Nathalie Bourg

Mutations in the proteolytic enzyme calpain 3 cause limb-girdle muscular dystrophy type 2A.

Limb-girdle muscular dystrophies (LGMDs) are a group of inherited diseases whose genetic etiology has yet to be elucidated. The autosomal recessive forms (LGMD2) constitute a genetically heterogeneous group with LGMD2A mapping to chromosome 15q15.1-q21.1. The gene encoding the muscle-specific calcium-activated neutral protease 3 (CANP3) large subunit is located in this region. This cysteine protease belongs to the family of intracellular calpains. Fifteen nonsense, splice site, frameshift, or missense calpain mutations cosegregate with the disease in LGMD2A families, six of which were found within La Réunion island patients. A digenic inheritance model is proposed to account for the unexpected presence of multiple independent mutations in this small inbred population. Finally, these results demonstrate an enzymatic rather than a structural protein defect causing a muscular dystrophy, a defect that may have regulatory consequences, perhaps in signal transduction.

β–sarcoglycan: characterization and role in limb–girdle muscular dystrophy linked to 4q12

β–sarcoglycan, a 43 kDa dystrophin–associated glycoprotein, is an integral component of the dystrophin–glycoprotein complex. We have cloned human β–sarcoglycan cDNA and mapped the β–sarcoglycan gene to chromosome 4q12. Pericentromeric markers and an intragenic polymorphic CA repeat cosegregated perfectly with autosomal recessive limb–girdle muscular dystrophy in several Amish families. A Thr–to–Arg missense mutation was identified within the β–sarcoglycan gene that leads to a dramatically reduced expression of β–sarcoglycan in the sarcolemma and a concomitant loss of adhalin and 35 DAG, which may represent a disruption of a functional subcomplex within the dystrophin–glycoprotein complex. Thus, the β–sarcoglycan gene is the fifth locus identified (LGMD2E) that is involved in autosomal recessive limb–girdle muscular dystrophy.

Calpain 3 deficiency is associated with myonuclear apoptosis and profoundperturbation of the IκBα/NF-κB pathway in limb-girdle musculardystrophy type 2A

Nature Med. 5, 503– 511 (1999). The top left corner of Fig. 1b on page 505 was cropped so that you could not view the calpain 3-stained nuclei in endomysia space. The corrected figure is shown below. We regret this error.

Beta-sarcoglycan: characterization and role in limb-girdle muscular dystrophy linked to 4q12.

β–sarcoglycan, a 43 kDa dystrophin–associated glycoprotein, is an integral component of the dystrophin–glycoprotein complex. We have cloned human β–sarcoglycan cDNA and mapped the β–sarcoglycan gene to chromosome 4q12. Pericentromeric markers and an intragenic polymorphic CA repeat cosegregated perfectly with autosomal recessive limb–girdle muscular dystrophy in several Amish families. A Thr–to–Arg missense mutation was identified within the β–sarcoglycan gene that leads to a dramatically reduced expression of β–sarcoglycan in the sarcolemma and a concomitant loss of adhalin and 35 DAG, which may represent a disruption of a functional subcomplex within the dystrophin–glycoprotein complex. Thus, the β–sarcoglycan gene is the fifth locus identified (LGMD2E) that is involved in autosomal recessive limb–girdle muscular dystrophy.

Calpain 3 Is Activated through Autolysis within the Active Site and Lyses Sarcomeric and Sarcolemmal Components

ABSTRACT Calpain 3 (Capn3) is known as the skeletal muscle-specific member of the calpains, a family of intracellular nonlysosomal cysteine proteases. This enigmatic protease has many unique features among the calpain family and, importantly, mutations in Capn3 have been shown to be responsible for limb girdle muscular dystrophy type 2A. Here we demonstrate that the Capn3 activation mechanism is similar to the universal activation of caspases and corresponds to an autolysis within the active site of the protease. We undertook a search for substrates in immature muscle cells, as several lines of evidence suggest that Capn3 is mostly in an inactive state in muscle and needs a signal to be activated. In this model, Capn3 proteolytic activity leads to disruption of the actin cytoskeleton and disorganization of focal adhesions through cleavage of several endogenous proteins. In addition, we show that titin, a previously identified Capn3 partner, and filamin C are further substrates of Capn3. Finally, we report that Capn3 colocalizes in vivo with its substrates at various sites along cytoskeletal structures. We propose that Capn3-mediated cleavage produces an adaptive response of muscle cells to external and/or internal stimuli, establishing Capn3 as a muscle cytoskeleton regulator.

Genomic organization of the dysferlin gene and novel mutations in Miyoshi myopathy

Objective: Mutations in the skeletal muscle gene dysferlin cause two autosomal recessive forms of muscular dystrophy: Miyoshi myopathy (MM) and limb girdle muscular dystrophy type 2B (LGMD2B). The purpose of this study was to define the genomic organization of the dysferlin gene and conduct mutational screening and a survey of clinical features in 21 patients with defined molecular defects in the dysferlin gene. Methods: Genomic organization of the gene was determined by comparing the dysferlin cDNA and genomic sequence in P1-derived artificial chromosomes (PACs) containing the gene. Mutational screening entailed conformational analysis and sequencing of genomic DNA and cDNA. Clinical records of patients with defined dysferlin gene defects were reviewed retrospectively. Results: The dysferlin gene encompasses 55 exons spanning over 150 kb of genomic DNA. Mutational screening revealed nine novel mutations associated with MM. The range of onset in this patient group was narrow with a mean of 19.0 ± 3.9 years. Conclusion: This study confirms that the dysferlin gene is mutated in MM and LGMD2B and extends understanding of the timing of onset of the disease. Knowledge of the genomic organization of the gene will facilitate mutation detection and investigations of the molecular biologic properties of the dysferlin gene.

Efficient recovery of dysferlin deficiency by dual adeno-associated vector-mediated gene transfer

Deficiency of the dysferlin protein presents as two major clinical phenotypes: limb-girdle muscular dystrophy type 2B and Miyoshi myopathy. Dysferlin is known to participate in membrane repair, providing a potential hypothesis to the underlying pathophysiology of these diseases. The size of the dysferlin cDNA prevents its direct incorporation into an adeno-associated virus (AAV) vector for therapeutic gene transfer into muscle. To bypass this limitation, we split the dysferlin cDNA at the exon 28/29 junction and cloned it into two independent AAV vectors carrying the appropriate splicing sequences. Intramuscular injection of the corresponding vectors into a dysferlin-deficient mouse model led to the expression of full-length dysferlin for at least 1 year. Importantly, systemic injection in the tail vein of the two vectors led to a widespread although weak expression of the full-length protein. Injections were associated with an improvement of the histological aspect of the muscle, a reduction in the number of necrotic fibers, restoration of membrane repair capacity and a global improvement in locomotor activity. Altogether, these data support the use of such a strategy for the treatment of dysferlin deficiency.

NF-κB-dependent expression of the antiapoptotic factor c-FLIP is regulated by calpain 3, the protein involved in limb-girdle muscular dystrophy type 2A

Limb‐girdle muscular dystrophy type 2A (LGMD2A) is a recessive genetic disorder caused by mutations in the cysteine protease calpain 3 (CAPN3) that leads to selective muscle wasting. We previously showed that CAPN3 deficiency is associated with a profound perturbation of the NF‐NF‐κBB/INF‐κBBα survival pathway. In this study, the consequences of altered NF‐BNF‐κBB/IBNF‐κBBBα pathway were investigated using biological materials from LGMD2A patients. We first show that the antiapoptotic factor cellular‐FLICE inhibitory protein (C‐FLIP), which is dependent on the NF‐BNF‐κBB pathway in normal muscle cells, is down‐regulated in LGMD2A biopsies. In muscle cells isolated from LGMD2A patients, NF‐BNF‐κBB is readily acti vated on cytokine induction as shown by an increase in its DNA binding activity. However, we observed discrepant transcriptional responses depending on the NF‐BNF‐κBB target genes. IBNF‐κBBBα is expressed following NF‐BNF‐κBB activation independent of the CAPN3 status, whereas expression of C‐FLIP is obtained only when CAPN3 is present. These data lead us to postulate that CAPN3 intervenes in the regulation of the expression of NF‐BNF‐κBB‐dependent survival genes to prevent apoptosis in skeletal muscle. Deregulations in the NF‐BNF‐κBB pathway could be part of the mecha nism responsible for the muscle wasting resulting from CAPN3 deficiency.—Benayoun, B., Baghdiguian, S., Lajmanovich, A., Bartoli, M., Daniele, N., Gicquel, E., Bourg, N., Raynaud, F., Pasquier, M.‐A., Suel, L., Lochmuller, H., Lefranc, G., Richard, I. NF‐BNF‐κBB‐dependent expression of the antiapoptotic factor C‐FLIP is regulated by calpain 3, the protein involved in limb‐girdle muscular dystrophy type 2A. FASEB J. 22, 1521–1529 (2008)

Cardiac ankyrin repeat protein is a marker of skeletal muscle pathological remodelling

In an attempt to identify potential therapeutic targets for the correction of muscle wasting, the gene expression of several pivotal proteins involved in protein metabolism was investigated in experimental atrophy induced by transient or definitive denervation, as well as in four animal models of muscular dystrophies (deficient for calpain 3, dysferlin, α‐sarcoglycan and dystrophin, respectively). The results showed that: (a) the components of the ubiquitin–proteasome pathway are upregulated during the very early phases of atrophy but do not greatly increase in the muscular dystrophy models; (b) forkhead box protein O1 mRNA expression is augmented in the muscles of a limb girdle muscular dystrophy 2A murine model; and (c) the expression of cardiac ankyrin repeat protein (CARP), a regulator of transcription factors, appears to be persistently upregulated in every condition, suggesting that CARP could be a hub protein participating in common pathological molecular pathway(s). Interestingly, the mRNA level of a cell cycle inhibitor known to be upregulated by CARP in other tissues, p21WAF1/CIP1, is consistently increased whenever CARP is upregulated. CARP overexpression in muscle fibres fails to affect their calibre, indicating that CARP per se cannot initiate atrophy. However, a switch towards fast‐twitch fibres is observed, suggesting that CARP plays a role in skeletal muscle plasticity. The observation that p21WAF1/CIP1 is upregulated, put in perspective with the effects of CARP on the fibre type, fits well with the idea that the mechanisms at stake might be required to oppose muscle remodelling in skeletal muscle.

β-Sarcoglycan: genomic analysis and identification of a novel missense mutation in the LGMD2E Amish isolate

The sarcoglycan complex is involved in the etiology of four autosomal recessive limb-girdle muscular dystrophies (LGMD2C-F). A missense mutation (T151R) in the beta-sarcoglycan gene on chromosome 4q12 has been shown to cause a mild form of LGMD2E in 11 families from a Southern Indiana Amish community sharing a common haplotype. We now report that two sibs from another Amish family with mild LGMD2E are compound heterozygotes for chromosome 4q12 markers. In order to characterize the genetic defect in this new family, we determined the genomic organization of the beta-sarcoglycan gene. A second missense mutation (R91C) has now been identified in this LGMD2E Amish family. This mutation is also present in the homozygous state in another family of probable Amish ancestry. Finally, analysis of all the components of the dystrophin-glycoprotein complex was performed for the first time on a biopsy from a patient homozygous for the beta-sarcoglycan mutation (T151R). Interestingly, in addition to the loss of the entire sarcoglycan complex, we detected a reduction of alpha-dystroglycan which suggests a role for the sarcoglycan complex in stabilizing alpha-dystroglycan at the sarcolemma.