The exclusion of
such causes defines the spectrum of idiopathic EM. Based on a protein analysis performed in one affected patient, we identified
the gene encoding calpain-3, CAPN3, as a candidate for a subset of idiopathic EM.
Methods: We screened CAPN3 for mutations using DHPLC and direct sequencing in six unrelated patients, recruited for EM
diagnosed after histological examination of muscle biopsy samples, without any identified causative factor.
Results: We identified CAPN3 mutations in the six unrelated patients originally diagnosed with idiopathic EM.
Interpretation: Mutations in CAPN3 can cause EM. Thus, a subset of idiopathic EM is genetically determined, with an
autosomal recessive mode of inheritance. Patients presented with a triad that appears to be indicative of CAPN3 mutations: (1)
EM in the first decade, (2) elevated serum creatine phosphokinase levels (isolated or with little corresponding weakness), and (3)
inconstant peripheral hypereosinophilia. However, that EM represents a distinct phenotype associated to CAPN3 mutations or,
rather, an early histopathological picture of LGMD2A must be further evaluated. Our findings should be of interest toward
further investigating the role of calpain-3 in skeletal muscle. Furthermore, patients with idiopathic EM should undergo calpain-3
protein analysis and be considered for subsequent molecular analysis of the CAPN3 gene.
Ann Neurol 2006;59:905–911
Eosinophilic myositis (EM) constitutes a group of rare
disorders characterized by eosinophilic infiltration of the
skeletal muscles, peripheral blood, and/or bone marrow
hypereosinophilia associated with inflammatory lesions
of the muscle tissue. The diagnosis is based on histological examination of muscle sections after biopsy.
In the evaluation of EM, the diagnosis of idiopathic
EM, in which no causative factor is identified, is retained after exclusion of parasite infections, systemic
disorders of hypereosinophilia, or the intake of drugs
or L-tryptophan.1
Calpain-3 is a muscle-specific protein, belonging to a
family of intracellular nonlysosomal proteases. Mutations in CAPN3 (15q15.1-15.3), the gene encoding
calpain-3, are responsible for the most prevalent form
of autosomal recessive limb-girdle muscular dystrophies
(AR-LGMDs), namely, LGMD2A (MIM #253600),
also referred to as calpainopathy.2 In the absence of
significant clues at the clinical level, calpain-3 protein
analysis on muscle specimens remains a first-line test to
point to the right diagnosis.3 Subsequent molecular
analysis of the CAPN3 gene is then warranted to confirm the diagnosis on even more solid grounds.
The precise function of calpain-3 within the muscle
fiber remains only partially understood, but it could
play a role in the inhibitory protein ␬B␣ (I␬B␣) metabolism and regulate the nuclear factor-␬B (NF-␬B)
survival pathway in skeletal muscle.4,5 It also has been
From the 1Département de Génétique Médicale, Laboratoire de Génétique Moléculaire, Hôpital d’Enfants de la Timone, Marseille,
France; 2Servicio de Neurologia and Unidad Experimental, Hospital
Donostia, San Sebastian, Basque Country, Spain; 3Service de Neuropathologie, Hôpital Neurologique, Lyon, France; 4Service de Pédiatrie et Néonatalogie, CHI, Annemasse, France; 5Unidad de Neuropediatrı́a, Hospital Universitario Miguel Servet, Zaragoza;
6
Servicio de Neurologia Infantil, Hospital Virgen del Camino, Pamplona; 7Servicio de Anatomı́a Patológica, Hospital Doce de Octubre, Madrid, Spain; 8Institut National de la Sante et de la Recherche Médicale U582 Institut de Myologie, Hôpital de la PitiéSalpétrière, Paris; 9Laboratoire d’Anatomie Pathologique, Hôpital
Pellegrin; 10Service de Neurologie, Hôpital Haut-Lévêque, Bordeaux, France; 11Servicio de Anatomı́a Patológica, Hospital Virgen
del Camino, Pamplona, Spain; 12Laboratoire de Biochimie Génétique, Hôpital Cochin; 13APHP, Hôpital Marin Hendaye; and 14Institut National de la Sante et de la Recherche Médicale U491, Faculté de Médecine Timone, Université de la Méditerranée, Marseille,
France.
Received Nov 2, 2005, and in revised form Jan 12, 2006. Accepted
for publication Feb 10, 2006.
Published online Apr 10, 2006 in Wiley InterScience
(www.interscience.wiley.com). DOI: 10.1002/ana.20833
Address correspondence to Dr Levy, Département de Génétique
Médicale, Hôpital Timone, 264 rue Saint-Pierre, 13385 Marseille
Cedex 5, France. E-mail: nicolas.levy@medecine.univ-mrs.fr
© 2006 American Neurological Association
Published by Wiley-Liss, Inc., through Wiley Subscription Services
905
Table. Clinical and Laboratory Findings in the Patients with Idiopathic Eosinophilic Myositis Included in This Study, and
Overview of Identified CAPN3 Mutations
Patient
No.
Sex
Age at
Diagnosis
(yr)
PEM1
M
4
PEM2
M
9
PEM3
M
7
PEM4
F
11
PEM5
F
3
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Annals of Neurology
Clinical and
Laboratory Findings
Isolated hyperCKemia
(4,900IU/L). Normal psychomotor
development. Normal neurological
findings, except
slight motor clumsiness with tiptoe
working. No anomalies on electromyogram (EMG). No
associated biological
inflammatory syndrome. Mild hypereosinophilia
(913/␮l). Negative
serologies for trichinosis. No uptake of
L-tryptophan.
Normal psychomotor
development. Normal neurological
findings, except
slight motor clumsiness. HyperCKemia
(7,517IU/L) and
mild hypereosinophilia (707/␮l).
Negative serologies
for trichinosis.
Isolated hyperCKemia
(6,743IU/L). Normal psychomotor
development. Normal neurological
findings, except increased motor fatigability. Negative
serologies for Toxocara and Fasciola.
Weakness of lower
limbs. HyperCKemia (10,000IU/L).
No hypereosinophilia on PBC.
Muscle testing at
onset: psoas 3/5,
adductor 4/5, triceps suralis 3/5,
quadriceps 5/5,
tibialis anterior 4/5,
tibialis posterior
4/5, twins 4/5, soleus 4/5. Muscle
testing at age 16
years: psoas 3/5,
adductor 3/5, gluteus 3/5.
Isolated hyperCKemia
(10,377IU/L). Pitiriasis Guibert. Normal psychomotor
development. Normal neurological
findings. Hypereosinophilia (1,945/
␮L). Negative serologies for
trichinosis. At age
9 years, difficulty
to walk on heels
and slight bilateral
aquilae retraction.
Vol 59
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June 2006
Muscle Biopsy Findings
Mutation 1
Mutation 2
Focal inflammatory lesions with necrosis and
polymorphic infiltrates
included eosinophils.
No evidence of parasites. Focal expression
of MHC-I. No alteration of dystrophin,
␣-sarcoglycan,
␥-sarcoglycan, dysferlin,
and merosine on immunohistological examinations. Complete absence of the calpain-3
p94, p60, and p30 on
Western blot analysis.
Exon 8:
c.1079G⬎A
(p.Trp360X)a
Exon 16:
c.1838delA
(p.Lys613ArgfsX49)b
Inflammatory lesions with
abundant eosinophilic
infiltration. No evidence of parasites. No
dystrophic features; no
alteration of dystrophin, sarcoglycan, and
merosine on immunohistological examinations.
IVS 5:
c.802-9G⬎Ac
Exon 22:
c.2306G⬎C
(p.Arg769Pro)d
Mild myopathic changes
with focal inflammatory lesions with abundant eosinophilic infiltration involving
necrotic fibers. Normal
pattern of dystrophin,
sarcoglycan, and merosine on immunohistological examinations.
No evidence of parasites.
Inflammatory reaction
around necrotic and
nonnecrotic fibers.
Inflammation collects
at endomysial site with
sometimes perivascular
infiltrate without destruction of walls of
arterioles and venules.
Cells consist of lymphocytes, histiocytes,
and few plasma cells.
Numerous eosinophilic
leucocytes are present.
No evidence of parasites.
Exon 1:
c.302G⬎A
(p.Arg101Lys)e
Exon 5:
c.664G⬎A
(p.Gly222Arg)f
Focal inflammatory lesions with necrosis and
polymorphic infiltrates
composed mainly of
eosinophils. No evidence of parasites. No
dystrophic features.
Exon 22:
2362_2363delinsTCATCT
(p.Arg788delinsSerSerfsX11)g
Exon 5:
c.664G⬎A
(p.Gly222Arg)f
Exon 22:
2362_2363delinsTCATCT
(p.Arg788delinsSerSerfsX11)g
Exon 22:
2362_2363delinsTCATCT
(p.Arg788delinsSerSerfsX11)g
Table. Continued
Patient
No.
Sex
Age at
Diagnosis
(yr)
PEM6
M
11
Clinical and Laboratory
Findings
Weakness of lower limbs
and difficulties to
walk (tiptoe walking
since the age of 2
years). Bilateral scapular winging. No Gowers’ sign. Bilateral
aquilae retraction.
Muscle testing at 11
years: 4/5 upper
limbs, adductor 3/5,
quadriceps 5/5, ischiotibialis 4/5, distal
muscles 4/5. HyperCKemia (8616IU/L).
Eosinophilia at upper
normal limit (490/
␮l). Bilateral and
symmetrical adipous
infiltration of adductor majoris and ischiotibialis on muscle
magnetic resonance
imaging.
Muscle Biopsy Findings
Mutation 1
Mutation 2
Mild myopathic changes
with irregular fiber size.
Focal inflammatory infiltration with necrosis
of involved fibers. The
inflammatory infiltration is composed of B
lymphocytes, histiocytes, and CD15positive granulocytes,
most of them eosinophils.
Exon 22:
2362_2363delinsTCATCT
(p.Arg788delinsSerSerfsX11)g
Exon 22:
2362_2363delinsTCATCT
(p.Arg788delinsSerSerfsX11)g
DHPLC ⫽ denaturing high performance liquid chromatography; MHC-1 ⫽ major histocompatibility complex class I; PBC ⫽ peripheral
blood count.
a
Previously reported in Richard and colleagues.2
b
Previously reported in Kramerova and colleagues.17
Previously reported in Piluso and colleagues.18
c
d
e
Not previously reported.
Previously reported in Saenz and colleagues.3
Previously reported in Urtasun and colleagues9 and Richard and colleagues.20
Previously reported in Urtasun and colleagues9 and Richard and colleagues.19
f
g
demonstrated that calpain-3 interacts with other members of the myofibrillar protein complex such as
titin.6,7
This study was first based on a routine protein analysis performed by Western blot in one affected patient,
which identified calpain-3 as a potential candidate of a
genetic cause in a subset of patients with idiopathic
EM. We report here the identification of inherited
pathogenic mutations in the CAPN3 gene in six unrelated patients originally diagnosed with idiopathic EM.
Subjects and Methods
Participants
We (A.L.M., H.T., J.L.P-S., E.Y., J.J.P., X.F., J.A.U., and
N.L.) personally examined all probands. They all underwent
a detailed interview, which included questions to parents
from affected children (Patients PEM1-6). All participants
provided written informed consent, and approval was obtained from the ethics committees of the institutions involved. Patients PEM1, PEM2, PEM3, PEM4, PEM5, and
PEM6 were included as being affected with EM, and diagnosis was based on histological examination of muscle biopsy
samples. Parasitic infection was excluded by serology
(PEM1-3 and PEM5) and indirect immunofluorescence on
muscle sections.
Procedures
Skeletal muscle biopsies
were taken from affected muscle and analyzed by histochemistry and immunocytochemistry. Parts of each muscle biopsy
were immediately frozen in cooled isopentane and stored in
liquid nitrogen at ⫺80°C until processing. Histoenzymological studies were conducted according to protocols described
previously.8,9
HISTOPATHOLOGICAL STUDIES.
A multiplex Western blot was
performed on muscle tissue of Patient PEM1 in a systematic
screening: protein electrophoresis was performed with a Protean II vertical gel system (BioRad, Hercules, CA), as described previously.10 The following antibodies were used:
Calp3d/2C4 (anti-exon 1 and 3 of calpain-3) and Calp3c/
12A2 (anti-exon 3 and 8 of calpain-3); NCL-DYS1 and
NCL-DYS2 (anti-dystrophin); NCL-Hamlet (anti-dysferlin);
and NCL-a-SARC and NCL-g-SARC (anti–␣-sarcoglycan
and anti–␥-sarcoglycan, respectively). All antibodies were
bought from Novocastra (Newcastle upon Tyne, United
Kingdom).
WESTERN BLOT ANALYSIS.
MUTATIONAL SCREENING. Genomic DNA was extracted
from peripheral blood samples obtained from all patients.
Specifically designed primer pairs were used to polymerase
chain reaction amplify the 24 CAPN3 exons and their re-
Krahn et al: CAPN3 Mutations in Idiopathic EM
907
spective intronic boundaries. For patients PEM1 and PEM2,
DHPLC analyses were performed on a WAVE System
3500HT apparatus (Transgenomic, Courtaboeuf, France),
according to the manufacturer’s recommendations. Temperature conditions were used as determined by the Navigator
software, or whenever indicated, and modified with respect
to the peak shape. Polymerase chain reaction amplicons presenting with abnormal elution patterns were sequenced directly. For Patients PEM3, PEM4, PEM5 and PEM6, molecular analysis was performed as described elsewhere.3
Polymerase chain reaction products were sequenced on
both strands by use of a terminator procedure (PE Biosystems, Foster City, CA), and sequencing reactions were
loaded on ABI310 automated sequencing analyzer (Applied
Biosystems, Courtaboeuf, France). Obtained results were analyzed using the Sequencher software (Gene Codes Corp.,
Ann Arbor, MI) and compared with the human CAPN3
gene sequence (g.DNA #AF209502.1).
Results
We included patients with EM of undetermined cause
from six families of caucasian origin. In all families,
parents never complained of any symptoms and no
muscle defect could be suspected. Despite the absence
of consanguinity in all families (f ⫽ 0), the clinical
clues and the genealogical distribution suggested an autosomal recessive mode of inheritance in our cohort of
patients.
Age at diagnosis ranged from 3 to 11 years. Patients
presented initially with hyperCKemia (4,900 –
10,377IU/L), either isolated (Patients PEM1, PEM3,
and PEM5) or in association with slight motor clumsiness (Patient PEM2) or weakness of the lower limbs
(Patients PEM4 and PEM6). Hypereosinophilia
(⬎500/␮l) was noted on the peripheral blood count of
Patients PEM1, PEM2, and PEM5 (707–1,945/␮l).
More detailed clinical information is given in the Table. All patients underwent a muscle biopsy. Muscle
tissue samples showed inflammatory lesions with infiltrates composed mainly of eosinophils, consistent with
the diagnosis of EM (Fig 1). During a random causative screening of myopathic patients in our unit, Patient PEM1 exhibited a loss of calpain-3 bands
(namely, p94, p60, and p30; Fig 2) as evidenced by
Western blotting. This led us to include this patient
for molecular analysis of the CAPN3 gene. The other
proteins tested with antibodies against dystrophin, dysferlin, ␣, and ␥ sarcoglycan were all preserved.
For Patients PEM2, PEM3, PEM4, PEM5 and
PEM6 Western blot analysis had not been performed
initially and could not be done subsequently due to
lack of available muscle tissue. Meanwhile, these patients were also included for molecular analysis of the
CAPN3 gene based on the phenotypic similarities on
histological examination of muscle biopsy samples with
Patient PEM1. The clinical case of Patient PEM2 has
been published previously.11
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Annals of Neurology
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Molecular analysis of the CAPN3 gene showed two
mutations causing heterozygous changes in Patient
PEM1: one nonsense and one frameshift mutations in
exons 8 (c.1079G⬎A, p.Trp360X) and 16 (c.1838delA,
p.Lys613ArgfsX49), respectively (Fig 3A; see the Table).
For Patient PEM2, molecular analysis showed two sequence variations. The first variation, in intron 5 (c.8029G⬎A), was previously reported as causing LGMD2A,
and we validated this hypothesis, demonstrating a splicing defect associated to this variation (data not shown).
The second variation is a novel missense mutation in
exon 22 (c.2306G⬎C; see Fig 3B). Although different
at the complementary DNA level, it leads to a previously
reported amino-acid change (p.Arg769Pro) (see the Leiden Muscular Dystrophy pages Web site for more information: http://www.dmd.nl).
Patient PEM3 is compound heterozygote for two
missense mutations (c.302A⬎G, p.Arg101Lys and
c.664G⬎A, p.Gly222Arg; see Fig 3C). Patients PEM4,
PEM5, and PEM6 carry the c.2362_2363delins
TCATCT (p.Arg788delinsSerSerfsX11) frameshift mutation, the latter being at the homozygous state in Patients PEM4 and PEM6, whereas Patient PEM5 is
compound heterozygote with the c.664G⬎A missense
mutation in trans (see Figs 3D–F). Mutations identified in all patients are summarized in the Table.
Each of the two mutations identified in Patients
PEM1, PEM3, and PEM6 was inherited from one of
their unaffected heterozygous mothers or fathers (data
not shown). This confirms the autosomal recessive inheritance of CAPN3 mutations in EM. Samples from
parents of Patients PEM2, PEM4, and PEM5 were not
available for analysis.
Discussion
Here, we report the identification of inherited diseasecausing mutations of the CAPN3 gene in patients presenting with idiopathic EM. The initial observation of
idiopathic EM being associated with CAPN3 mutations in one patient has been subsequently confirmed
in five additional unrelated patients. All patients had a
consistent and similar phenotype of EM on histological
examination of muscle biopsy samples (see Fig 1).
Compound heterozygous or homozygous CAPN3 mutations were identified in the six unrelated patients
originally diagnosed with idiopathic EM (summarized
in the Table).
Mutations were identified in exons 1, 5, 8, 16, and
22 and in intron 5. This does not point to a specific
mutational pattern in EM; the recurrence of the
“Basque” mutation (c.2362_2363delinsTCATCT) is
most likely related to a recruitment bias.
Inflammatory features may be observed in some
muscular dystrophies, such as facioscapulohumeral
muscular dystrophy12 and dysferlinopathies.13 How-
Fig 1. Histological findings on muscle biopsy samples of the patients. (A) Patient PEM1 (hematoxylin and eosin staining): focal
inflammation with necrosis and regeneration (left); polymorphic inflammatory infiltrate mainly composed of eosinophils (middle);
focal membranary MHC-I staining (right). (B) Patient PEM3 (hematoxylin and eosin staining): mild myopathic changes with focal
inflammatory lesions with abundant eosinophilic infiltration involving necrotic fibers. (C) Patient PEM4 (transverse cryostat section): endomysial inflammation surrounding several necrotic and nonnecrotic muscular fibers, with focal perivascular distribution
(left); necrotic muscle fibers surrounded by predominant inflammatory cells consisting of lymphocytes and macrophages, with small
percentage of eosinophils (right). (D) Patient PEM5 (hematoxylin and eosin staining): focal inflammatory lesions with necrosis and
polymorphic infiltrates composed mainly of eosinophils. (E) Patient PEM6 (hematoxylin and eosin): mild myopathic changes with
irregular fiber size. Focal inflammatory infiltration with necrosis of involved fibers. The inflammatory infiltration is composed of B
lymphocytes, histiocytes, and CD15-positive granulocytes, most of them eosinophils. Findings for Patient PEM2 have been published
elsewhere.11
ever, in both cases, the inflammatory infiltrate is nonspecific; in particular, no eosinophils may be observed.
The six patients included in this study were children
who underwent muscle biopsy at an early stage of their
muscular disease. Eosinophilic infiltration thus could
be detected as an early and transient feature in calpainopathies, because it was not present in biopsies performed in older patients. Thus, histological examination of muscular tissue samples from patients suspected
to be affected with LGMD2A should include the evaluation of eosinophilic infiltration. Also, a retrospective
lecture of histological sections should be of interest for
patients diagnosed with “typical” LGMD2A, to evalu-
ate the presence of eosinophils on initial muscle biopsy
samples. Furthermore, the presence of inflammatory
features raises the question of an eventual beneficial effect of antiinflammatory therapies at early stages of the
disease.
In our study, three patients (PEM1, PEM2, and
PEM5) presented with hypereosinophilia on their peripheral blood count. Among our large series of patients affected with “typical” LGMD2A, clinical
records allowed us to retrieve hypereosinophilia in
three. However, in these patients, records of hypereosinophilia were available only during the symptomatic
stage of the disease. Whether transient hypereosino-
Krahn et al: CAPN3 Mutations in Idiopathic EM
909
Fig 2. Western blot analysis of muscle proteins in Patient
PEM1, performed during a systematic causative screening and
showing a complete absence of the calpain-3 bands p94, p60,
and p30 (arrows). C ⫽ control subject; P ⫽ Patient PEM1;
DYS ⫽ dystrophin; CAPN3 ⫽ calpain-3; ␣-SGC ⫽ ␣-sarcoglycan; ␥-SGC ⫽ ␥-sarcoglycan.
philia at early/presymptomatic stages of calpainopathy
is a more frequent feature remains unclear. This should
be better characterized by a systematic consideration of
hypereosinophilia in patients presenting with clinical
symptoms of muscular dystrophy or elevated serum
creatine phosphokinase levels, or both.
In this study’s patients, parasitic infections such as
trichinosis were excluded as being the cause of EM.
Nonetheless, it could be of interest to evaluate whether
benign parasitic infections might act as a triggering factor in genetically determined muscular dystrophy processes such as calpainopathies.
In summary, our patients presented with a triad that
appears to be indicative of CAPN3 mutations and, possibly, further development of LGMD2A: (1) EM in
the first decade, (2) elevated serum creatine phosphokinase levels (isolated or with little corresponding weakness), and (3) inconstant peripheral hypereosinophilia.
We recommend mutational analysis of the CAPN3
gene in cases of idiopathic EM. This should be of great
interest to evaluate whether EM is exclusive of specific
mutations or is an obligatory stage of every case of calpainopathy. Finding EM in cases with mutations that
will surely lead to “typical” LGMD2A does not exclude
cases in which EM will not be followed by an evolutive
course of a calpainopathy. In this regard, it must be
remembered that the mutations described in patients
with “typical” LGMD2A appear to be clustered in specific exons.14 This might be biased by the underdiagnosis of several mutations associated with a benign
phenotype (ie, hyperCPKemia, myalgia) not suggestive
of a calpainopathy.
The involvement of CAPN3 mutations in EM
should be of interest to further investigate the function
of the muscle-specific calpain-3 protein. Calpains may
play a role in Fas/Fas-associated death domain–induced
interleukin-1␣ (IL-1␣) release by cleavage of pro–IL-
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Annals of Neurology
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1␣.15 In addition, increase of IL-5 has been correlated
to local accumulation of eosinophils in EM muscle.16
Several reports have pointed out a possible role of
calpain-3 in apoptotic processes.4,5 Apoptotic myonuclei were detected in muscular biopsy specimens of
LGMD2A patients, and apoptosis was found to be correlated with altered subcellular distribution of I␬B␣
and NF-␬B, resulting in sarcoplasmic sequestration of
NF-␬B. Therefore, calpain-3 present in healthy muscle
as sarcoplasmic and nuclear forms may control I␬B␣
turnover and indirectly regulate NF-␬B–dependent expression of survival genes. More recent studies in the
calpain-3 knock-out mouse suggested a role for
calpain-3 in sarcomere formation and remodeling, by
targeting myofibrillar proteins for ubiquitination and
proteasomal degradation.17 These studies suggest that
accumulation of aged and damaged proteins can lead
to cellular toxicity and a cell stress response in
calpain-3 knock-out mouse muscles, and that these
characteristics are pathological features of LGMD2A.
Notably, muscles from these calpain-3 knock-out mice
present with inflammatory features.7 Therefore, it
should be interesting to further evaluate whether this
inflammatory histological phenotype resembles EM, by
Fig 3. Mutations in CAPN3 in Patients PEM1 (A), PEM2
(B), PEM3 (C), PEM4 (D), PEM5 (E), and PEM6 (F).
Forw. ⫽ forward sequence; Rev. ⫽ reverse sequence.
determining the proportion of eosinophils in the inflammatory infiltrates of these mice.
The presence of eosinophils in muscle in early stages
of myositis associated to CAPN3 mutations might reflect a compensatory role and coincide with the asymptomatic phase of the disease. In contrast, eosinophils
could mediate a T helper cell type 1 to type 2 immune
response and, consequently, lead to a particular inflammatory response in which apoptosis would be an essential feature of the process.
In summary, we report the first identification of mutations in the CAPN3 gene in patients with idiopathic
EM. Our results suggest that at least a subset of idiopathic EM is genetically determined, with an autosomal recessive mode of inheritance. This should allow
early genetic counseling in the families of affected patients. Furthermore, these data indicate that EM could
either correspond to a novel phenotype associated to
CAPN3 mutations or, more likely, a predystrophic evolutionary histopathological stage of LGMD2A. Supplementary patients affected with idiopathic EM are now
being further evaluated regarding the status of their
CAPN3 mutation. This will allow quantification of the
proportion of calpainopathies among this histopathological phenotype. In addition, our findings have implications for the routine diagnosis of patients with
EM of undetermined cause. Indeed, these should undergo investigations of calpain-3 expression on Western
blot analysis of muscle biopsy samples and be considered for subsequent molecular analysis of the CAPN3
gene.
This study was supported by the Assistance Publique des Hôpitaux
de Marseille, the French network for molecular exploration of neuromuscular disorders (DHOS/OPRC, 02707, N.L.), the Association
Française contre les Myopathies (AFM, A.U.), the foundation
ILUNDAIN, A.L.M. Spanish Research Grants (FIS02/1246; FIS
05/1467, A.L.M.), and a fellowship of the Department of Health of
the Basque Country (M.G.).
We are extremely grateful to the patients and their families for their
invaluable cooperation. We thank J. F. Pellissier and A. De SandreGiovannoli for helpful discussions on pathological features of EM
and critical comments and discussions on this manuscript.
References
1. Pickering MC, Walport MJ. Eosinophilic myopathic syndromes. Curr Opin Rheumatol 1998;10:504 –510.
2. Richard I, Broux O, Allamand V, et al. Mutations in the proteolytic enzyme calpain 3 cause limb-girdle muscular dystrophy
type 2A. Cell 1995;81:27– 40.
3. Saenz A, Leturcq F, Cobo AM, et al. LGMD2A: genotypephenotype correlations based on a large mutational survey on
the calpain 3 gene. Brain 2005;128:732–742.
4. Baghdiguian S, Martin M, Richard I, et al. Calpain 3 deficiency
is associated with myonuclear apoptosis and profound perturbation of the IkappaB alpha/NF-kappaB pathway in limb-girdle
muscular dystrophy type 2A. Nat Med 1999;5:503–511.
5. Baghdiguian S, Richard I, Martin M, et al. Pathophysiology of
limb girdle muscular dystrophy type 2A: hypothesis and new
insights into the IkappaBalpha/NF-kappaB survival pathway in
skeletal muscle. J Mol Med 2001;79:254 –261.
6. Haravuori H, Vihola A, Straub V, et al. Secondary calpain3
deficiency in 2q-linked muscular dystrophy: titin is the candidate gene. Neurology 2001;56:869 – 877.
7. Kramerova I, Kudryashova E, Tidball JG, Spencer MJ. Null
mutation of calpain 3 (p94) in mice causes abnormal sarcomere
formation in vivo and in vitro. Hum Mol Genet 2004;13:
1373–1388.
8. Romero NB, Marsac C, Paturneau-Jouas M, et al. Infantile familial cardiomyopathy due to mitochondrial complex I and IV
associated deficiency. Neuromuscul Disord 1993;3:31– 42.
9. Urtasun M, Saenz A, Roudaut C, et al. Limb-girdle muscular
dystrophy in Guipuzcoa (Basque Country, Spain). Brain 1998;
121(pt 9):1735–1747.
10. Anderson LV, Davison K, Moss JA, et al. Characterization of
monoclonal antibodies to calpain 3 and protein expression in
muscle from patients with limb-girdle muscular dystrophy type
2A. Am J Pathol 1998;153:1169 –1179.
11. Pena Segura JL, Adrados I, Jimenez Bustos JM, et al. [Eosinophilic myositis in a 9 year old boy]. Rev Neurol 2001;33:
960 –963.
12. Arahata K, Ishihara T, Fukunaga H, et al. Inflammatory response in facioscapulohumeral muscular dystrophy (FSHD):
immunocytochemical and genetic analyses. Muscle Nerve 1995;
2:S56 –S66.
13. Gallardo E, Rojas-Garcia R, de Luna N, et al. Inflammation in
dysferlin myopathy: immunohistochemical characterization of
13 patients. Neurology 2001;57:2136 –2138.
14. Fanin M, Fulizio L, Nascimbeni AC, et al. Molecular diagnosis
in LGMD2A: mutation analysis or protein testing? Hum Mutat
2004;24:52– 62.
15. Schaub FJ, Liles WC, Ferri N, et al. Fas and Fas-associated
death domain protein regulate monocyte chemoattractant
protein-1 expression by human smooth muscle cells through
caspase- and calpain-dependent release of interleukin-1alpha.
Circ Res 2003;93:515–522.
16. Murata K, Sugie K, Takamure M, et al. Eosinophilic major
basic protein and interleukin-5 in eosinophilic myositis. Eur
J Neurol 2003;10:35–38.
17. Kramerova I, Kudryashova E, Venkatraman G, Spencer MJ.
Calpain 3 participates in sarcomere remodeling by acting upstream of the ubiquitin-proteasome pathway. Hum Mol Genet
2005;14:2125–2134.
18. Piluso G, Politano L, Aurino S, et al. The extensive scanning of
the calpain-3 gene broadens the spectrum of LGMD2A phenotypes. J Med Genet 2005;42:686 – 693.
19. Richard I, Brenguier L, Dincer P, et al. Multiple independent
molecular etiology for limb-girdle muscular dystrophy type 2A
patients from various geographical origins. Am J Hum Genet
1997;60:1128 –1138.
20. Richard I, Roudaut C, Saenz A, et al. Calpainopathy: a survey
of mutations and polymorphisms. Am J Hum Genet 1999;64:
1524 –1540.
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