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Unformatted text preview: © 1999 Oxford University Press Human Molecular Genetics, 1999, Vol. 8, No. 5 871–877 Identical mutation in patients with limb girdle
muscular dystrophy type 2B or Miyoshi myopathy
suggests a role for modifier gene(s)
Tracey Weiler1, Rumaisa Bashir3, Louise V. B. Anderson4, Keith Davison4,
Jennifer A. Moss4, Stephen Britton3, Edward Nylen1, Sharon Keers3,
Elizabeth Vafiadaki3, Cheryl R. Greenberg2, Kate M. D. Bushby3 and
1Department of Biochemistry and Molecular Biology, and 2Departments of Pediatrics and Child Health and
Human Genetics, University of Manitoba, 770 Bannatyne Avenue, Winnipeg, Manitoba R3E 0W3, Canada,
3School of Biochemistry and Genetics, University of Newcastle upon Tyne, Newcastle upon Tyne NE1 7RU, UK
and 4Neurobiology Department, University of Newcastle upon Tyne, Newcastle upon Tyne NE2 4HH, UK
Received December 21, 1998; Revised and Accepted February 28, 1999 DDBJ/EMBL/GenBank accession nos AF075575, AF137333 and AF3600028 ABSTRACT INTRODUCTION Limb girdle muscular dystrophy type 2B (LGMD2B)
and Miyoshi myopathy (MM), a distal muscular dystrophy, are both caused by mutations in the recently
cloned gene dysferlin, gene symbol DYSF. Two large
pedigrees have been described which have both types
of patient in the same families. Moreover, in both
pedigrees LGMD2B and MM patients are homozygous
for haplotypes of the critical region. This suggested
that the same mutation in the same gene would lead to
both LGMD2B or MM in these families and that
additional factors were needed to explain the development of the different clinical phenotypes. In the present
paper we show that in one of these families Pro791 of
dysferlin is changed to an Arg residue. Both the
LGMD2B and MM patients in this kindred are homozygous for this mutation, as are four additional patients
from two previously unpublished families. Haplotype
analyses suggest a common origin of the mutation in
all the patients. On western blots of muscle, LGMD2B
and MM patients show a similar abundance in dysferlin
staining of 15 and 11%, respectively. Normal tissue
sections show that dysferlin localizes to the sarcolemma while tissue sections from MM and LGMD patients
show minimal staining which is indistinguishable
between the two types. These findings emphasize the
role for the dysferlin gene as being responsible for
both LGMD2B and MM, but that the distinction between
these two clinical phenotypes requires the identification of additional factor(s), such as modifier gene(s). The limb girdle muscular dystrophies (LGMDs) are a heterogenous group of muscle disorders characterized by predominant
weakness and wasting of muscles of the pelvic and shoulder
girdle. There is broad clinical heterogeneity and this is parallelled
by genetic heterogeneity. Both autosomal dominant and recessive
forms occur and to date there is evidence for at least 12 different
loci (1,2). The second form of autosomal recessive LGMD
identified was mapped to chromosome 2p13 and named limb
girdle muscular dystrophy type 2B (LGMD2B) (3). Another
myopathy affecting initially distal muscles, especially the gastrocnemius, was also mapped to 2p13 (4) and is known as
Miyoshi myopathy (MM) (5). The similar map positions of these
two myopathies raised the possibility that they might be allelic
variants of the same gene (4). Support for the idea that both
diseases were caused by the same gene came from the description
of two large families in which both clinical phenotypes occurred
in each kindred (6,7). Moreover, both types of patient were
homozygous for one haplotype in each family, and it was
postulated that the diseases were not only caused by the same
gene but also the same mutation (6). If correct, the different
phenotypes had to be the result of additional factors. Recently, a
single gene responsible for both LGMD2B and MM was cloned.
It codes for a 237 kDa protein with homology to the Caenorhabditis elegans spermatogenesis factor fer-1, a protein believed
to have a role in membrane fusion (8). The gene responsible for
these dystrophies was therefore named dysferlin (DYSF) (9,10).
Identification of the gene for LGMD2B/MM allowed us to test
the hypothesis that the same mutation in DYSF can cause both
LGMD2B and MM. With the development of an antibody to
dysferlin (11), it was of particular interest to see whether
mutations leading to LGMD2B and/or MM could be distinguished on the basis of dysferlin protein expression in skeletal
muscle. Here we report that, in the large Canadian aboriginal *To whom correspondence should be addressed. Tel: +1 204 789 3701; Fax: +1 204 789 3900; email@example.com 872 Human Molecular Genetics, 1999, Vol. 8, No. 5 Figure 1. Segregation of the DYSF mutation and core haplotypes in three aboriginal kindreds. (A) Large kindred with both LGMD2B and MM patients. (B) Two
small aboriginal families, apparently unrelated to the large pedigree, with four LGMD patients. Patients 1 and 2 have MM (hatched symbols), patients 3–9 and 11–14
have LGMD (black symbols) and patient 10 is pre-clinical (checked symbol). +/+ indicates that the individual does not have the C2745G mutation in DYSF, +/–
indicates that the individual is heterozygous for the mutation and –/– indicates that the individual is homozygous for the C2745G mutation. Cy172-H32, intragenic
marker 5′ of the mutation (9); 104-sat, intragenic marker 3′ of the mutation (10). Numbers of alleles indicate their sizes in bp. kindred with both LGMD2B and MM patients, all affected
individuals are homozygous for one missense mutation, which
results in similar reductions of dysferlin expression in both types
of patient. As a result, the development of the different
phenotypes, at least in the kindred described here, cannot be
explained on the basis of allelic diversity, but require additional
factor(s), such as modifier gene(s). Observation of the same
mutation in patients of two different aboriginal families surrounded by the same core haplotype suggests that this mutation
had a common origin.
Mutation detection in Canadian aboriginal pedigrees
The aboriginal kindreds under study have 14 affected individuals
including 13 symptomatic patients and one preclinical patient
(Fig. 1). Eleven patients are following a course of a fairly rapidly
progressive LGMD with onset in the teens and wheelchair
requirement by the third decade of life. Two other patients show
a milder distal myopathy compatible with MM. All patients have
grossly elevated serum creatine kinase (CK) levels (6). The
preclinical patient (12 years of age) has asymmetric hypertrophy
of the left calf and grossly elevated CK levels, but only very mild
proximal muscle weakness.
Patients 8 and 9 (Fig. 1A) were selected for mutation analysis
because patient 8 is one of three patients who were reported to be
heterozygous for the haplotype of the candidate region and
therefore expected to be heterozygous for the disease-causing
mutation, and patient 9 was reported to be homozygous for the
common haplotype spanning a >4 cM region between D2S291
and D2S286 (6). A search for mutations in DYSF was performed on amplified exons using single strand conformation polymorphism (SSCP) and heteroduplex analyses. Despite the haplotype
findings in these two patients, both were found to be homozygous
for a C→G transversion found in both patients at position 2745
of DYSF (Fig. 2). This mutation changes the proline at position
791 to arginine and abolishes an HpaII site, thus allowing for easy
mutation detection (Fig. 2B) in the entire pedigree. Pro791 is a
conserved residue between dysferlin and fer-1, the C.elegans
protein mentioned above (8) (alignment not shown).
The mutation segregates in an autosomal recessive fashion in
the entire pedigree (Fig. 1A). It was not seen in 100 unrelated
control chromosomes nor in other dysferlin-associated muscular
dystrophies studied to date. The same mutation was found in four
other LGMD patients from two apparently unrelated families
from another remote inbred aboriginal community (Fig. 1B). The
diagnosis of dysferlin-associated muscular dystrophy has therefore been confirmed in these families as well.
Haplotype analyses were performed on the patients and their family
members with four microsatellites flanking the dysferlin gene, two
DYSF intragenic markers and the DYSF C2745G mutation (Fig.
1A). As expected, patients 1–5, 9 and 10 are homozygous for the
same D2S292–D2S443–Cy172-H32–DYSF C2745G–104-sat–
D2S291–D2S2110 haplotype [184-247-199-(–)-156-184-139]. Patients 6–8 are heterozygous for D2S292, D2S443 and D2S2110.
However, these patients are homozygous for the Cy172-H32–DYSF
C2745G–104-sat–D2S291 core-haplotype [199-(–)-156-184]. The
four patients (nos 11–14) from the other Canadian aboriginal
kindreds are also homozygous for D2S292–D2S443–
Cy172-H32–DYSF C2745G–104-sat–D2S291–D2S2110 haplotype 873
Human Molecular Genetics, 1999, Vol.No.No. 5
Nucleic Acids Research, 1994, Vol. 22, 8, 1 873 DISCUSSION Figure 2. DYSF mutation detection. (A) Protein schematic with location of
exon (hatched) containing the C2745G mutation (I), peptide used for generating
the antibody NCL-hamlet (solid black) and membrane spanning and retention
domain (checked). (B) Sequence of exon harbouring the C2745G mutation
(grey box) and location of HpaII sites (underlined and italic). (C) Detection of
the DYSF mutation (C2745G). A 292 bp fragment containing the exon
illustrated in (B), amplified from intronic primers and restricted with HpaII.
Lane 1, pGEM DNA marker (Promega, Madison, WI); lanes 2 and 3, DNA
from an unaffected individual that does not carry the mutation (paternal
grandfather of patient 2, Fig. 1A); lanes 4 and 5, DNA from a carrier (first
sibling of patient 2, Fig. 1A); lanes 6 and 7, DNA from an LGMD patient (no.
8, Fig. 1A). DNA in lanes 2, 4 and 6 is unrestricted; DNA in lanes 3, 5 and 7
is restricted with HpaII. [186-243-199-(–)-158-184-139]. However, the alleles of markers
D2S292, D2S443 and 104-sat differ from the common diseasecarrying chromosome (Fig. 1B).
Dysferlin protein expression in LGMD2B and MM
A monoclonal antibody was generated to a peptide close to the
C-terminal end of dysferlin. The peptide corresponds to amino
acids 1999–2016 of the dysferlin protein which comprises 2080
amino acids (Fig. 2A). Details of dysferlin characterization with
this antibody are described in the accompanying paper by
Anderson et al. (11). On tissue sections of control muscle,
dysferlin localizes to the sarcolemma (Fig. 3A). The pattern is
very similar to that seen with dystrophin staining (Fig. 3B). The
preclinical patient (no. 10, Fig. 1A) with this mutation shows
patchy sarcolemmal staining in some fibres (Fig. 3C). Dystrophin
staining of the same patient shows a normal pattern at the
sarcolemma (Fig. 3D). In addition, muscle sections from one MM
patient (no. 1, Fig. 1A) and one LGMD2B patient (no. 12, Fig.
1B), both homozygous for the Pro791Arg mutation, were stained
for dysferlin. They show weak and variable dysferlin staining on
a small proportion of fibres (Fig. 3E and F).
On western blots, control muscle shows the expected band at
230 kDa for dysferlin (Fig. 4). The band is reduced to ∼33% of
normal control muscle in the preclinical patient (Fig. 4, lane 2),
11% in the MM patient (Fig. 4, lane 3) and 15% in the LGMD2B
patient (Fig. 4, lane 4). Essentially, the expression levels of
dysferlin protein in tissue sections and on western blots are
indistinguishable between the patient with LGMD2B and the one
with MM. The recent cloning of the LGMD2B/MM gene, DYSF, and the
current work have proven that both LGMD2B and MM in this
large Canadian aboriginal pedigree are caused by the same gene
(9,10) and by the same mutation in this gene.
There is strong supporting evidence that the Pro791Arg
mutation is disease-causing in the patients studied here. This
includes evidence that: (i) the mutation segregates correctly for an
autosomal recessive disease in this large kindred and the two
other small families; (ii) the mutation has not been seen on 100
control chromosomes; (iii) MM and LGMD2B patients homozygous for the mutation show an identical reduction of dysferlin
protein; (iv) the Pro→Arg change should have dramatic effects on
the conformation of the protein; and (v) this Pro residue is
conserved between the C.elegans protein fer-1 and dysferlin.
Surprisingly, patients 6–8 (Fig. 1A), who had been reported to
be heterozygous for the critical haplotype and, therefore, were
expected to be compound heterozygotes (6), are also homozygous for the Pro791Arg mutation. This points to a common
ancestry in all the patients of the pedigree and to ancient
recombination events closely flanking the chromosomal segment
around the disease gene in the mother of patients 6–8. This
interpretation is supported further by the haplotype analysis using
microsatellites within and very closely flanking the dysferlin
gene, which indicate that patients 6–8 are homozygous for the
same core haplotype as all other patients in this family (Fig. 1A).
The presence of four patients (nos 11–14, Fig. 1B) in an
apparently unrelated aboriginal community homozygous for the
same Cy172-H32–DYSF C2745G–104-sat–D2S2110 haplotype
suggests that there may be common ancestry between these two
communities as well. The 158 bp allele of the intragenic marker
104-sat in these patients differs from the disease-associated allele
of the large family at that locus by 2 bp. As the alleles flanking
this marker are identical to those on the disease-associated
haplotype, it is most likely that a mutation of the 104-sat
microsatellite had occurred.
There appears to be a slight discrepancy between the very
minimal staining of dysferlin at the membrane in tissue sections
of patients and the clearly visible 230 kDa band on western blots
that represents between 11 and 15% of the staining intensity of
control muscle. While this may in part just reflect the aggregate
staining of all dysferlin molecules in one band, it may also be an
indication of a possible misfolding of the protein that may
interfere with correct integration into the membrane. Whatever
the reason, it is clear from this study and the accompanying paper
that the antibody to dysferlin will offer a marked enrichment of
the tools for the very difficult field of LGMD diagnostics (12,13).
The results presented here also point out that it appears unlikely
that one will be able to distinguish LGMD2B and MM on the
basis of dysferlin protein expression. This does not rule out the
possibility that a large series of biopsies from different muscles
of LGMD2B and MM patients might still show differences
between MM and LGMD2B. However, such a study is clearly not
The higher level of dysferlin detected in the preclinical patient
(33%; Fig. 4, lane 2) appears to agree with the lack of symptoms
at this stage of the disease. Given the limited number of biopsies
studied, it is unknown whether these levels will continue to
decline as the disease develops or whether such different dysferlin
levels could represent variations one may encounter even in 874 Human Molecular Genetics, 1999, Vol. 8, No. 5 Figure 3. Immunocytochemical labelling of normal and dystrophic human skeletal muscle sections. (A) Normal control section labelled with NCL-hamlet to dysferlin;
(B) normal control labelled with 1/10 Dy8/6C5 to dystrophin; (C) preclinical patient (no. 10, Fig. 1A) labelled with NCL-hamlet; (D) same preclinical patient labelled
with 1/10 Dy8/6C5 to dystrophin; (E) patient with MM (no. 1, Fig. 1A) labelled with NCL-hamlet; (F) LGMD2B patient (no. 12, Fig. 1B) labelled with NCL-hamlet.
All patients are homozygous for the Pro791Arg mutation. patients with the same mutation. Given the large size of the
dysferlin gene (>150 kb with 55 exons), we cannot rule out that
additional mutations in DYSF may cause the variation in
expression, nor can we rule out, for that matter, that such second
mutations could determine the LGMD2B or MM phenotype.
LGMDs characteristically show strong clinical heterogeneity
(1,14). Contributing to this heterogeneity are 12 different genes
and their many different mutations (1,2). Furthermore, even in
families with identical mutations of the same gene, clinical
heterogeneity has been observed. This is generally typified by
varying degrees of severity (15,16). In the case of LGMD2B and
MM, the different clinical phenotypes led originally to the
designation of different disease entities, which are characterized
by the primary involvement of proximal or distal muscle groups,
respectively. Mapping of these two diseases to the same
chromosomal region suggested that they may be allelic (4,7),
similar to Duchenne and Becker muscular dystrophies (17). The difference, however, between LGMD2B and MM is not just one
of severity as is generally seen in the other examples described for
allelic diversity, but in the primary muscle group involvement.
The fact that an identical mutation has been described in patients
who vary with respect to severity and initial muscle involvement
clearly points out that additional factors, such as modifier gene(s),
must be involved in modifying the clinical phenotype. Modifier
genes or multiple genes have been postulated previously for
LGMDs (6,9,15,16,18,19). Given that the principal difference in
the clinical phenotype between MM and LGMD2B patients is in
the type of muscles involved initially, finding a modifier gene
might reveal the mechanism for the muscle involvements,
characteristic for specific types of muscular dystrophy. The
clear-cut differences in clinical phenotype of the LGMD2B and
MM patients in the pedigree described here and in a second,
similarly large, family reported from Russia (7) may hold the clue
for identifying such modifier gene(s). 875
Human Molecular Genetics, 1999, Vol.No.No. 5
Nucleic Acids Research, 1994, Vol. 22, 8, 1 875 Mutation detection Figure 4. Western blot of human skeletal muscle biopsies. Lane 1, normal
control; lane 2, preclinical patient (no. 10, Fig. 1A); lane 3, MM patient (no. 1,
Fig. 1A); lane 4, LGMD2B patient (no. 12, Fig. 1B); lane 5, normal control
muscle labelled with different antibodies to generate molecular size markers
[1/10 Dy8/6C5 to dystrophin at 400 kDa, 1/300 NCL-hamlet to dysferlin at
230 kDa, 1/10 Calp3d/2C4 to calpain 3 at 94 and 30 kDa (20) and 1/2
Ad1/20A6 to α-sarcoglycan at 50 kDa]. Lanes 1–4 were labelled with 1/300
anti-dysferlin antibody, NCL-hamlet. The lower panel shows the corresponding
myosin heavy chain (MHC) bands from the Coomassie blue stained gel. The
outline of this band can be seen at 200 kDa in lane 1. The MHC bands on the
gel are used to indicate how much muscle protein, as opposed to fat and fibrous
connective tissue, is loaded in each lane. Densitometric estimates of dysferlin/
myosin abundance were expressed as a percentage of the normal sample in lane
1; the values are: lane 2, 32.9%; lane 3, 10.9%; lane 4, 15.8%. SSCP analysis and sequencing was performed as previously
described (10). A total of 36% of the coding region of DYSF has
been tested in two patients (nos 8 and 9). The only change detected
was at nt 2745. To detect the Pro791Arg mutation, intronic primers
62.2F (5′-GGCCTTATGTTGGGAAAAATACGA-3′) and 62.2R
(5′-AGTCAGAGGTCAGCTCACGGTGTG-3′) were used to amplify a 292 bp product using the following conditions: (i) 94_C for
4 mins; (ii) 30 cycles of 94_C for 30 s, 55_C for 1 min and 72_C
for 1.5 mins; and (iii) 72_C for 10 mins. This sequence has two
HpaII sites. The first site is located 10 bp downstream of the
intron–exon boundary at nt 2745 surrounding the mutation
(C2745G). The second site is located 90 bp downstream of the
intron/exon boundary at nt 2824 serving as a convenient internal
control for complete digestion. The 292 bp PCR product was
restricted with HpaII and fragments were separated on an 8%
acrylamide gel and stained with ethidium bromide. After digestion,
three fragments of 80, 100 and 112 bp were detected in DNA from
normal controls; four fragments of 80, 100, 112 and 192 bp were
detected in DNA from carriers; and two fragments of 100 and 192
bp were detected in DNA from patients homozygous for the
C2745G mutation. One hundred control chromosomes of individuals from Northern England were tested for this mutation.
Numbering of base pair positions is as described by Liu et al. (9),
GenBank accession no. AF075575 for dysferlin cDNA.
DNA from patients and their immediate family was genotyped for
two dysferlin intragenic microsatellites, Cy172-H32 (9) and
104-sat (10) as well as four microsatellites surrounding DYSF.
Haplotypes were constructed using the known map order:
tel-D2S292– D2S443–Cy172-H32–DYSF C2745G–104-sat–
D2S291– D2S2110-cen (9,10) assuming a minimal number of
Sequence alignments MATERIALS AND METHODS
The pedigrees showing 13 affected individuals, one preclinical
patient and five consanguinity loops are presented in Figure 1A
and B. The pedigrees as illustrated have been slightly modified
for reasons of confidentiality. Onset of weakness was noted in
adolescence. Features of proximal or distal wasting and weakness
were demonstrated at presentation. There was no evidence of
involvement of extraocular or facial muscles. Two of the patients
(nos 1 and 2) predominantly manifested the MM phenotype with
distal wasting, weakness, grossly elevated CK and slow progression of disease with clinically evident proximal weakness.
Eleven had an LGMD phenotype (patients 3–9 and 11–14) but
with some distal wasting. All LGMD patients, except patients
12–14, are currently non-ambulatory. Patient 10 is still in a
preclinical stage, except for asymmetric calf hypertrophy,
minimal proximal muscle weakness and a dystrophic muscle
biopsy. No cardiac disease was evident clinically in the affected
individuals but only two have had formal assessments with
electrocardiography and echocardiography. All but five obligate
carrier parents were available for study and all had normal muscle
strength. Details have been described elsewhere (6). Protein sequence alignments were performed with dysferlin (GenBank accession no. 1373333) and fer-1 (GenBank accession no.
3600028) using CLUSTALW 1.7 from the Institute for Biomedical
Computing (http://www.ibc.wustl.edu/service/msa/index.html ),
ALIGN from the Genestream Resource Centre (http://
vega.crbm.cnrs-mop.fr/bin/align-guess.cgi ) and BLASTP (Blast 2
Sequences) from National Centre for Biotechnology Information
The monoclonal antibody NCL-hamlet was generated against a
peptide near the membrane spanning domain at the C-terminus of
dysferlin comprised of amino acids 1999–2016 (Fig. 2A) as
described in the accompanying paper by Anderson et al. (11).
Open muscle biopsies were obtained from three patients. A
gastrocnemius biopsy of a 12-year-old preclinical patient homozygous for Pro791Arg with asymptomatic hypertrophy of her
left calf (no. 10, Fig. 1A), a gastrocnemius biopsy from a
20-year-old patient (no. 1, Fig. 1A) with MM, and a deltoid
biopsy from a 26-year-old LGMD2B patient (no. 12 in Fig. 1B). 876 Human Molecular Genetics, 1999, Vol. 8, No. 5 Each biopsy was divided into three portions. The third sample
was snap frozen in isopentane which had been cooled in liquid
nitrogen. This portion was used for immunocytochemistry and
western blot analysis.
Western blot analysis
Polyacrylamide gel electrophoresis and western blotting were
performed as described previously (20). All tissue samples were
weighed frozen, and homogenized in 19 vol of electrophoresis
treatment buffer, giving a loading concentration of ∼200 mg in 30
ml. For the densitometric analysis, dried gels and blots were
scanned at 400 d.p.i. on an Epson GT8000 flatbed scanner using
white light for gels, and blue for blots. Each image was stored as
a bit-map where 8 bits = 1 pixel, and each pixel was graded from
0 (pure black) to 255 (pure white) on a 256 greyscale. Customized
software written for the Optimas v5.2 image analysis package
was used for the densitometric analysis. In this analysis, greyscale
values were converted to OD units using a Kodak SR-37
step-wedge so that the scanner was calibrated as a true
densitometer. The outline of each band was defined by a software
algorithm involving background measurements and the greyscale
value for each pixel within the band was automatically converted
to an OD value, producing a volume OD measurement for the
band. Pathological muscle samples contain a variable amount of
fat and fibrous connective tissue; therefore, myosin heavy chain
staining on the post-blotted gel was used as an indication of how
much true muscle protein was loaded in each sample. Thus, the
volume OD value for each dysferlin band was divided by the
corresponding value for myosin heavy chain in that sample to
produce a density value that was ‘normalized volume OD’ (20).
The normalized volume OD for each patient was then expressed
as a percentage of the average value for three control samples run
on the same blot, resulting in an estimate of protein abundance
expressed as ‘percentage of normal’ for each patient sample. Very
little tissue was available for most patient samples, so it was not
possible to undertake the analysis multiple times for statistical
analysis. The method outlined here, while not perfect, represents
the best attempt we were able to make at obtaining meaningful
results from a single shot experiment.
Immunocytochemistry at the light level was performed as
described previously (21) and in the accompanying paper by
Anderson et al. (11), with 6 µm unfixed frozen sections,
monoclonal primary antibodies and 1/100 Dako R260 rhodamine
conjugated secondary antibody. The primary antibodies were
used undiluted, or at the dilutions specified in each figure legend.
CK, creatine kinase; DYSF, gene symbol for dysferlin; LGMD,
limb girdle muscular dystrophy; LGMD2B, limb girdle muscular
dystrophy type 2B; MHC, myosin heavy chain; MM, Miyoshi
myopathy; SSCP, single strand conformation polymorphism.
We thank the families for their participation in this study, the
referring physicians and Ms Louise Dilling for assistance in sample collection. This work has been supported by grants from
the Medical Research Council of Canada, the Muscular Dystrophy Association of Canada, the Manitoba Health Research
Council, the Children’s Hospital Foundation of Manitoba, the
Muscular Dystrophy Group of Great Britain and Northern
Ireland, the Medical Research Council of Great Britain, the
Association Française contre les Myopathies and the Italian
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- Spring '10