Download The 10 autosomal recessive limb-girdle muscular - Genoma

Neuromuscular Disorders 13 (2003) 532–544
www.elsevier.com/locate/nmd
Review
The 10 autosomal recessive limb-girdle muscular dystrophies
Mayana Zatz*, Flavia de Paula, Alessandra Starling, Mariz Vainzof
Human Genome Research Center, Departamento de Biologia, Instituto de Biociências, Universidade de São Paulo,
Rua do Matão 277, Cidade Universitária, CEP 05508-900 Sao Paulo, Brazil
Received 14 December 2002; received in revised form 13 April 2003; accepted 18 April 2003
Abstract
Fifteen forms of limb-girdle muscular dystrophies (5 autosomal dominant and 10 autosomal recessive) have already been found. The
10 genes responsible for the autosomal recessive forms, which account for more than 90% of the cases, had their product identified. This review
will focus on the most recent data on autosomal recessive-limb-girdle muscular dystrophy and on our own experience of more than 300 patients
studied from 120 families who were classified (based on DNA, linkage and muscle protein analysis) in eight different forms of autosomal
recessive-limb-girdle muscular dystrophy. Genotype – phenotype correlations in this highly heterogeneous group confirm that patients with
mutations in different genes may be clinically indistinguishable. On the other hand, for most forms of autosomal recessive-limb-girdle muscular
dystrophy a discordant phenotype, ranging from a relatively severe course to mildly affected or asymptomatic carriers may be seen in patients
carrying the same mutation even within the same family. A gender difference in the severity of the phenotype might exist for some forms of
autosomal recessive-limb-girdle muscular dystrophy, such as calpainopathy and telethoninopathy but not for others, such as dysferlinopathies
or sarcoglycanopathies. Understanding similarities in patients affected by mutations in different genes, differences in patients carrying the same
mutations or why some muscles are affected while others are spared remains a major challenge. It will depend on future knowledge of gene
expression, gene and protein interactions and on identifying modifying genes and other factors underlying clinical variability.
q 2003 Elsevier B.V. All rights reserved.
Keywords: Limb-girdle muscular dystrophy; Genotype–phenotype correlation; Inter and intrafamilial variability
1. Introduction
The limb-girdle muscular dystrophies (LGMDs) are a
heterogeneous group of genetically determined progressive
disorders of skeletal muscle with a primary or predominant
involvement of the pelvic or shoulder-girdle musculature.
The clinical course in this group is characterized by normal
intelligence and great variability, ranging from severe forms
with onset in the first decade and rapid progression to milder
forms with later onset and a slower course [1,2].
At least 15 genes, 5 autosomal dominant (AD) and 10
autosomal recessive (AR), responsible for LGMD have
been mapped. Linkage analysis indicates that there is further
genetic heterogeneity both for AD as well as for AR-LGMD
[3]. The AD forms are relatively rare and represent less than
10% of all LGMDs. This was confirmed in our population
since among a large cohort of more than 100 Brazilian
families with multiple affected cases only four showed a
* Corresponding author. Tel.: þ55-11-3091-7563; fax: þ 55-11-30917419.
E-mail address: [email protected] (M. Zatz).
0960-8966/03/$ - see front matter q 2003 Elsevier B.V. All rights reserved.
doi:10.1016/S0960-8966(03)00100-7
typical AD inheritance (unpublished observations). A comprehensive review of all forms of LGMD becomes each
time more difficult and therefore this review will focus on
the AR forms.
The first AR-LGMD, LGMD2A, was mapped to 15q in
1991 [4] in a group of patients from Réunion Island. Shortly
thereafter, linkage analysis in Brazilian families showed
evidence of genetic heterogeneity [5]. Indeed, nine additional forms of AR-LGMD were mapped in the last decade,
most recently LGMD2J.
The protein products of these 10 genes have been
identified (Fig. 1). They are: calpain-3 for LGMD2A [6],
dysferlin for LGMD2B [7,8], a-sarcoglycan (SG) for
LGMD2D [9,10], b-SG for LGMD2E [11,12], g-SG for
LGMD2C [13,14], d-SG for LGMD2F [15 – 17], the
sarcomeric protein telethonin for LGMD2G [18,19],
TRIM32 for LGMD2H [20], fukutin-related protein
(FKRP) for LGMD2I [21] and titin for LGMD2J ([22]
Bjarne Udd, personal communication).
Genotype – phenotype correlation studies have been
reported for the different forms of LGMD in an attempt to
enhance our comprehension on the underlying pathological
M. Zatz et al. / Neuromuscular Disorders 13 (2003) 532–544
533
Fig. 1. Schematic representation of proteins from the sarcolemma, the sarcomere, the cytosol and the nucleus, involved in the process of muscle degeneration in
the several forms of LGMDs.
mechanisms, to better characterize each of the subgroups
and more importantly to try to identify modifier genes or
epigenetic factors which might modulate the clinical course
in patients who carry the same pathological mutation.
2. LGMD2A—calpainopathy
LGMD2A is caused by mutations in the human
CAPN3 gene, which codes for calpain-3, the skeletal
muscle-specific member of the calpain family [6]. The
demonstration of the involvement of the muscle-specific
calcium-activated neutral protease 3 in LGMD2A was the
first example of an enzymatic rather than a structural protein
defect causing a progressive muscular dystrophy. Calpainopathy is the most frequent form of LGMD in several populations including the Brazilian one, accounting for about
30% of the identified cases. Clinically, LGMD2A is characterized by symmetrical and selective proximal atrophy with
no cardiac or facial disturbance and normal intelligence.
However, the course is highly variable [2,23]. Calf hypertrophy is apparently rare in European patients but not
uncommon in Brazilian LGMD2A families where affected
sibs may show a discordant pattern.
Elevated serum creatine kinase (CK), particularly in the
active phase of the disorder has been reported for LGMD2A
patients by us and others [1,2,6,23,25]. However, most
recently we have identified patients, from three unrelated
families, who carried mutations in the calpain-3 gene and
had normal serum CK (three cases) or a neurogenic pattern
on electromyography (two cases) [Starling et al., submitted
for publication; Paula et al., in preparation]. These
unexpected observations suggest that the spectrum of
variability in calpainopathy might be broader than suspected
and that a normal CK should not be considered an exclusion
criteria for LGMD2A even in ambulant patients.
A wide intra and interfamilial clinical variability ranging
from severe to milder forms was reported in a multicenter
study of 163 European patients [23]. The mean age at onset
was 13.7 (ranging from 2 to 40 years old) and the mean age
at loss of walking ability was 17.3 years (range 5 –39 years)
after onset with no sex difference in age at onset or
progression. In the British population, a slightly younger
age at onset in female than in male patients was observed
[24], although the sample was relatively small (seven males
and six females). We have analysed 93 (47 females and 46
males, from 38 families) Brazilian patients who were
characterized at the molecular and most of them at the
protein level as well [25]. Eighteen patients (, 19%) were
confined to a wheelchair (mean age 21.7 years, range
12 – 45). When we compared both sexes, it was observed
that, the mean age at onset and ascertainment did not differ
significantly between males and females. However, a
statistically significant difference was seen for loss of
ambulation (observed for 12 males but only 6 females)
which occurred on an average earlier in males (mean
age 18.6, ranging from 12 to 32 years old) than in
females (29.7 years, ranging from 18 to 45 years old).
534
M. Zatz et al. / Neuromuscular Disorders 13 (2003) 532–544
This observation suggests a more rapid progression in
LGMD2A affected males than in females Brazilian patients.
Interestingly, in a recent screening of calpain-3 deficiency
through muscle protein analysis in Italian patients [26] there
was a statistically greater proportion of affected males (43)
than females (23). This gender disproportion caught our
attention. One possible explanation is that there were more
severely affected males than females who were therefore
more likely to be ascertained in accordance to our data.
The CAPN-3 gene comprises 24 exons and covers a
genomic region of 50 kb. It is expressed as a 3.5 kb transcript, and a 94 kDa translated protein. Screening of mutations in a large set of European, Brazilian and Japanese
patients has revealed more than 100 distinct pathogenic
CAPN3 mutations distributed along the entire length of the
gene ([23,25,27], Leiden database at http://www.dmd.nl/
capn3_home.htlm).
Most of the mutations represent private variants,
although some mutations were found more frequently in
isolated communities, with a high degree of inbreeding
[23,28,29]. In a recent study of 21 Japanese LGMD2A
patients, 10 of 13 identified mutations were not found in
other populations (including six novel ones). Three of them,
accounting for 71% of the cases, were recurrent mutations
[27]. In the Brazilian population, we identified two recurrent
null mutations (R110X and 2362-2363AG . TCATCT)
and seven novel pathogenic mutations. Interestingly, 41 of
the identified mutations (, 80%) were concentrated in only
six exons (1, 2, 4, 5, 11 and 22), which have important
implications for diagnostic purposes. This observation may
be particularly helpful for patients who are severely affected
and in whom getting a muscle biopsy may be difficult or for
patients who show normal results for all known LGMD
proteins including calpain-3 amount [25,30].
3. Protein analysis
Calpain-3, a muscle-specific 94 kDa calcium-activated
neutral protease 3, binds to titin. It is a cysteine protease,
which plays a role in the disassembly of sarcomeric proteins, but it may also have a regulatory role in modulation
of transcription factors. Calpainopathy seems to result
from the loss of proper substrate processing activity of
calpain-3 [31].
Western blot (WB) analysis of muscle calpain-3 in
LGMD2A patients can show a total, partial, or more rarely,
no apparent deficiency at all, with no direct correlation
between the amount of calpain and the severity of the
phenotype. Very low levels or no expression of calpain-3
were seen in European and Brazilian patients with a clinical
course varying from mild to severe [30,32]. Normal or
almost normal 94 kDa calpain-3 bands were found in
some patients with missense mutations [25,30] or more
recently in a Brazilian patient compound heterozygote for a
missense/null mutation (unpublished observation).
The analysis of calpain-3 in other forms of muscular
dystrophy revealed no apparent alteration in sarcoglycanopathy [32,33] or telethoninopathy patients [33]. However,
a secondary reduction of calpain-3 was first reported in
LGMD2B patients, suggesting a possible association
between calpain-3 and dysferlin [32,34,35]. Subsequently,
other studies have shown a secondary reduction of calpain-3
in other forms of LGMD such as LGMD2I [22], and
2q-linked muscular dystrophy [36 – 38], which requires
further studies.
The functional role of calpain-3 is still under discussion
but several pathogenic hypotheses have been proposed to
explain why mutations in this gene cause muscular dystrophy: calpain-3 could have a protective effect and be
involved in muscle detoxification preventing a degradation
of the muscle fibers. Alternatively it could have a role in the
proper processing and assembly of the structural scaffold of
the muscle cell. It has been suggested also that it may play a
significant role in intracellular signal transduction systems
[39 –42]. According to Baghdiguian et al. [43], calpain-3
deficiency would be associated with myonuclear apoptosis
and a profound perturbation of the IkBa/NF-kB pathway.
4. Genotype – phenotype correlations
Although there is a marked inter and intrafamilial heterogeneity in the severity of the clinical course in LGMD2A
muscular dystrophy [2,23,44] it has been suggested that on
an average missense mutations are usually associated to a
milder phenotype than null mutations [23,27,30].
We have compared Brazilian patients in whom both
mutations were identified [25], who were classified in three
groups according to the nature of the mutation: (1) patients
with missense mutations on both alleles or one missense and
one in frame deletion; (2) patients who were compound
heterozygous for missense/null mutations and (3) patients
carrying null mutations (frameshift or stop codon mutations) or splicing site changes on both alleles. Comparison
among the three groups showed that on an average the ages
at onset and ascertainment were significantly higher in
group (1) than for both groups (2) and (3) in accordance
with Richard et al. [23]. However, the mean ages of onset
and ascertainment did not differ between groups (2) and
(3) which suggests that one null mutation is enough to
determine a more severe phenotype.
No direct correlation has been observed between the
protein amount and the severity of the phenotype or type of
mutation although null mutations were more often associated with total absence of muscle calpain-3 while missense
mutation with partial deficiency. Almost normal 94 kDa
calpain-3 amount was found in about 10% of LGMD2A
Brazilian patients (3 among 31 unrelated patients in whom
muscle biopsies were available for protein studies) ([25] and
unpublished observation). However, their clinical course
was not milder which suggests that although present
M. Zatz et al. / Neuromuscular Disorders 13 (2003) 532–544
the protein is non-functional. In addition, the observation
that patients carrying two pathogenic mutations may have
an apparently normal calpain amount also suggests that the
relative proportion of calpainopathy screened through
protein analysis might be underestimated.
5. LGMD2B—dysferlinopathies
This form of LGMD includes Miyoshi myopathy
(MM), a distal muscle disorder that preferentially affects
the gastrocnemius muscle or LGMD type 2B with
characteristic proximal weakness at onset. Although the
initial presentation may be different the distinction
between patients with distal or proximal onset is very
difficult after many years of progression. This form of
LGMD arises from mutations in the dysferlin gene, which
contains over 55 exons and spans a region of 150 kb [7,8,
45]. The clinical picture is on an average less severe than
all other AR-LGMD forms although there is a wide intra
and interfamilial variability [46,47]. We also observed this
in 81 Brazilian patients (40 males and 41 females) from
26 families. Calf hypertrophy is rare but is also observed
in some cases. Interestingly, patients often lose their
ability to walk on tiptoes before the ability to walk on
their heels, a feature which might be helpful for
differential diagnosis. Confinement to wheelchair may
occur, on an average 10 – 20 years after onset of the
disease but on an average it is less frequent and occurs
later than among calpainopathy patients [2,44]. Among
Brazilian patients, about 10% (five males and three
females) were confined to a wheelchair (mean age
37.8 ^ 9.02, ranging from 27 to 50 years). Cardiac and
respiratory muscles are not involved in either MM or
LGMD2B and patients have normal intelligence. Serum
CK is always elevated among these patients even in the
pre-clinical stages. LGMD2B may be the most prevalent
form (35 –45%) in some populations such as in the Cajun/
Arcadian population of North America [35]. In the
Brazilian population it is the second more prevalent
form of AR-LGMD accounting for , 22% of the
classified forms.
6. Protein analysis
Dysferlin is a ubiquitously expressed 230 kDa molecule
that is localized in the periphery of skeletal muscle fibers,
linked to the sarcolemmal membrane [48]. It is expressed in
early stages of development [22,35,48] and can be detected
also in blood, skin and in chorionic villus biopsy [35,49].
Given the homology of dysferlin to a nematode
spermatogenesis factor that is required for successful
membrane fusion, it has been suggested that the lack of
dysferlin might cause faulty myotube fusion and impair
muscle regeneration [7,48].
535
Protein analyses in LGMD2B have shown a total
deficiency of dysferlin, both through IF and WB. Although
a partial deficiency has been reported in LGMD2B patients
from UK [48], dysferlin deficiency seems to be specific to
LGMD2B in our patients [35].
A reduced sarcolemmal expression along with accumulation of intracellular staining has been described in skeletal
muscle of patients with disruption in the dystrophin–
glycoprotein complex or DGC [50]. In our experience,
however, the analysis of dysferlin has shown a normal
localization and molecular weight (MW) in Duchenne
(DMD) and sarcoglycanopathy patients, suggesting no
interaction between dysferlin and the DGC [35]. A possible
association between dysferlin and caveolin3 has recently
been described [51].
Following the cloning of dysferlin, two additional
human genes, with strong homology to dysferlin, coding
for myoferlin and cardioferlin, were identified indicating
the existence of a family of ferlin-related proteins [50]. It
has been suggested that myoferlin, a plasma membrane
protein highly expressed in cardiac and skeletal muscle,
could act as a possible modifier of the muscular dystrophy
phenotype [52]. However, our studies in LGMD2B patients
showed no correlation between myoferlin expression and
the severity of the phenotype, suggesting that this protein
does not seem to modify the clinical course of dysferlin
deficient patients [35].
7. Genotype – phenotype correlations
Due to the large size of the dysferlin gene, and the
absence of any apparent mutation hotspot region, few
mutations have been identified to date [49]. Therefore the
diagnosis is mainly performed through linkage (in large
pedigrees) or muscle dysferlin deficiency in patients’
muscle biopsies. Only six mutations were identified to
date in seven Brazilian families (two homozygous and five
in heterozygous state) [53]. A novel splicing mutation was
reported in a LGMD2B family associated with inflammation [54]. A striking feature associated with dysferlinopathy which remains unexplained is why patients carrying
the same mutation, even within the same family, differ not
only in severity but also in the distribution of muscle
weakness [55].
Interestingly, in the animal model for dysferlinopathy
[56] it was observed that males have on an average a more
severe course than females (Reginald Bittner, personal
communication). However, in opposition to calpainopathy,
no gender difference was seen among our Brazilian patients
(unpublished observation) for age of onset, ascertainment or
wheelchair confinement. The identification of new mutations in the dysferlin gene is of great interest to enhance our
comprehension on the underlying mechanism associated
with such a variable phenotype.
536
M. Zatz et al. / Neuromuscular Disorders 13 (2003) 532–544
8. Sarcoglycanopathies
Among
AR-LGMD,
the
sarcoglycanopathies
(LGMD2C – 2F) represent a subgroup caused by mutations
in one of the genes that encode g-, a-, b- and d-SGs,
respectively [9,11 – 13,16]. Severe clinical Duchenne-like
presentations tend to be more common among SG patients.
Serum CK is always greatly elevated, in particular
when patients are still ambulant. Onset usually occurs
early in the childhood and confinement to a wheelchair
before the age of 16 [2,44,57– 60]. Nevertheless, milder
courses in LGMD2C –2E patients that harbor missense or
even nonsense mutations have also been found by us and
others [11,14,61,62]. The SGs are transmembrane glycoproteins which, together with sarcospan, dystrophin, dystroglycans, syntrophins and a-dystrobrevin, constitute the
DGC [63 – 66]. In addition to the four SGs that comprise
the SG – sarcospan subcomplex of the DGC in the striated
muscle [67], a fifth SG, named 1-SG, is expressed in a wide
variety of tissues [68,69]. In the smooth muscle, 1-SG
replaces a-SG as an integral component of a unique
SG –sarcospan complex composed of 1-, b-, g- and d-SGs
and sarcospan [70]. Recently, it has been shown that
mutations in 1-SG cause myoclonus-dystonia syndrome,
an AD non-degenerative central nervous system disorder
[71,72].
Until now, 77 distinct pathogenic mutations have been
found in SG patients: 41 in the a-SG gene, 20 in the b-SG
gene, 10 in the g-SG gene and 6 in the d-SG gene (Leiden
Database at http://www.dmd.nl/capn3_home.htlm). We
have been able to confirm at the molecular level a
diagnosis of sarcoglycanopathy in 39 Brazilian families
with the following relative frequency: 23% for LGMD2C,
40% for LGMD2D, 23% for LGMD2E and 14% for
LGMD2F. As a group, sarcoglycanopathies account for
about 1/3 of the classified forms of AR-LGMD in our
population. Interestingly the four subtypes are well
represented in Brazil, in contrast to what has been observed
in other countries. While in Europe and North America, the
great majority of the patients deficient for the SG proteins
are affected by LGMD2D [57,73,74], LGMD2C corresponds to almost 100% of the sarcoglycanopathies in
Northern Africa [75]. In addition, LGMD2F seems to be
very rare all over the world [44,57,60]. Probably, the
relative balance among the different forms in Brazil may
be attributed to the high degree of miscegenation of our
population and increased consanguinity particularly for
some groups. Indeed, the rate of consanguinity in
LGMD2F is 100%, compared to values between 43 and
75% in the other three forms. Therefore, the higher
prevalence of LGMD2F among Brazilian sarcoglycanopathies may reflect both a founder effect as well as the inbred
nature of this sample.
Out of the 18 distinct SG mutations that are segregating
in the Brazilian LGMD2C –2F families, seven were found
to be recurrent [76] and three of them account for more than
60% of the disease alleles. These are c.521delT in the g-SG
gene, c.656delC in the d-SG gene and R77C in a-SG, which
were detected in 100, 80 and 64% of the LGMD2C, 2F and
2D alleles, respectively. The alterations g-SG/c.521delT
and d-SG/c.656delC have probably been introduced in our
population by African-Brazilians, since all patients in each
of these two subgroups share a common haplotype [14,77].
On the other hand, the R77C mutation is associated with at
least three distinct haplotypes in Brazil [78]. Actually, this
mutation is the most frequent alteration in LGMD2D alleles
in other populations [60,73,79 –81], and this cytosine, in a
CpG island, has been considered a mutational hotspot
[81]. Nevertheless, the percentage of the R77C mutation
among the LGMD2D alleles in our country is apparently the
highest reported so far.
Among the remaining 15 mutations identified by us, the
great majority lie either in the a- or in the b-SG genes, while
only one lies in d-SG. As most of the reports described in the
literature [57,60,73,79 – 81], we found only missense mutations associated to LGMD2D. They are spread across five
exons and all of them result in amino acid substitutions
localized in the extracellular domain of the protein, which
corresponds to its largest portion. With regard to b-SG,
although missense, frameshift and splicing mutations were
detected, 75% of them lie in exons 3 and 4.
9. Analysis of the SG proteins
The DGC acts as a linker between the cytoskeleton of the
muscle cell and the extracellular matrix, providing mechanical support to the plasma membrane during myofiber
contraction [65,82]. Besides this structural function, there is
now increasing evidence that the DGC might play a role in
cellular communication, as highlighted by its interaction
with signaling molecules such as calmodulin, nitric oxide
synthase and Grb2 [66,83].
In the majority of muscle biopsies from patients with a
mutation in one of the SG genes, the primary loss or
deficiency of any one of the four SGs, in particular of band d-SG, leads to a secondary deficiency of the whole
subcomplex [2,44,84 –86]. However, exceptions may occur,
such as minor deficiencies of g-SG with partial preservation
of the other three SG in LGMD2C [67] or the partial
deficiency of only a-SG with the retention of the other three
in LGMD2D [85,87]. The observation of a complete deficiency of one SG with partial deficiency of the others may
help to indicate which gene should be first screened for
mutations.
Patients with SG mutations may also have a secondary
reduction in dystrophin, particularly those with primary
g-SG deficiency, suggesting that g-SG might interact more
directly with dystrophin [86]. In addition, this observation
should be taken into account for differential diagnosis with
X-linked Becker dystrophy in patients with no identified
mutation.
M. Zatz et al. / Neuromuscular Disorders 13 (2003) 532–544
Animal models lacking a-, b-, g- and d-SG have been
described and have provided new insights into the pathogenesis of muscular dystrophy as well as in the function of
the SG complex. Based on these studies there are now
evidences that this complex may be an independent
signaling module [84].
10. Genotype –phenotype correlations
Some frequent alterations, like R77C in the a-SG [44,
79,80,86,88] and c.521delT in the g-SG genes [14], have
been associated with both severe and mild forms with
discordant phenotypes among affected sibs. More recently,
this was observed by us in two brothers homozygous for a
novel mutation c.2T . C in the b-SG gene both showing
an identical immunohistochemical profile for the SG
complex [76].
Brazilian patients harboring null mutations in both
alleles of one of the SG genes as well as a drastic decrease
of the entire SG complex usually but not always show a
severe phenotype. In one of our patients in whom muscle
analysis revealed deficiency of the four SGs, no mutation
was found suggesting that the pathogenic mutation(s) may
be located in unexplored non-coding regions in one of the
SG genes or in another known or unknown gene encoding or
interfering with a protein of the DGC [89].
Most patients with missense changes in both alleles also
show a dramatic reduction of the primary protein together
with variable deficiency of the secondary SGs. This pattern
has been associated usually with a severe clinical course in
LGMD2E and 2F but with both mild and severe
presentations in LGMD2D. Within this last subgroup,
clinical variability among patients homozygous for the
same mutation seems to correlate with the residual amount
of g-SG in the muscle, despite the absence of the other
three SGs in all of them [86]. However, normal or almost
normal levels of the primarily involved SG in the muscle
have been found in some rare LGMD2C and LGMD2D
patients [67,81,87].
Interestingly there were significantly more females (61)
than males (40) in our samples of 101 sarcoglycanopathy
patients (from 39 families). One possible explanation is
that affected females are already ascertained with a
probable diagnosis of LGMD while severely affected
males in whom a muscle biopsy could not be obtained
may be erroneously classified as Xp21 muscular dystrophy.
Indeed when the probands were excluded the proportion of
affected females: males (34F:28M) did not differ significantly from the expected. Comparison between genders
showed no statistically significant difference for age of
onset (8.0 ^ 3.0 for females and 8.4 ^ 3.6 for males) or
wheelchair confinement (13.0 ^ 2.8 for females and
14.0 ^ 7.6 for males).
537
11. LGMD2G—telethoninopathy
LGMD2G is a rare form of AR-LGMD, which was
mapped in Brazilian patients in 1997 [18]. Subsequently
it was shown that this form of AR-LGMD is caused by
mutations in the gene encoding the sarcomeric protein
telethonin. Patients from four unrelated Brazilian genealogies had null mutations in the telethonin gene and
deficiency of this protein in muscle [19]. Screening of
mutations in the telethonin gene in other populations did
not allow so far the identification of any other case of
telethoninopathy. This is surprising since the genealogy,
which led to the mapping of the LGMD2G gene, is of Italian
origin and affected patients in this family are compound
heterozygotes for two different null mutations. In addition,
mutations in heterozygous state were identified among
Italian patients [90]. On the other hand, recently, a partial
deficiency of muscle telethonin was identified in patients
from two unrelated families from United States but no
mutation was identified in the telethonin gene suggesting
that this probably represents a secondary effect [91].
12. Protein analysis
Telethonin is a sarcomeric protein of 19 kDa, present in
the Z disk of the sarcomere of the striated and cardiac
muscle [92]. It was found to be one of the substrates of the
serine kinase domain of titin. The specific function of
telethonin and its interaction with the other proteins from
the muscle is still unknown.
Protein analysis in six Brazilian patients, from four
unrelated families, carrying the same frameshift mutation
in the telethonin gene showed deficiency of the protein
telethonin in muscle. Additional protein studies in these
patients showed normal expression for dystrophin, the four
SGs, dysferlin, calpain-3 and titin. Immunofluorescence
analysis for a-actinin-2 and myotilin showed a normal cross
striation pattern, suggesting that at least part of the Z-line of
the sarcomere is preserved. Ultrastructural analysis confirmed the maintenance of the integrity of the sarcomeric
architecture. Thus, as mutations in the telethonin gene do
not seem to alter the sarcomere integrity, the underlying
pathogenic mechanism is still under investigation [33].
However, since this form of LGMD has been confirmed
exclusively in Brazilian patients carrying the same mutation, the existence and effect of other mutations in the
telethonin gene are still unknown.
13. Genotype – phenotype correlations
A wide spectrum of inter and intrafamilial clinical
variability was observed in 14 patients (six males and eight
538
M. Zatz et al. / Neuromuscular Disorders 13 (2003) 532–544
females) from four families. All showed proximal involvement and marked weakness but seven had atrophy in the
distal muscles of the legs (with a phenotype resembling
MM) while six patients had calf hypertrophy which was
asymmetric in three unrelated male patients. The age at
onset ranged from 9 to 15 years old. Five of them lost
the ability to walk within the third or fourth decade,
while eight, currently aged 23 –44 years, are still ambulant.
Serum CK was increased 3 to 30-fold. Heart involvement
was observed in five of nine assessed patients. Interestingly,
the course seems milder on an average in affected females
than males. In three families, the oldest sisters were less
severely affected than their younger sibs. In the fourth
one, although the affected female is the youngest one she is
also apparently showing a milder course than her older
brother.
14. LGMD2H
This form of LGMD has been exclusively observed in
Manitoba Hutterites [93 –95] where more than 60 affected
individuals have been identified [20,22]. It is characterized
as a mild form of LGMD with proximal weakness, serum
CK levels more than four times above normal and either a
electromyography or a muscle biopsy consistent with a
myopathic disorder [1]. The age of onset is around
the second or third decade of life with a slow progression.
Most patients remain ambulatory into the sixth decade of
life. They have normal intelligence and there is no evidence
of cardiac or facial involvement [94,95].
The LGMD2H form was mapped at 9q [96] and recently
the gene responsible for this form has been cloned [20]. It is
a putative E3-Ubiquitin-ligase gene, previously called
‘HT2A’ [97] which encodes a member of the TRIM family
of proteins, TRIM32. The missense change V4, which
changes codon 487 of TRIM32 from aspartate to asparagine
(D487N) is responsible for the phenotype found in the
Hutterites [20]. This mutation occurs in a domain considered to be the NHL domain [98]. Since the aspartate is
one of the most conserved amino acids in this domain such
mutation could result in a significant alteration of the
structure of TRIM32, which has a RING-finger domain, a
B1 box, a coiled-coil domain and six NHL domains [20].
Frosk et al. suggest that TRIM32 function might be tagging
protein(s) for degradation in the proteosome. Target proteins would be recognized through the NHL domains by
protein– protein interactions, and this function is abolished
in LGMD2H patients [20].
15. LGMD2I
LGMD2I, linked to chromosome 19q13.3, was first
described in a large consanguineous Tunisian family [99].
In the following year, Brockington et al. [100] identified a
new gene, FKRP gene that mapped at the same locus of the
LGMD2I. This gene was identified through homology to the
Fukuyama congenital muscular dystrophy (FCMD) gene (a
severe progressive muscular weakness from early infancy
and marked central nervous system involvement [101]).
The FKRP gene consists of four exons, but the entire
coding sequence is located within exon 4. Mutations in
this gene were shown to cause LGMD2I as well as the
congenital muscular dystrophy 1C (CMD1C) [21,100].
CMD1C is a severe condition (age at onset in the first
few weeks of life and inability to walk) while LGMD2I has
a milder and variable course (age at onset in the second or
third decade of life and slower progression), both with
preserved intelligence and elevated serum CK. In both
disorders, weakness and wasting of the shoulder-girdle
muscles and primary restrictive respiratory and cardiac
involvement have been reported [21,100,102].
Although first identified in Tunisia, LGMD2I was
subsequently found to be rare in this country but the
commonest form in several European countries. Among
German patients, LGMD2I has been shown to represent the
most frequent differential diagnosis between DMD and
BMD ([22]; Thomas Voit, personal communication).
Interestingly, some LGMD Hutterite families did not
appear to be linked to the chromosome 9 locus. Indeed when
the putative disease-causing mutation (D487N in TRIM32)
for LGMD2H was identified, none of the affected individuals in these families carried this mutation, indicating
genetic heterogeneity. More recently, DNA sequence analysis of the FKRP gene in these patients revealed that they
carry the missense C826A mutation [20]. Surprisingly this
mutation has been shown to be the most common mutation
among LGMD2I patients. The presence of both LGMD2H
and LGMD2I in the Hutterite population further emphasizes
the importance of taking into account the possibility of
genetic heterogeneity in gene mapping studies even in
genetically isolated population.
The function of the FKRP is still unknown but it has
sequence similarities to a family of proteins involved in the
glycosylation of cell surface molecules [100]. Direct protein
quantification of FKRP was not reported yet, since there is
no available antibody to date. However, some interesting
secondary protein alterations have been reported in
LGMD2I and CMD1C patients. These include a secondary
laminin a2 reduction, mainly on immunoblots, reduced
WB labeling for laminin b1 and secondary reduction of
calpain-3. A variable reduction of a-DG expression was
also observed in the skeletal muscle biopsy of affected
individuals, with a reduction in the MW on immunoblotting,
which could indicate a glycosylation defect associated to
this disease, a novel pathogenic mechanism in muscular
dystrophy [100,102]. Until a proper antibody against FKRP
is developed, muscle proteins analysis showing a relative
secondary deficiency of a-DG and laminin a2 might
suggest a diagnosis of LGMD2I, which should be confirmed
through molecular analysis.
M. Zatz et al. / Neuromuscular Disorders 13 (2003) 532–544
16. Genotype –phenotype correlations
Similar to severe DMD and the milder allelic form of
BMD, both caused by mutations in the dystrophin gene, it
was shown that mutations in the FKRP gene could cause
two clinically distinct disorders with overlapping symptoms
resulting in an intermediate phenotype. At the most severe
end of the spectrum, children never achieve independent
ambulation or may even be associated to severe mental
retardation as recently shown in two unrelated Turkish
patients [103]. At the other end of the spectrum patients
may be asymptomatic until their fifth or sixth decades. The
common C826A mutation was found in the majority of
the mildly affected patients. Intermediate cases often are
compound heterozygote for this mutation and another missense or stop mutation. The most severe cases were found to
be compound heterozygotes for two other missense mutations or one missense and one stop codon mutation. No
patients have yet been reported with two null type mutation
[22]. However, similar to most forms of LGMD there is
intrafamilial variability in the presence of other complications such as respiratory or cardiac failure or in the
severity of the phenotype [104].
The common prevalent C826A missense mutation was
found in most European LGMD2I families associated frequently within two distinct haplotypes, suggesting few
independent mutation events [21]. Our research group has
identified 16 clinically affected patients from 13 Brazilian
LGMD2I genealogies who carry mutations in the FKRP
gene (de Paula et al., manuscript in preparation). Five
families share the recurrent C826A change (four in
homozygous and one in heterozygous state). Eight missense
pathogenic changes (not found in at least 100 normal
controls) were identified in the other patients. With
exception of one affected female, deceased at age 33, the
patients from these 11 genealogies (seven females and four
males), aged 25– 51, are still ambulant. In one additional
isolated affected male, with an intermediate clinical course,
a FKRP mutation was identified in just one allele. The most
severe course was observed in three affected sisters who
are compound heterozygous for one missense and one stop
codon mutation. The two older sisters had a Duchenne-like
course, deceased at age 14 and 15 but the younger sister can
still walk short distances at age 24. On the other end of
the spectrum four individuals from two unrelated families,
carrying novel homozygous missense mutations were
completely asymptomatic [105]. It is important to point
out that these cases would remain undetected if they did
not have clinically affected relatives, which suggests that
the frequency of mutations in the FKRP gene may be
underestimated.
In short, genotype– phenotype correlation studies for the
FKRP gene are still emerging. However, the identification
of pathogenic mutations in patients from different populations suggests that the spectrum of clinical variability
539
associated with mutations in the FKRP gene seems to be
wider than for other forms of LGMD [106].
17. LGMD2J
A new form of LGMD has been recently classified
as LGMD2J based on the description of a Finnish family
with patients homozygous for a titin mutation, which in
heterozygous states causes AD tibial muscular dystrophy
([38,107], Bjarne Udd, personal communication). The
coding region of the titin gene has 363 exons, which encodes
a giant structural protein of the sarcomere [108]. The titin
molecule has several ligand-binding sites along its structure.
Two of the titin ligands, the muscle-specific protease calpain
and the telethonin are of special interest because they are
mutated in LGMD2A [6] and LGMD2G [19].
An interesting question is whether we should classify
patients carrying two mutated titin alleles as affected by an
AR form of LGMD, that is LGMD2J, or as homozygous
for AD tibial muscular dystrophy. The observation that
homozygous patients have a different phenotype, a more
severe-childhood-onset-LGMD ([38] Bjarne Udd, personal
communication) when compared with the dominant form
(where clinical symptoms occur at age 35 – 45 or much
later) suggests that the first alternative is probably more
appropriate.
18. Relative proportions of the different forms
The relative proportion of the different AR-LGMD
forms varies in different populations. For example in
North Africa and Turkey, sarcoglycanopathies seem to be
the most prevalent forms while in Europe, LGMD2A is
more common in some countries such as Italy or Spain and
LGMD2I in others such as England [88,109]. Apparently
LGMD2H has been found so far only among Hutterites [20]
while the incidence of LGMD2J around the world is still
unknown.
In Brazil the relative proportion of the different forms
among classified patients (through DNA and/or muscle
protein analysis) from 120 families was found to be: 32%
for LGMD2A, 22% for LGMD2B, 32% for the sarcoglycanopathy group, 3% for LGMD2G and 11% for
LGMD2I. Therefore, the most prevalent form is
LGMD2A and the less common are telethoninopathy and
d-SG although both forms have been identified in our
population (Table 1).
19. Main remarks and conclusions
For differential diagnosis, the following observations
should be considered. Among patients with a milder course,
those with distal atrophy, inability to walk on tiptoes
540
M. Zatz et al. / Neuromuscular Disorders 13 (2003) 532–544
Table 1
Key clinical findings in Brazilian patients and relative proportions of the 10 AR-LGMD around the world as well as in the Brazilian population
LGMD
Protein
Age at onset
Loss of
ambulation in
most cases
Serum CK fold
increased
Gender
distribution
Relative
proportions
Cardiac
involvement
Diagnosis
LGMD2A
Calpain-3
First to second
decade
Second to fifth
decade
Normal to .60
May be more
severe in
males than
females
Rare
DNA and
muscle
protein
LGMD2B
Dysferlin
Second to sixth
decade
Third to sixth–
seventh
decade
6 to .150
No gender
difference
Rare
Muscle protein
LGMD2C
g-SG
First to third
decade
Second to third
decade
10 to .100
No gender
difference
Common
DNA and
muscle
protein
LGMD2D
a-SG
First to third
decade
Second to
fourth or fifth
decade
10 to .100
No gender
difference
Common
DNA and
muscle
protein
LGMD2E
b-SG
First to third
decade
Second to
10 to .100
fourth decade
No gender
difference
Common
DNA and
muscle
protein
LGMD2F
d-SG
First decade
Second decade
10 to .100
No gender
difference
Common
LGMD2G
Telethonin
Second to third
decade
Third to fifth
decade
1.5–25
Males more
severely
affected
DNA and
muscle
protein
DNA and
muscle
protein
LGMD2H
TRIM32
Second to third
decade
Relatively
common in
many
countries;
32% in
Brazil
Relatively
common in
many
countries;
22% in
Brazil
Reported in
many
countries;
very
common in
North Africa;
23% of
Brazilian SG
The most
frequent SGglycanopathy
in many
countries;
40% of
Brazilian SG
Reported in
many
countries;
23% of
Brazilian SG
Relatively rare
14% of
Brazilian SG
Described only
in Brazil—
3% of the
cases
Described only
in Hutterites
?
DNA
LGMD2I
CMD1C
FKRP
The commonest
AR-LGMD
in many
countries;
13% in
Brazil
Common
DNA
LGMD2J
Titin
Described in
Finland; still
unknown
?
DNA
Most ambulant On an average
No reported
in the sixth
fourfold
gender
decade
above normal
difference
20 to .100
No reported
From birth to
Extremely
gender
fourth or fifth
variable from
difference
decade
early
childhood to
absence of
weakness in
fourth decade
First to third
Normal to two- No reported
decade
threefold
gender
difference
and very high serum CK levels are more likely to have
LGMD2B. However, the clinical presentation may be very
similar in patients with the different forms of AR-LGMD.
Those with a severe DMD-like course and proximal
Common
weakness are more likely to belong to the sarcoglycanopathy, LGMD2I or calpainopathy subgroups [22,23,44].
Testing for defective protein expression is a very useful
tool for directing the search for gene mutations. For
M. Zatz et al. / Neuromuscular Disorders 13 (2003) 532–544
example, we start mutation screening in the a-SG gene
in patients with deficiency of any component of the SG
complex since this is the most prevalent sarcoglycanopathy
in our population. Among the non-sarcoglycanopathies, the
absence of dysferlin or telethonin on muscle biopsy strongly
suggests a diagnosis of LGMD2B or LGMD2G, respectively. However, results on calpain analysis should be interpreted with caution since normal or only slight reductions
may be found in LGMD2A patients while secondary
deficiencies may occur in other forms of MD. When no
muscle is available, the choice of what gene should be
screened first for mutations will depend on the relative
frequency of each form in different populations.
Among the 10 AR-LGMD forms already identified,
with exception of LGMD2H and 2J, all others are well
represented in the Brazilian population. This allows
some interesting comparative observations for the eight
AR-LGMD subtypes present in Brazil, such as:
(a) Inter and intrafamilial clinical variability was observed
among patients from all groups with exception of
LGMD2F where all patients reported so far present a
severe phenotype.
(b) Calf hypertrophy was observed in patients from all
groups, but is rare among LGMD2B patients.
(c) Heart involvement was observed in Brazilian patients
from all groups in particular in sarcoglycanopathies
and LGMD2I patients. It is rare among calpainopathy
and dysferlinopathy patients.
(d) On an average, LGMD2B (dysferlin) patients were the
less affected but some missense mutations in the FKRP
gene may be associated with a very mild phenotype or
even non-penetrance in the fourth decade.
(e) In addition to sarcoglycanopathies, a severe Duchennelike phenotype may be seen among LGMD2I and
LGMD2A patients.
(f) Serum CK activities showed on an average the highest
values in sarcoglycanopathies and dysferlinopathy
patients and are elevated in all ambulant patients. In
the telethonin group serum CK may range from
borderline to 30-fold increase.
(g) Although most ambulant LGMD2A patients have
elevated serum CK, some may have normal serum
levels even in the active phase of the disease.
(h) Some LGMD2G (telethonin) patients had distal
atrophy with a phenotype resembling MM while the
others had calf hypertrophy with a typical LGMD
presentation.
(i) A gender difference in the severity of the clinical
course, affecting more males than females, may exist
for calpainopathy and telethoninopathy but apparently
not for dysferlinopathy and sarcoglycanopathy.
(j) The spectrum of clinical variability associated with
mutations in the FKRP gene seems to be wider than for
other forms of LGMD ranging from severe congenital
forms to absence of symptoms in the fourth decade.
541
Understanding how clinically indistinguishable phenotypes may be observed among patients with different forms
of LGMD, while a discordant phenotype may be seen in
unrelated patients carrying the same mutation and even
among affected sibs remains a major challenge. It will
depend on future knowledge on gene expression, gene and
protein interactions, protein processing and on identifying
modifying genes or other mechanisms underlying clinical
variability.
Acknowledgements
The collaboration of the following persons is gratefully
acknowledged: Dr Maria Rita Passos-Bueno, Dr Eloisa
S. Moreira, Dr Rita C.M. Pavanello, Dr Ivo Pavanello,
Dr Edmar Zanotelli, Dr Acary S.B. Oliveira, Dr Helga Silva,
Dr Suely K. Marie, Marta Cánovas, Antonia Cerqueira,
and Constancia Urbani. Special thanks to all the patients who
collaborated with this study. We would also like to thank
very much the following researchers, who kindly provided us
with specific antibodies: Dr Louise Anderson, Dr Elizabeth
McNally, Dr Carsten Bonnemann, Dr Louis M. Kunkel, Dr
Vincenzo Nigro, Dr Jeff Chamberlain, Dr Georgine Faulkner. We are also grateful to the two anonymous reviewers
for their valuable suggestions. This work was supported by
grants from FAPESP-CEPID, PRONEX and CNPq.
References
[1] Bushby KMD. The limb-girdle muscular dystrophies: multiple
genes, multiple mechanisms. Hum Mol Genet 1999;8:1875–82.
[2] Zatz M, Vainzof M, Passos-Bueno MR, et al. Limb-girdle muscular
dystrophy: one gene with different phenotypes, one phenotype with
different genes. Curr Opin Neurol 2000;13(5):511–7. [Review].
[3] Starling AL, Vainzof M, Pavanello R, et al. Evidence of further
genetic heterogeneity for both autosomal dominant and autosomal
recessive limb-girdle muscular dystrophy. Am J Hum Genet 2001;
69(4):1942. [Suppl 1].
[4] Beckmann JS, Richard I, Hillarie D, et al. A gene for limb girdle
muscular dystrophy maps to chromosome 15 by linkage. C R Acad
Sci Paris 1991;132:141–8.
[5] Passos-Bueno MR, Richard I, Vainzof M, et al. Evidence of genetic
heterogeneity for the autosomal recessive adult forms of limb-girdle
muscular dystrophy following linkage analysis with 15q probes in
Brazilian families. J Med Genet 1993;30(5):385–7.
[6] Richard I, Broux O, Allamand V, et al. A novel mechanism leading
to muscular dystrophy: mutations in calpain 3 cause limb girdle
muscular dystrophy type 2A. Cell 1995;81:27–40.
[7] Bashir R, Britton S, Stratchan T, et al. A novel mammalian gene
related to the C. elegans spermatogenesis factor fer-1 is mutated in
patients with limb-girdle muscular dystrophy type 2B (LGMD2B).
Nat Genet 1988;20:37–42.
[8] Liu J, Aoki M, Illa I, et al. Dysferlin, a novel skeletal muscle gene, is
mutated in Miyoshi myopathy and limb girdle muscular dystrophy.
Nat Genet 1998;20:31–6.
[9] Roberds SL, Leturcq F, Allamand V, et al. Missense mutations in the
adhalin gene linked to autosomal recessive muscular dystrophy. Cell
1994;78:625–33.
542
M. Zatz et al. / Neuromuscular Disorders 13 (2003) 532–544
[10] McNally EM, Yoshida M, Mizuno Y, Ozawa E, Kunkel LM. Human
adhalin is alternatively spliced and the gene is located on
chromosome 17q21. Proc Natl Acad Sci 1994;91:9690 –4.
[11] Lim LE, Duclos F, Allamand V, et al. b-Sarcoglycan (43 DAG):
characterization and involvement in a recessive form of limb-girdle
muscular dystrophy linked to chromosome 4q12. Nat Genet 1995;11:
257–65.
[12] Bönnemann CG, Modi R, Noguchi S, et al. Mutations cause
autosomal recessive muscular dystrophy with loss of the sarcoglycan
complex. Nat Genet 1995;11:266–73.
[13] Noguchi S, McNally EM, Ben Othmane K, et al. Mutations in the
dystrophin-associated protein g-sarcoglycan in chromosome 13
muscular dystrophy. Science 1995;270:819 –22.
[14] McNally E, Passos-Bueno MR, Bonnemann C, et al. Mild and severe
muscular dystrophy caused by a single g-sarcoglycan mutation. Am
J Hum Genet 1996;59:1040 –7.
[15] Passos-Bueno MR, Moreira ES, Vainzof M, Marie SK, Zatz M.
Linkage analysis in autosomal recessive limb-girdle muscular
dystrophy (AR-LGMD) maps a sixth form to 5q33-34 (LGMD2F)
and indicates that there is at least one more subtype of AR LGMD.
Hum Mol Genet 1996;5:815–20.
[16] Nigro V, Moreira ES, Piluso G, et al. The 5q autosomal recessive
limb-girdle muscular dystrophy (LGMD2F) is caused by a mutation
in the d-sarcoglycan gene. Nat Genet 1996;14:195–6.
[17] Nigro V, Piluso G, Belsito A, et al. Identification of a novel
sarcoglycan gene at 5q33 encoding a sarcolemmal 35 kDa
glycoprotein. Hum Mol Genet 1996;5:1179– 86.
[18] Moreira ES, Vainzof M, Marie SK, Sertié AL, Zatz M, Passos-Bueno
MR. The seventh form of autosomal recessive limb-girdle muscular
dystrophy is mapped to 17q11–12. Am J Hum Genet 1997;61:
151–9.
[19] Moreira ES, Wiltshire TJ, Faulkner G, et al. Limb-girdle muscular
dystrophy type 2G (LGMD 2G) is caused by mutations in the gene
encoding the sarcomeric protein Telethonin. Nat Genet 2000;24:
163–6.
[20] Frosk P, Weiler T, Nylen E, et al. Limb-girdle muscular dystrophy
type 2H associated with mutation in TRIM32, a putative
E3-ubiquitin-ligase gene. Am J Hum Genet 2002;70(3):663–72.
[21] Brockington M, Yuva Y, Prandini P, et al. Mutations in the fukutinrelated protein gene (FKRP) identify limb girdle muscular dystrophy
2I as a milder allelic variant of congenital muscular dystrophy
MDC1C. Hum Mol Genet 2001;10(25):2851–9.
[22] Bushby KMD, Beckmann JS. The 105th ENMC sponsored workshop: pathogenesis in the non-sarcoglycan limb-girdle muscular
dystrophies, Naarden, April 12 –14, 2002. Neuromuscul Disord
2003;13:80–90.
[23] Richard I, Roudaut C, Saenz A, et al. Calpainopathy: a survey of
mutations and polymorphisms. Am J Hum Genet 1999;64:1524– 40.
[24] Pollitt C, Anderson LV, Pogue R, Davison K, Pyle A, Bushby KM.
The phenotype of calpainopathy: diagnosis based on a multidisciplinary approach. Neuromuscul Disord 2001;11(3):287– 96.
[25] Paula F, Vainzof M, Passos-Bueno MR, et al. Clinical variability in
calpainopathy: what makes the difference? Eur J Hum Genet 2002;
10(12):825 –32.
[26] Fanin M, Pegoraro E, Matsuda-Asada C, Brown RH, Angelini C.
Calpain-3 and dysferlin protein screening in patients with limbgirdle dystrophy and myopathy. Neurology 2001;1:660–5.
[27] Chae J, Minami N, Jin Y, et al. Calpain 3 gene mutations: genetic and
clinico-pathologic findings in limb-girdle muscular dystrophy.
Neuromuscul Disord 2001;11(6–7):547– 55.
[28] Urtasun M, Saenz A, Roudaut C, et al. Limb-girdle muscular
dystrophy in Guipuzcoa (Basque Country Spain). Brain 1998;
121(Pt 9):1735–47.
[29] Fardeau M, Eymard B, Mignard C, Tome FM, Richard I, Beckmann
JS. Chromosome 15-linked limb-girdle muscular dystrophy: clinical
phenotypes in Reunion Island and French metropolitan communities.
Neuromuscul Disord 1996;6(6):447–53.
[30] 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(4):1169–79.
[31] Sorimachi H, Beckmann JS. Defects of non-lysosomal proteolysis:
calpain3 deficiency. In: Karpati G, editor. Structural and molecular
basis of skeletal muscle diseases. Basel: ISN Neuropath Press; 2002.
p. 148–53.
[32] Vainzof M, Anderson LVB, Moreira ES, et al. Characterization of
the primary defect in LGMD2A and analysis of its secondary effect
in other LGMDs. Neurology 2000;54:A436.
[33] Vainzof M, Moreira ES, Passos-Bueno MR, et al. The effect of
telethonin deficiency in LGMD-2G and its expression in other forms
of muscular dystrophies and congenital myopathies. BBA—Gene
Basis Dis 2002;1588(1):33–40.
[34] Anderson LV, Harrison RM, Pogue R, et al. Secondary reduction in
calpain 3 expression in patients with limb girdle muscular dystrophy
type 2B and miyoshi myopathy (primary dysferlinopathies).
Neuromuscul Disord 2000;10:553–9.
[35] Vainzof M, Pavanello RCM, Anderson LVB, Moreira ES, PassosBueno MR, Zatz M. Dysferlin analysis in autosomal recessive limbgirdle muscular dystrophies (AR-LGMD). J Mol Neurosci 2001;17:
71 –80.
[36] Haravuori H, Vihola A, Straub V, et al. Secondary calpain3
deficiency in 2q-linked muscular dystrophy: titin is the candidate
gene. Neurology 2001;56(7):869–77.
[37] Garvey SM, Rajan C, Lerner AP, Frankel WN, Cox GA. The muscular
dystrophy with myositis (mdm) mouse mutation disrupts a skeletal
muscle-specific domain of titin. Genomics 2002;79(2):146 –9.
[38] Hackman P, Richard I, Vihola A, et al. Tibial muscular dystrophy
(TMD), 2q31 linked myopathy: sequencing and functional studies of
the titin (TTN) gene. J Neurol Sci 2002;199(Suppl 1):S35.
[39] Carafoli E, Molinari M. Calpain: a protease in search of a function?
Biochem Biophys Res Commun 1998;247(2):193 –203. [Review].
[40] Ono Y, Sorimachi H, Suzuki K. Structure and physiology of calpain,
an enigmatic protease. Biochem Biophys Res Commun 1998;245(2):
289 –94.
[41] Suzuki K, Sorimachi H. A novel aspect of calpain activation. FEBS
Lett 1998;433:1–4. [Review].
[42] Beckmann JS, Fardeau M. Limb-girdle muscular dystrophies. In:
Emery Alan EH, editor. Neuromuscular disorders: clinical and
molecular genetics. Chichester: Wiley & Sons Ltd; 1998. p. 123 –56.
[43] 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(5):503–11.
[44] Passos-Bueno MR, Vainzof M, Moreira ES, Zatz M. The seven
autosomal recessive limb-girdle muscular dystrophy (LGMD): from
LGMD2A to LGMD2G. Am J Med Genet 1999;82:392–8.
[45] Matsuda C, Aoki M, Hayashi Y, Arahata K, Brown RH. Dysferlin is
a surface membrane-associated protein that is absent in Miyoshi
myopathy. Neurology 1999;53:1119 –22.
[46] Illa I, Serrrano-Munuera C, Gallardo E, et al. Distal anterior
compartment myopathy: a dysferlin mutation causing a new
muscular dystrophy phenotype. Ann Neurol 2001;49:130– 4.
[47] Illarioshkin SN, Ivanova-Smolenskaya IA, Greenberg CR, et al.
Identical dysferlin mutation in limb girdle muscular dystrophy type
2B and dystal myopathy. Neurology 2000;55:1931 –3.
[48] Anderson LV, Davison K, Moss JA, et al. Dysferlin is a plasma
membrane protein and is expressed early in human development.
Hum Mol Genet 1999;8:855–61.
[49] Ho M, Gallardo E, McKenna-Yasek D, De Luna N, Illa I, Brown Jr RH.
A novel, blood-based diagnostic assay for limb girdle muscular
dystrophy 2B and Miyoshi myopathy. Ann Neurol 2002;51(1):129–33.
[50] Piccolo F, Moore SA, Ford GC, Campbell KP. Intracellular
accumulation and reduced sarcolemmal expression of dysferlin in
limb-girdle muscular dystrophies. Ann Neurol 2000;48(6):902–12.
M. Zatz et al. / Neuromuscular Disorders 13 (2003) 532–544
[51] Matsuda C, Hayashi YK, Ogawa M, et al. The sarcolemmal proteins
dysferlin and caveolin-3 interact in skeletal muscle. Hum Mol Genet
2001;10(17):1761–6.
[52] Davis DB, Delmonte AJ, Ly CT, McNally EM. Myoferlin, a
candidate gene and potential modified of muscular dystrophy. Hum
Mol Genet 2000;9:217–26.
[53] Paula F, Vainzof M, Moreira ES, et al. Novel dysferlin mutations in
Brazilian LGMD2B patients. Am J Hum Genet 2001;69(4):639.
[Suppl 1].
[54] McNally EM, Ly CT, Rosenmmann H, et al. Splicing mutation in
dysferlin produces limb-girdle muscular dystrophy with inflammation. Am J Med Genet 2000;91:305–12.
[55] Weiler T, Bashir R, Anderson LV, et al. Identical mutation in
patients with limb girdle muscular dystrophy type 2B or Miyoshi
myopathy suggests a role for modifier gene(s). Hum Mol Genet
1999;8(5):871–7.
[56] Bittner RE, Anderson LV, Burkhardt E, et al. Dysferlin deletion in
SJL mice (SJL-Dysf) defines a natural model for limb girdle
muscular dystrophy 2B. Nat Genet 1999;23(2):141 –2.
[57] Fanin M, Duggan DJ, Mostacciuolo ML, et al. Genetic epidemiology
of muscular dystrophies resulting from sarcoglycan gene mutations.
J Med Genet 1997;34:973–7.
[58] Vainzof M, Passos-Bueno MR, Pavanello RC, Marie SK, Oliveira
AS, Zatz M. Sarcoglycanopathies are responsible for 68% of severe
autosomal recessive limb-girdle muscular dystrophy in the Brazilian
population. J Neurol Sci 1999;164:44– 9.
[59] Bushby KM. Making sense of the limb-girdle muscular dystrophies.
Brain 1999;122:1403– 20.
[60] Duggan DJ, Gorospe JR, Fanin M, Hoffman EP, Angelini C.
Mutations in the sarcoglycan genes in patients with myopathy.
N Engl J Med 1997;336:618–24.
[61] Duclos F, Broux O, Bourg N, et al. Beta-sarcoglycan: genomic
analysis and identification of a novel missense mutation in the
LGMD2E Amish isolate. Neuromuscul Disord 1998;8:30–8.
[62] Passos-Bueno MR, Moreira ES, Marie SK, et al. Main clinical
features for the three mapped autosomal recessive limb-girdle
muscular dystrophies and estimated proportion of each form in
13 Brazilian families. J Med Genet 1996;33:97 –102.
[63] Yoshida M, Ozawa E. Glycoprotein complex anchoring dystrophin
to sarcolemma. J Biochem 1990;108:748 –52.
[64] Ervasti JM, Campbell KP. Membrane organization of the
dystrophin –glycoprotein complex. Cell 1991;66:1121– 31.
[65] Ozawa E, Noguchi S, Mizuno Y, Hagiwara Y, Yoshida M. From
dystrophinopathy to sarcoglycanopathy: evolution of a concept of
muscular dystrophy. Muscle Nerve 1998;21:421–38.
[66] Rando TA. The dystrophin –glycoprotein complex, cellular signaling, and the regulation of cell survival in the muscular dystrophies.
Muscle Nerve 2001;24:1575– 94.
[67] Crosbie RH, Lebakken CS, Holt KH, et al. Membrane targeting
and stabilization of sarcospan is mediated by the sarcoglycan
subcomplex. J Cell Biol 1999;145:153–65.
[68] Ettinger AJ, Feng G, Sanes JR. Epsilon-sarcoglycan, a broadly
expressed homologue of the gene mutated in limb-girdle muscular
dystrophy 2D. J Biol Chem 1997;272:32534–8.
[69] McNally EM, Ly CT, Kunkel LM. Human epsilon-sarcoglycan is
highly related to alpha-sarcoglycan (adhalin), the limb girdle
muscular dystrophy 2D gene. FEBS Lett 1998;422:27 –32.
[70] Straub V, Ettinger AJ, Durbeej M, et al. Epsilon-sarcoglycan
replaces alpha-sarcoglycan in smooth muscle to form a unique
dystrophin – glycoprotein complex. J Biol Chem 1999;274:
27989–96.
[71] Zimprich A, Grabowski M, Asmus F, et al. Mutations in the gene
encoding epsilon-sarcoglycan cause myoclonus-dystonia syndrome.
Nat Genet 2001;29:66 –9.
[72] Muller B, Hedrich K, Kock N, et al. Evidence that paternal
expression of the epsilon-sarcoglycan gene accounts for reduced
[73]
[74]
[75]
[76]
[77]
[78]
[79]
[80]
[81]
[82]
[83]
[84]
[85]
[86]
[87]
[88]
[89]
[90]
[91]
[92]
543
penetrance in myoclonus-dystonia. Am J Hum Genet 2002;71(6):
1303– 11.
Angelini C, Fanin M, Freda MP, Duggan DJ, Siciliano G, Hoffman
EP. The clinical spectrum of sarcoglycanopathies. Neurology 1999;
52:176–9.
Duggan DJ, Manchester D, Stears KP, Mathews DJ, Kart C,
Hoffman EP. Mutations in the delta-sarcoglycan gene are a rare
cause of autosomal recessive limb-girdle muscular dystrophy
(LGMD2). Neurogenetics 1997;1:49–58.
Othmane KB, Speer M, Stauffer J, et al. Evidence for linkage
disequilibrium in chromosome 13-linked Duchenne-like muscular
dystrophy (LGMD2C). Am J Hum Genet 1995;57:732–4.
Moreira ES, Vainzof M, Suzuki OT, Pavanello RCM, Zatz M,
Passos-Bueno MR. Genotype/phenotype correlations on 35 Brazilian
families with sarcoglycanopathies, including the description of three
novel mutations. J Med Genet 2003;40(2):E12.
Moreira ES, Vainzof M, Marie SK, Nigro V, Zatz M, Passos-Bueno
MR. A first missense mutation in the delta sarcoglycan gene
associated with a severe phenotype and frequency of limb-girdle
muscular dystrophy type 2F (LGMD2F) in Brazilian sarcoglycanopathies. J Med Genet 1998;35:951–3.
Passos-Bueno MR, Moreira ES, Vainzof M, et al. A common
missense mutation in the adhalin gene in three unrelated Brazilian
families with a relatively mild form of autosomal recessive limbgirdle muscular dystrophy. Hum Mol Genet 1995;4:163– 7.
Piccolo F, Roberds SL, Jeanpierre M, et al. Primary adhalinopathy:
a common cause of autosomal recessive muscular dystrophy of
variable severity. Nat Genet 1995;10:243–5.
Carrié A, Piccolo F, Leturcq F, et al. Mutational diversity and
hotspots in the a-sarcoglycan gene in autosomal recessive muscular
dystrophy (LGMD2D). J Med Genet 1997;34:470– 5.
Eymard B, Romero NB, Leturcq F, et al. Primary adhalinopathy
(a-sarcoglycanopathy): clinical, pathologic, and genetic correlation
in 20 patients with autosomal recessive muscular dystrophy.
Neurology 1997;48:1227–34.
Campbell KP, Kahl SD. Association of dystrophin and an integral
membrane glycoprotein. Nature 1989;338:259– 62.
Cohn RD, Campbell KP. Molecular basis of muscular dystrophies.
Muscle Nerve 2000;23:1456 –71.
Hack AA, Groh ME, McNally EM. Sarcoglycans in muscular
dystrophy. Microsc Res Tech 2000;48:167–80.
Bönnemann CG. Limb-girdle muscular dystrophies: an overview.
J Child Neurol 1999;14:31 –3.
Vainzof M, Passos-Bueno MR, Moreira ES, et al. The sarcoglycan
complex in the six autosomal recessive limb-girdle (AR-LGMD)
muscular dystrophies. Hum Mol Genet 1996;5:1963–9.
Vainzof M, Moreira ES, Canovas M, et al. Partial a-sarcoglycan
deficiency associated with the retention of the SG complex in a
LGMD2D family. Muscle Nerve 2000;23:984 –8.
Bushby KMD, Beckmann JS. Diagnostic criteria for the limb-girdle
muscular dystrophies: report of the ENMC workshop on limb-girdle
muscular dystrophies. Neuromuscul Disord 1995;5:71–4.
Cartegni L, Chew SL, Krainer AR. Listening to silence and
understanding nonsense: exonic mutations that affect splicing. Nat
Rev Genet 2002;3(4):285–98.
Politano LP, Nigro V, Aurino S, et al. Multiple heterozygosity
for different muscle genes is not rare among individuals with
pauci-symptomatic hyperckmia. Neuromuscul Disord 2002;12
(7–8):733. [Seventh International Congress of the World Muscle
Society].
Iannaccone ST, Muirhead D, Elmonoufy N, et al. Partial Telethonin
deficiency in severely affected north American LGMD patients.
Neuromuscul Disord 2002;12:732. [Seventh International Congress
of the World Muscle Society].
Valle G, Faulkner G, De Antoni A, et al. Telethonin, a novel
sarcomeric protein of heart and skeletal muscle. FEBS Lett 1997;
415:163–8.
544
M. Zatz et al. / Neuromuscular Disorders 13 (2003) 532–544
[93] Halliday W, Greenberg CR, Wrogemann K, et al. Genetic
heterogeneity of limb girdle muscular dystrophy in Manitoba
Hutterites. Am J Hum Genet 1998;63(Suppl):A392. [Abstract 2280].
[94] Shokeir MH, Kobrinsky NL. Autosomal recessive muscular
dystrophy in Manitoba Hutterites. Clin Genet 1976;9(2):197–202.
[95] Weiler T, Greenberg CR, Nylen E, et al. Limb girdle muscular
dystrophy in Manitoba Hutterites does not map to any of the known
LGMD loci. Am J Med Genet 1997;72(3):363–8.
[96] Weiler T, Greenberg CR, Zelinski T, et al. A gene for autosomal
recessive limb-girdle muscular dystrophy in Manitoba Hutterites
maps to chromosome region 9q31-q33: evidence for another LGMD
locus. Am J Hum Genet 1998;63:140 –7.
[97] Fridell RA, Harding LS, Bogerd HP, Cullen BR. Identification of a
novel human zinc finger protein that specifically interacts with the
activation domain of lentiviral Tat proteins. Virology 1995;209(2):
347–57.
[98] Slack FJ, Ruvkun G. A novel repeat domain that is often associated
with RING finger and B-box motifs. Trends Biochem Sci 1998;
23(12):474 –5.
[99] Driss A, Amouri R, Ben Hamida C, et al. A new locus for autosomal
recessive limb-girdle muscular dystrophy in a large consanguineous
Tunisian family maps to chromosome 19q13.3. Neuromuscul Disord
2000;10(4–5):240–6.
[100] Brockington M, Blake DJ, Prandini P, et al. Mutations in the fukutinrelated protein gene (FKRP) cause a form of congenital muscular
dystrophy with secondary laminin alpha2 deficiency and abnormal
glycosylation of alpha-dystroglycan. Am J Hum Genet 2001;69(6):
1198– 209.
[101] Fukuyama Y, Kawazura M, Haruna H. A peculiar form of congenital
progressive muscular dystrophy: report of fifteen cases. Paediatr
Univ Tokyo 1960;4:5 –8.
[102] Brockington M, Blake DJ, Brown SC, Muntoni F. The gene for a
novel glycosyltransferase is mutated in congenital muscular
dystrophy MDC1C and limb girdle muscular dystrophy 2I.
Neuromuscul Disord 2002;12(3):233– 4.
[103] Topaloglu H, Brockington M, Yuva Y, et al. FKRP gene mutations
cause congenital muscular dystrophy, mental retardation and
cerebellar cysts. Neurology 2003;60:988– 92.
[104] Prandini P, Boito C, Bakay M, Angelini C, Hoffman E, Pegoraro E.
Discordant clinical phenotype in a family affected with LGMD2I.
J Neurol Sci 2002;199(Suppl 1).
[105] Paula F, Starling A, Jazedje T, et al. Fukutin related protein gene
mutations in Brazilian limb girdle muscular dystrophy families.
Neuromuscul Disord 2002;12(7 – 8). [Seventh World Muscular
Society Congress].
[106] Mercuri E, Brockington M, Straub V, et al. Phenotypic spectrum
associated with mutations in the fukutin-related protein gene. Ann
Neurol 2003;53:537–42.
[107] Haravuori H, Makela-Bengs P, Udd B, et al. Assignment of the tibial
muscular dystrophy locus to chromosome 2q31. Am J Hum Genet
1998;62(3):620 –6.
[108] Labeit S, Kolmerer B. Titins: giant proteins in charge of muscle
ultrastructure and elasticity. Science 1995;270(5234):293–6.
[109] Poppe M, Cree L, Bourke J, et al. The phenotype of limb-girdle
muscular dystrophy type 2I. Neurology 2003;60:1246 –51.