Limb girdle muscular dystrophy in a sibling pair with a homozygous Ser606Leu mutation in the alternatively spliced IS2 region of calpain 3

Abstract

Previous family studies revealed a large number of calpain 3 ( CAPN3 ) mutations that cause recessive forms of limb girdle muscular dystrophy (LGMD2A) with selective atrophy of the proximal limb muscles. Correlations between the nature and site of a particular mutation and its corresponding phenotype, however, can only be established from homozygous mutations, which are particularly rare in the alternatively spliced NS, IS1 and IS2 regions of CAPN3. Here we identified a sibling pair with LGMD2A-type muscular dystrophy caused by a homozygous Ser606Leu (S606L) substitution in the IS2 linker domain. Normal protein levels, unaltered myofibrillar targeting and conserved calcium-induced autocatalytic activity of the mutated protein could be demonstrated in muscle biopsies from one patient. Despite this inconspicuous modification of the IS2 linker between domains III and IV, both patients developed signs and symptoms of the disease within their second decade of life. The unexpected severity of the clinical manifestation points to the high relevance of the calpain 3-specific IS2 segment between domains III and IV. We conclude that the structural motif around the Ser606 residue represents an important functional site that may regulate the transient activation and limited proteolysis of calpain 3.

Calpain 3, the “gatekeeper” of proper sarcomere assembly, turnover and maintenance

“…Pathogenicity was also shown to be associated with loss of “proper proteolytic activity” as some mutations have been shown to reduce the ability of CAPN3 to proteolyze potential substrates such as fodrin and HSP60 or to autolytically self-cleave [42-44]. However, loss of enzymatic activity is not the case for some pathogenic LGMD2A missense mutations and therefore, it suggests that the latter may simply prevent substrate specificity or that CAPN3 may perform other as yet unidentified non-proteolytic roles (eg [45-47]).…”

“…The fact that IS1 and IS2 [34, 45, 56] also harbor pathogenic mutations (including null-type mutations) suggests that it is the mature full-length product of CAPN3 (i.e., p94 as it was initially named by its discoverers [20]) that constitutes the (main) etiologic target of LGMD2A: indeed, these mutations are not likely to affect the alternatively spliced isoforms lacking these particular sequences, leaving only mature full-length CAPN3 as the defective entity. This is further corroborated by the observation that (i) while the alternatively spliced isoforms are synthesized during development and muscle maturation, they are absent in fully differentiated muscles and (ii) that LGMD2A patients or animal models with obliterated calpain 3 genes, are born with apparently normally constituted muscles, yet the disease settles in progressively as they grow and age.…”

Calpain 3: a key regulator of the sarcomere?

“…The predominant product of this gene is encoded by 24 exons corresponding to a 3316‐bp mRNA and is principally expressed in adult skeletal muscle in fast‐twitch and slow‐twitch fibers [2,6]. Accordingly, the phenotype of LGMD2A affects both types of fiber [7].…”

“…To add more complexity, it has been shown that S606L, a mutation located in IS2, leads to a normal calpain 3 level, with autolytic activity as well as correct subcellular localization [7]. Interestingly, in vitro analysis of other LGMD2A missense mutations (S744G and R769Q) indicated that they retain autolytic activity as well [15].…”

Limb-Girdle Muscular Dystrophy Type 2A Resulting From c.C479G and c.G1818A Mutations in the Calpain-3 Gene

“…4 Although c.G1818A has not been previously reported, the homozygous presence of a missense mutation at c.C1817T was previously reported on 2 siblings with LGMD2A onset in the second decade of life. 9 Thus, defects on the IS2 sequence also result in disease. There is growing evidence that synonymous changes contribute to disease.…”