(last modified September 26, 2004)
Links to other databases:
Gene Symbol nomenclature Locus Link db OMIM Gene Map GDB
The human calpain-3 gene (Gene Symbol CAPN3, alias p94) localizes to chromosome 15q15.1-15.3 and has 24 exons spanning some 53 kb of DNA. The gene is exceptional by having many rather small exons; 10 exons are only 58-86 bp, while exons 12, 15 and 14 are even smaller, 12, 18 and 37 bp resp. Most introns vary in size between 0.2-2.6 kb. Exceptional are introns 18 and 20, measuring below 100 bp, and intron 1, with a size of 24.3 kb covering about half of the gene.
Markers around the gene include D5S514, D5S779, CAPN3, D5S782, D5S780 and D5S778.
|Exon||Exon size (bp)||Intron size (kb)||5' cDNA position||Splice after||Remarks|
|1||>310||24.368||0||5' UTR / 309 bp coding|
|24||>...||-||2440||-||.. / 3' UTR|
Intron sizes were derived from GenBank files NM_000070 and AC012651, containing the human CAPN3 cDNA and genomic sequences respectively.
The calpain-3 gene is predominantly expressed in skeletal muscle tissue as a 3.5 kb transcript. Additional transcripts, derived from alternative splicing and the use of alternative promoter have also been identified.
Calpain-3 (CAPN3) is a muscle-specific member of the calpain family, a group of nonlysosomal calcium-dependent cysteine proteases whose functional role is largely unknown. CAPN3 is a 94 kDa protein containing four main domains and three short unique inserted sequences, NS, IS1 and IS2;
IS2 contains a titin-binding site and and a nuclear location signal (NLS).
To elucidate the molecular mechanism of LGMD2A, Ono et al. (1998) constructed nine CAPN3 missense mutations found in LGMD2A and analysed the functional consequences. All mutants completely or almost completely lost their proteolytic activity against a potential substrate, fodrin. Some of the mutants retained their autolytic activity and/or their connectin/titin binding ability, indicating that these properties are not involved in the LGMD-2A phenotype. The results of Ono et al. (1998) provide strong evidence that LGMD-2A results from the loss of proteolysis of substrates by calpain-3, suggesting a novel molecular mechanism leading to muscular dystrophies.
Links to other databases: OMIM: 253600 Human Gene Mutation Database
Limb-girdle muscular dystrophy type 2A (LGMD-2A, MIM 253600) is an autosomal recessive neuromuscular disorder. LGMD-2A is characterized mainly by symmetrical and selective atrophy of the pelvic, scapular and trunk muscles, elevated serum creatine kinase and a necrotic regeneration pattern on muscular biopsies. Calf hypertrophy is rare and there is no mental, cardiac or facial disturbance. In most cases symptoms occur in childhood, progress gradually and frequently, 10-20 years after onset, patients become unable to walk (Beckmann & Fardeau 1998....). In comparison to patients with sarcoglycanopathies (LGMD2C-2F), LGMD-2A patients do not have the marked quadriceps femoris involvement, calf hypertrophy is less frequent and macroglossia is never seen (Richard 1999).
The gene for LGMD-2A was mapped to a 1 cM region of chromosome 15q51.1-15.3, both using linkage and linkage-disequilibrium analysis (Beckmann , Allamand ). A detailed characterization of the genes in this region ultimately lead to the identification of the gene involved, calpain-3 (Chiannilkulchai , Richard ). Mutations in LGMD-2A patients in the CAPN3 gene were first identified by Richard et al. .
The most extensive mutation study published so far (Richard ) was based on the analysis of 181 families from 19 countries. It reports the identification of 97 distinct pathogenic mutations (4 nonsense, 32 deletions / insertions, 8 splice site and 53 missense mutations), together with 12 polymorphisms and 5 unclassified variants. In 24 of the 181 families only one mutant allele could be identified. A study of Ono et al. (1998) provided strong evidence that LGMD-2A results from the loss of proteolysis of substrates by calpain-3, suggesting a novel molecular mechanism leading to muscular dystrophies.
Mutations were distributed along the entire length of the CAPN3 gene. A small hot spot is present in exon 21 and exon 11 has an excess of missens mutations. Although some mutations are found more frequently, most represent private variants. Some mutations were found on only one haplotype, suggesting a common ancestry. These include c.550delA, c.946-1G>A in families from Reunion Island, c.1611C>A (Tyr537X) in Turkish and metropolitan French families, c.2105C>T (Ala702Val) in Turkish and metropolitan French families, c.2306G>A (Arg769Gln) in the Amish community and metropolitan French families and c.2362AG>TCATCT in Basques and families from Reunion Island.
Genotype / phenotype analysis (Richard ) revealed no evident correlations between mutations and the clinical phenotype except for a quite homogeneous age at onset (15 years) for patients carrying two null mutations. These patients usually lost walking ability before the age of 40. In contrast, age at onset in other patient categories varied largely from as early as 2.5 and as late as 40 years of age.
To streamline the genetic analysis, Richard et al... developed a LGMD type 2 screening protocol. The first step uses a fluorescent set of markers bracketing the LGMD2A-2F disease loci to identify the LGMD-type involved. In a second step, haplotype analysis in combination with the geographic origin of the patient is used to infer a potential ancestral origin thereby pointing to specific mutation(s).
Using Western blot analysis of LGMD2A-patients, Fanin et al. (2003) observed that a considerable proportion (20%) had a normal CAPN3 expression. They noticed that while in control muscle the calpain-3 Ca2+-dependent autocatalytic activity was evident within 5 minutes, in all mutant patient samples the protein was not degraded, indicating that the normal autocatalytic function had been lost. Using this functional test, they were able to show the pathogenetic role of several missense mutations (incl. Arg489Gln, Arg490Gln and Arg490Trp).
Calpains are intracellular calcium-modulated nonlysosomal cysteine proteases (responding to Ca2+-signals) removing portions of protein substrates, thereby irreversibly modifying their function (for review see Branca ). Members of the calpain protease family are present in a variety of organisms ranging from mammals to plants. Some calpains are ubiquitously expressed, while others are tissue specific. Calpains are thought to be involved in a multitude of physiological and pathological events, but their precise functions are only poorly understood. In two cases the alteration of a calpain has been identified as being responsible for a human disease; (i) calpain-3 causing limb girdle muscular dystrophy type 2A, and (ii) calpain-10 involved in non-insulin-dependent diabetes (OMIM:605286).
Titin and filamin C have been shown to be substrates of CAPN3 (Taveau ). In vivo CAPN3 colocalizes with these substrates at various sites along cytoskeletal structures (Taveau ). These authors 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.
Kramerova (2004) recently generated a Capn3 knockout mouse (C3KO). The mice were atrophic and contained small foci of muscular necrosis. Myogenic cells fused normally in vitro, but lacked well-organized sarcomeres (electron microscopy analysis). Titin distribution was normal in longitudinal sections but EM-analysis of muscle fibers showed misaligned A-bands. In vitro studies revealed that Capn3 can bind and cleave titin and that some human muscular dystrophy pathogenic mutations result in reduced affinity of CAPN3 for titin, which suggests a role for CAPN3 in myofibrillogenesis and sarcomere remodeling
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