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Homozygous mutations in caveolin-3 cause a severe form of rippling muscle disease.

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Homozygous Mutations in Caveolin-3
Cause a Severe Form of Rippling
Muscle Disease
Christian Kubisch, MD,1 Benedikt G. H. Schoser, MD,2 Monika v. Düring, MD,3 Regina C. Betz, MD,1,4
Hans-Hilmar Goebel, MD,5 Susanne Zahn, MSc,1 Antje Ehrbrecht, MSc,1 Jan Aasly, MD,6
Anja Schroers, MD,7 Nikola Popovic, MSc,7 Hanns Lochmüller, MD,2 J. Michael Schröder, MD,8
Thomas Brüning, MD,9 Jean-Pierre Malin, MD,7 Britta Fricke, MD,3 Hans-Michael Meinck, MD,10
Torberg Torbergsen, MD,11 Hartmut Engels, PhD,1 Bruno Voss, PhD,9 and Matthias Vorgerd, MD7
Heterozygous missense mutations in the caveolin-3 gene (CAV3) cause different muscle disorders. Most patients with
CAV3 alterations present with rippling muscle disease (RMD) characterized by signs of increased muscle irritability
without muscle weakness. In some patients, CAV3 mutations underlie the progressive limb-girdle muscular dystrophy
type 1C (LGMD1C). Here, we report two unrelated patients with novel homozygous mutations (L86P and A92T) in
CAV3. Both presented with a more severe clinical phenotype than usually seen in RMD. Immunohistochemical and
immunoblot analyses of muscle biopsies showed a strong reduction of caveolin-3 in both homozygous RMD patients
similar to the findings in heterozygous RMD. Electron microscopy studies showed a nearly complete absence of caveolae
in the sarcolemma in all RMD patients analyzed. Additional plasma membrane irregularities (small plasmalemmal discontinuities, subsarcolemmal vacuoles, abnormal papillary projections) were more pronounced in homozygous than in
heterozygous RMD patients. A stronger activation of nitric oxide synthase was observed in both homozygous patients
compared with heterozygous RMD. Like in LGMD1C, dysferlin immunoreactivity is reduced in RMD but more pronounced in homozygous as compared with heterozygous RMD. Thus, we further extend the phenotypic variability of
muscle caveolinopathies by identification of a severe form of RMD associated with homozygous CAV3 mutations.
Ann Neurol 2003;53:512–520
Hereditary rippling muscle disease (RMD, MIM
600332) is a rare autosomal dominant disorder characterized by signs of increased muscle irritability such as
percussion/pressure-induced rapid muscle contractions
(PIRCs), electrically silent wave-like contractions (rippling muscle), and muscle mounding on percussion.
This rather benign myopathy usually is not progressive
and not accompanied by dystrophic changes. Recently,
we identified missense mutations in the caveolin-3
(CAV3) gene in families with autosomal dominant
RMD1 and in one patient with sporadic RMD.2 Mutations in CAV3 also have been described in autosomal
dominant limb-girdle muscular dystrophy type 1C
(LGMD1C),3,4 in distal myopathy,5 and in children
with elevated creatine kinase (hyperCKemia) without
neuromuscular symptoms.6
It already has been suggested that CAV3 mutations
(G55S, C71W, R125H) also might cause autosomal
recessive LGMD.7,8 However, the presence of these alterations in healthy controls and the normal caveolin-3
(Cav-3) immunostaining at the sarcolemma in patients
with the G55S alteration show that these alterations
are benign polymorphisms.8 Evidence of autosomal recessive inheritance of RMD recently has been reported
From the 1Institute of Human Genetics, University of Bonn, Bonn;
2
Friedrich-Baur-Institute, Department of Neurology, LudwigMaximilians-University, Munich; 3Department of Anatomy, RuhrUniversity Bochum, Bochum, Germany; 4Department of Medical
Genetics, University of Antwerp, Antwerp, Belgium; 5Department
of Neuropathology, University of Mainz, Mainz, Germany; 6Department of Neurology, University of Trondheim, Trondheim,
Norway; 7Department of Neurology, Ruhr-University Bochum, Bochum; 8Department of Neuropathology, University Hospital,
Aachen; 9Institute for Occupational Medicine, Ruhr-University Bochum, Bochum; 10Department of Neurology, University of Heidel-
berg, Germany; and
Tromso, Norway.
512
© 2003 Wiley-Liss, Inc.
11
Department of Neurology, University of
Received Sep 20, 2002, and in revised form Nov 20. Accepted for
publication Nov 21, 2002.
Address correspondence to Dr Vorgerd, Department of Neurology,
Ruhr-University Bochum, Buerkle-de-la-Camp-Platz 1, D-44789
Bochum, Germany. E-mail: matthias.vorgerd@ruhr-uni-bochum.de
in two families from Oman who, in addition to PIRCs
and rippling muscle waves, showed cardiac involvement, short stature, and a delayed bone age.9 This phenotype clearly differs from CAV3 mutant RMD where
symptoms are restricted to the skeletal muscle.
Caveolae are small plasma membrane invaginations
that participate in membrane trafficking, transport, and
signal transduction.10 –12 Cav-3, the muscle-specific
caveolin and major protein of muscle caveolae, forms a
complex with the dystrophin-glycoprotein complex.
Cav-3 directly interacts with neuronal nitric oxide synthase (nNOS) by negatively regulating the catalytic activity of nNOS.13,14 We recently have shown that
cytokine-stimulated NOS activity is increased in C2C12
myotubes transfected with mutant CAV3.1 In agreement with this, transgenic mice expressing the P104L
CAV3 mutant in skeletal muscle showed an increase of
nNOS activity in skeletal muscle.15 In contrast, a severe reduction of nNOS expression has been shown in
LGMD1C4 and in Duchenne muscular dystrophy.16
In this study, we identified homozygous missense
mutations in CAV3 in two unrelated patients. Both
presented with an unusually severe form of RMD. Immunostaining and immunoblot analyses showed loss of
Cav-3. Electron microscopy studies demonstrated an
almost complete absence of caveolae in skeletal muscle
of both homozygous patients similar to the findings in
heterozygous RMD. Moreover, dysferlin immunostaining, studies of NOS activity, and ultrastructural studies
of additional plasma membrane irregularities showed
more pronounced alterations in homozygous patients
compared with RMD patients with heterozygous missense mutations.
Subjects and Methods
Subjects
The study included four unrelated patients with RMD (Table). The clinical phenotypes and mutations of the Norwegian (Patient 3, see Table) and German patient (Patient 4,
see Table) with heterozygous RMD have been described earlier.1,17,18 A previously unreported patient (Patient 1, see Table) reported severe muscle stiffness in his legs since the age
of 3 years. He was adopted from Colombia at the age of 18
months and his family history is unknown. In childhood, a
delayed motor development was recognized, and elevated CK
levels suggested a muscular dystrophy. On examination at
the age of 20 years, he was athletic with generalized hypertrophic skeletal muscles, especially the shoulder, truncal, and
thigh muscles. He had a moderate flexor contracture in the
ankle joint and tiptoe walking, but no muscle weakness. He
showed generalized PIRCs. He sometimes recognized spontaneous, short-lasting rolling muscle contractions in his
thighs after heavy exercise (rippling waves), but this could
not be demonstrated on several clinical examinations. Patient
2 reported a slowly progressive muscle weakness, muscle
pain, and stiffness in his legs starting not before the age of
26 years. On examination at the age of 29 years, he showed
weakness of proximal limb muscles, neck flexors, and ab-
Table. Genotype and Clinical Phenotype of Patients with Rippling Muscle Disease
Patient No.,
Gender, Age
(yr), Origin
Clinical Phenotype
Mutation
Inheritance
Pattern
Main Symptoms
Ripplinga
PIRCb
CK (U/L)
Others
1, M, 20,
Colombian
L86P
Homozygous
Severe permanent
muscle stiffness
(⫹)
⫹
1,500–5,000
2, M, 29,
Italian
A92T
Homozygous
Muscle weakness,
pain and stiffness
⫹
⫹
1526
3, M, 20,
Norwegian
A45T
Heterozygous
⫹
⫹
919
4, F, 37,
German
A45T
Heterozygous
Muscle cramps
and intermittent stiffness
Muscle cramps
and intermittent stiffness
dmm; contractures of
Achilles tendons;
no cm, normal
height
Weakness of proximal
limb muscles MRC
grade neck flexors
MRC grade 4 and
weakness of abdominal muscles;
no cm, normal
height
no cm, normal height
(⫹)
⫹
440
no cm, normal height
a
(⫹), Present only with stretching maneuvers on examination or reported by the patient; ⫹, easily elicited on examination;
⫹, easily elicited on examination.
b
PIRC ⫽ percussion/pressure-induced rapid muscle contractions; MRC ⫽ Medical Research Council (grading system of muscle weakness
ranging from 0 ⫽ paralysis to 5 ⫽ full strength); dmm ⫽ delayed motor milestones; cm ⫽ cardiomyopathy; CK ⫽ creatine kinase (normal:
⬍80U/L).
Kubisch et al: Caveolin-3 Gene
513
dominal muscles. In addition, he could demonstrate rippling
in his quadriceps muscle and had generalized PIRCs. His
parents came from Sicily, Southern Italy, and both were reported to be asymptomatic. They were not available for clinical examination nor for mutational analysis.
Mutational and Linkage Analysis
Genomic DNA was extracted from peripheral blood lymphocytes by a standard protocol and the two coding exons of
CAV3 were amplified and directly sequenced as previously
described.1 Alternative primers for exon 2 amplification were
CAV3 Ex2F2: 5⬘-ctt ctg tga gtt gag gct tcc-3⬘; CAV3 Ex2R2:
5⬘-atc atg ggg tat gga gca gtc-3⬘; CAV3 Ex2F3: 5⬘-agg tta
acc tga cct cta ggg-3⬘; CAV3 Ex2R3: 5⬘-cat tgt gct tct gtg
gct gg-3⬘. Genotyping was performed with highly polymorphic fluorescence marked microsatellite markers. The order
of markers was derived from the UCSC Genome Browser
(April 2002 freeze; http://genome.ucsc.edu/) and Ensembl
(http://www.ensembl.org/). Genotyping was done on an ABI
3100 automated sequencer, and alleles were scored manually
using genotyper.
Fluorescence In Situ Hybridization
The caveolin-3–containing BAC RP11-128A5 (GenBank accession number AC068312, size 170,348bp) was hybridized
simultaneously with the hybridization control PAC 196F4
(subtelomeric 3q29). BAC and PAC DNA was isolated according to a standard protocol. Probe DNA was digested
with DNAse I (Roche, Mannheim, Germany). DNA fragments were purified using QIAquick PCR purification kits
(Qiagen, Chatsworth, CA). Chemical labeling of the probes
was performed with the Universal Linkage System (ULS;
Kreatech Diagnostics, Amsterdam, The Netherlands). BAC
RP11-128A5 DNA was labeled with dGreen-ULS and PAC
196F4 with Cy3-ULS. The labeling reactions were purified
using QIAquick PCR purification kits and coprecipitated
with ethanol in the presence of ⫻25 excess human c0t1
DNA (Gibco-BRL, Karlsruhe, Germany). The probe mixture
was dissolved in 60% deionized formamide, 2 ⫻ standard
saline citrate, 50mM sodium phosphate, pH 7.0, 10% dextran sulphate. Fluorescence in situ hybridization (FISH) was
performed as described by Tanke and colleagues19 with minor modifications. The chromosomes were counterstained by
incubation in a 4,6-diamidino-2-phenylindole-2 HCl solution (10ng/ml). After dehydration in an ethanol series, the
slides were embedded in antifade solution (Citifluor AF1;
Agar Scientific, Stansted, UK). Imaging and analysis were
conducted on an Axioplan 2 imaging fluorescence microscope (Zeiss, Jena, Germany) equipped with a Sensys
charged-coupled device camera (Photometrics, Tucson, AZ)
and a Cytovision workstation (Applied Imaging, Newcastle
upon Tyne, UK).
Immunohistochemistry and Western Blotting
Skeletal muscle sections were prepared from control tissue
and RMD Patients 1, 2, and 3 (see Table) according to standard techniques. A panel of proteins was examined immunohistochemically, including caveolin-1, -2, and -3 (Trans-
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duction Laboratories, Lexington, KY), ␣-dystroglycan
(Upstate Biotechnology, Lake Placid, NY), ␤-dystroglycan,
dystrophin, dysferlin, laminin ␣2 (Novocastra, Newcastle
upon Tyne, UK), and nNOS (Sigma, St. Louis, MO). Proteins from muscle homogenates of RMD Patients 1, 2 and 3
(see Table) were subjected to electrophoresis and Western
blotting. Equal amounts of total protein were separated by
electrophoresis through a 7% (for dysferlin) or 12% (for
Cav-3) polyacrylamide gel and transferred to nitrocellulose
membranes. Blots were incubated with monoclonal antibodies to dysferlin (dilution 1:80; Novocastra) or Cav-3 (dilution 1:5,000; Transduction Laboratories), followed by a
horseradish peroxidase–labeled secondary antibody to mouse
IgG (dilution 1:1,250; DAKO, Glostrup, Denmark). Blots
were developed using the ECL detection system (Amersham
Pharmacia Biotech, Uppsala, Sweden).
NADPH Diaphorase (NDP) Activity Assay
Ten-micrometer frozen muscle sections of RMD Patients 1,
2 and 3 were fixed in 2% paraformaldehyde in phosphatebuffered saline, pH 7.4, at 2 hours. After rinsing in
phosphate-buffered saline, the sections were incubated in
0.2% Triton X-100 for 10 minutes at 37°C. The subsequent
reaction was performed for 1 hour in a dark, humidified
chamber at 37°C in 0.2% Triton X-100, 0.1mM NADPH,
and 0.16mg/ml nitro blue tetrazolium solution. The reaction
was terminated by washing with phosphate-buffered saline.
Electron Microscopy
Skeletal muscle samples of RMD Patients 1 to 4 and of controls were fixed in 2.5% glutaraldehyde and 1% paraformaldehyde in 0.1M phosphate buffer. Tissue samples were postfixed with 2% OsO4, dehydrated in graded ethanol, and
embedded in Araldite. Alternating series of semithin and ultrathin (gold/silver) sections were cut on a Reichert-Jung Ultracut E. Ultrathin sections were stained with uranyl acetate
followed by lead citrate and analyzed in a Philips 400 or a
Philips 420 transmission electron microscope, operating at
80kV.
Results
Novel Homozygous CAV3 Missense Mutations
in Patients with a Severe Form of Rippling
Muscle Disease
Patients 1 and 2 met the diagnostic criteria for RMD
(see Table), which prompted us to search for CAV3
mutations. Moreover, Patient 1 had severe muscle stiffness since early childhood and contractures of the
Achilles tendons leading to a gait disturbance. Patient
2 had slowly progressive muscle weakness beginning in
early adulthood. In both patients, symptoms were restricted to skeletal muscles, and there was no evidence
of a pathological involvement of the heart and bone.
Direct sequencing of the two coding exons showed ap-
Fig 1. (A) Mutations identified in exon 2 of CAV3 in two patients with rippling muscle disease (RMD). Direct sequencing showed
Patient 1 carrying a homozygous T3 C base transition at nucleotide position 215 of CAV3 (top chromatograms) and Patient 2
carrying a homozygous G3 A transition at nucleotide 232 of CAV3 (bottom chromatograms). Underlined sequences highlight the
wild-type codon in the normal control as well as the homozygous mutation in the patients. The corresponding amino acid change is
shown above the codon. (B) Microsatellite analyses for the CAV3 locus in both homozygous RMD patients. In the first column, the
marker and gene names are given in telomeric-centromeric order; the second column shows the physical position of the microsatellites
and the CAV3 gene according to the UCSC Genome Browser (April 2002 freeze). The right two columns show the marker alleles
for both patients. (C) Results of FISH analysis in Patients 1 (left) and 2 (right). Green hybridization signals of BAC RP11-128A5
(harboring the CAV3 gene) in band 3p25 of both chromosomes 3 demonstrate that there is no microdeletion of this region. Control
signals in 3q29 are shown in red.
parently homozygous missense mutations within exon
2 of CAV3. In Patient 1, the mutation was a T3 C
transition at nucleotide position 215 (L86P; Fig 1A). A
homozygous G3 A transition at nucleotide position
232 within codon 92 was found in Patient 2 (A92T;
see Fig 1A). The mutations were not found in 120
German control chromosomes. These novel mutations
were confirmed with two additional nonoverlapping
primer pairs (not shown). Both alterations are located
in the membrane-associated domain of Cav-3 and af-
Kubisch et al: Caveolin-3 Gene
515
fect amino acids that are conserved in human, rat, and
mouse.
Because it was not possible to obtain DNA samples
of further family members, we could not directly prove
whether the patients are indeed homozygous for these
alterations. A possible alternative would be compound
heterozygosity for the missense mutations and a larger
deletion on the other allele, which because of insufficiency of additional genes may be responsible for the
more pronounced phenotype. To investigate this possibility, we performed microsatellite analyses and FISH
on human chromosome 3p25. In Patient 1, we demonstrated homozygosity for a region between D3S1515
and D3S1286 spanning at least 9MB of genomic sequence (see Fig 1B). FISH analysis with the 170kb
spanning BAC clone harboring the CAV3 gene showed
two signals on 3p25 (see Fig 1C). This finding excludes a larger deletion and together with the microsatellite data supports that the identified missense mutation is homozygous by descent. In contrast, in
Patient 2 we did not find homozygosity for the microsatellites. The known surrounding markers, that is,
D3S1489 (approximately 160kb telomeric of CAV3)
and D3S3691 (approximately 52kb centromeric), were
heterozygous (see Fig 1B). FISH analysis showed two
equally intense signals on 3p25 excluding a larger deletion in Patient 2 (see Fig 1C), although we cannot
definitely rule out a smaller deletion harboring CAV3
or a part of it on one allele. Typing of three novel
polymorphic microsatellite markers 7kb telomeric,
within intron 1, and 5kb centromeric of CAV3 showed
homozygosity in Patient 2 and heterozygosity in a control (not shown). Although there is no overt consanguinity of the parents in Patient 2, both come from a
small town in Sicily, making it likely that the observed
missense mutation is indeed homozygous and identical
by descent.
By Immunohistochemistry, Cav-3 Is Absent and
Dysferlin Reduced in the Sarcolemma in Rippling
Muscle Disease
Muscle biopsies of Patients 1, 2, and 3 showed variation in fiber size with marked muscle fiber hypertrophy
Fig 2. (A) Immunofluorescence analysis of caveolin-3 (Cav-3) and dysferlin in muscle biopsies of patients with homozygous and heterozygous rippling muscle disease (RMD). In all patients there are few fibers with abnormal patchy plasma membrane staining for
Cav-3 and diminished but not absent dysferlin staining in the sarcolemma. (B) Immunoblot analysis of muscle biopsies from the
homozygous and heterozygous RMD patients. Samples containing identical amounts of protein were subjected to immunoblotting
with anti–Cav-3 and anti–dysferlin antibodies. (lane 1) Normal control; (lanes 2 and 3) homozygous RMD patients; (lane 4) heterozygous RMD patient. In all RMD patients dysferlin was normal, whereas Cav-3 was completely absent.
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and atrophic muscle fibers and an increase in central
nuclei. These findings were more pronounced in homozygous patients. Signs of degeneration, for example,
necrosis and connective tissue proliferation, were not
observed. Skeletal muscle fibers of all RMD patients
showed an almost complete loss of Cav-3 expression in
the sarcolemma (Fig 2A). There was no upregulation
of Cav-1 and Cav-2 (not shown). All RMD patients
showed a complete loss of Cav-3 by immunoblot analysis (see Fig 2B). Dysferlin immunoreactivity was reduced in the sarcolemma in most fibers of the homozygous RMD patients. We also found a slight reduction
of dysferlin in the sarcolemma in the heterozygous
RMD patient that was less pronounced than in the homozygous RMD patients (see Fig 2A). In immunoblot
analysis, all RMD patients showed normal expression
of dysferlin (see Fig 2B). All other membrane proteins
tested by immunohistochemistry were unchanged, indicating a preserved dystrophin–glycoprotein complex
(not shown).
NADPH Diaphorase Activity Is Increased in Rippling
Muscle Disease
Immunofluorescence analysis showed normal nNOS
immunoreactivity in all RMD patients (Fig 3). Like
nNOS, NDP activity in skeletal muscle fibers also was
concentrated in the sarcolemma and colocalized with
nNOS.20 In the NDP assay, almost all muscle fibers of
the homozygous RMD patients showed a more intense
signal in the sarcolemma. Moreover, in both homozygous patients we detected a reticular NDP-positive
staining within the sarcoplasm. In the heterozygous
RMD patient, the level of NDP staining in large muscle fibers was between those levels of homozygous
RMD and controls, and there was no sarcoplasmic
staining (see Fig 3).
By Electron Microscopy, Caveolae Are Numerically
Reduced in Rippling Muscle Disease
In controls, skeletal muscle fibers exhibited irregularly
distributed caveolae in the sarcolemma (Fig 4). Mutations in CAV3 resulted in an almost complete absence
of caveolae in skeletal muscle fibers of all RMD patients. All RMD patients displayed foci of electron
dense filamentous material irregularly distributed and
located closely attached to the cytoplasmic face of the
sarcolemma. In addition, all RMD patients showed
plasma membrane irregularities. First, there were small
membrane irregularities over which the basal lamina
was thickened. Second, the surface area was increased
because of irregular folds and ridges or finger-like processes. Third, invaginations of the sarcolemma with either flask-shaped or tubular profiles measuring from
230 to 1,000nm were present (see Fig 4). These
plasma membrane irregularities and subsarcolemmal
vacuoles appeared more pronounced in homozygous
than in heterozygous RMD patients.
Discussion
This study for the first time identified pathogenic homozygous missense mutations in CAV3 in two unrelated patients presenting with an unusually severe form
of RMD. In the homozygous patients, the clinical diagnosis of RMD was based on signs of increased muscle irritability. Yet, both patients presented additional
symptoms that are not characteristic for autosomal
dominant RMD, which suggested a distinct genetic ba-
Fig 3. Immunofluorescence analysis of neuronal nitric oxide synthase and NDP assay in muscle biopsies of the homozygous and heterozygous rippling muscle disease (RMD) patients. Muscle tissue from all RMD patients showed a normal sarcolemmal staining of
nNOS (top). NDP assay showed an increased sarcolemmal activity and a reticular sarcoplasmic staining in almost all myofibers of
the homozygous RMD patients. In the heterozygous RMD patient, there was a similar but less intense NDP-positive staining in
most of the large muscle fibers (bottom).
Kubisch et al: Caveolin-3 Gene
517
Fig 4. Electron microscopy analysis of skeletal muscle fibers from normal control and RMD patients. In normal controls, caveolae
appear as 50 to 80nm invaginations of the sarcolemma (arrows). In all rippling muscle disease (RMD) patients (Patients 1– 4),
CAV3 mutations lead to a loss of caveolae at the sarcolemma, whereas Cav-1 induced caveolae in endothelial cells of the capillaries
are present (black arrowheads in Pat. 4 panel). In all four RMD patients, irregular foci of filamentous material attached to the
cytoplasmic face of the sarcolemma are present (white arrowheads). Note the subsarcolemmal vacuoles (asterisks) in Patients 1, 2,
and 3. Pat. 1, ⫻46,000; Pat. 2, ⫻32,000; control; Pat. 3 and 4, ⫻60,000.
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sis. Indeed, we were able to identify apparently homozygous missense mutations in CAV3, which reflect
the more pronounced phenotype. Patient 1 has been
adopted in early infancy; therefore, no information
about his family is available. Concerning Patient 2, we
had to rely on his personal information, which does
not show any neuromuscular symptoms in his parents
or siblings. We therefore are not able to distinguish
whether this severe form of RMD is autosomal recessive RMD or whether it is the more severe presentation
of autosomal dominant RMD in homozygous patients.
In any way, the RMD patients with homozygous
CAV3 mutations are different from those with a recently described autosomal recessive form of RMD
with additional cardiac and skeletal symptoms,9 which
are not present in our patients. In this autosomal recessive form of RMD, no mutational analysis of the
CAV3 gene has been published. However, the multisystemic involvement suggests a different genetic origin.
We also asked whether the unique clinical and genetic findings are reflected by morphological and/or
functional differences. The strong reduction of Cav-3
in immunostaining and immunoblotting was similar in
homozygous and heterozygous RMD patients. These
results are consistent with electron microscopy findings
of nearly complete absence of caveolae in all RMD patients. This is in agreement with previous observations
that in Cav-3 knockout mice, and in patients with
LGMD1C caveolae are nearly absent at the sarcolemma.21–23 Therefore, pathogenic CAV3 mutations seem
to cause a reduction of the Cav-3 protein and of caveolae irrespective of the degree of neuromuscular symptoms.
In addition, we detected a reduction of dysferlin by
immunostaining more pronounced in the homozygous
RMD patients. It was shown that cav-3 coimmunoprecipitates with dysferlin and that in LGMD1C caused
by CAV3 mutations (R26Q, T63P) dysferlin was severely reduced in the sarcolemma.24 Our data are consistent with those findings and further indicate that
CAV3 mutations can affect anchoring of dysferlin to
the sarcolemma. Quantitative electron microscopy of
nonnecrotic muscle fibers from LGMD2B patients
with primary deficiency of dysferlin have shown plasmalemmal defects, sometimes covered by thickened
basal lamina, papillary projections of fibers, and small
subsarcolemmal vacuoles.25 Interestingly, the plasma
membrane irregularities of RMD patients we observed
are very similar and more pronounced in homozygous
than heterozygous subjects. Thus, the secondary alteration of dysferlin may be of functional significance and
may contribute to the plasma membrane defects in
RMD.
We demonstrated a normal expression of nNOS at
the sarcolemma in all RMD patients. This is in agreement with recent data from Cav-3 knockout mice,11
P104L transgenic mice,15 and previous studies in various other RMD muscle biopsies.1,2 Moreover, an increased NDP activity in RMD muscle was found that
was more pronounced in homozygous RMD patients.
In contrast, in LGMD1C and Duchenne muscular
dystrophy a significant reduction of nNOS expression
has been described.4,16 Interestingly, in mdx mice that
expressed normal levels of NO in muscle by an nNOS
transgene, the muscular dystrophy was ameliorated.26
Therefore, it can be speculated that normal nNOS expression and increased NO production may be functionally important in modifying the phenotype of
caveolinopathies.
All patients with autosomal dominant RMD and
CAV3 mutations showed signs of increased muscle irritability, and the clinical course is usually benign.
Both homozygous patients reported here were clinically
more severely affected than the hitherto described heterozygous RMD patients.17,18,27 This severe form of
RMD overlaps with the clinical symptoms of slowly
progressive weakness in LGMD1C.3,4 We suggest that
caveolinopathies present as a clinical continuum where
signs of increased muscle irritability and muscle weakness are the main findings. These symptoms may either
occur alone or appear simultaneously, possibly depending on the type of CAV3 mutation and modifying factors such as dysferlin and nNOS activity.
This work was supported by the Deutsche Forschungsgemeinschaft,
BONFOR, and the WiMed Bergmannsheil, Bochum.
R.C.B. holds a postdoctoral position at the Flemish Fund for Scientific Research (FWO-Vlaanderen).
We thank the patients for participation. We are very grateful to S.
Galuschka, S. Böhm, L. Augustinowski, K. Knippschild, I. Goebel,
A. Stiller, M. Bousfia, G. Reifenberg, M. Schlie, and I. Warlo for
excellent technical assistance. We also thank Dr K. Ricker for critical reading of the manuscript.
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