Analysis of the UK diagnostic strategy for limb girdle muscular dystrophy 2A.

Diagnosis of limb girdle muscular dystrophy type 2A can be complex due to phenotypic variability, lack of precision of protein analysis in muscle biopsies, and absence of mutational hot spots in the CAPN3 gene. The aim of this study was to review clinical and biopsy data from a group of patients with known CAPN3 genetic status to validate and refine our current diagnostic strategy, which combines clinical information and protein analysis to direct gene testing. We analysed 85 patients in whom CAPN3 gene sequencing had been performed. Forty-two patients had two mutations, 15 a single mutation and in 28 no mutation was found. We identified clinical features that clearly discriminated the LGMD2A patients. These were: presence of scapular winging, contractures and normal respiratory function. In addition, a typical pattern of muscle weakness on manual muscle testing could be confirmed. Interpretation of protein expression obtained by Western blot was complex and involved the analysis of a number of bands detected by two antibodies for calpain 3. Loss of all calpain 3 bands was 100% specific for LGMD2A, but this pattern was found in only 23%. Absence or reduction of the approximately 60 kDa bands was also highly specific for LGMD2A, while increased abundance was highly predictive of no mutations being found even where other bands were reduced, suggesting that this is the most sensitive marker of artefactual protein degradation. Twenty-three percent of the patients with two mutations had normal full-sized calpain 3 protein, consistent with the finding of mutations localized in parts of the gene likely or proven to be involved in autolytic activity. Clinical and biochemical findings in patients with only one mutation were similar to patients with two mutations, indicating that other gene analysis techniques should be used before excluding the diagnosis. Our analysis confirms that our strategy is still valid to prioritize genetic testing in this complex group of patients, provided patients with normal protein but a suggestive clinical phenotype are not excluded from genetic testing.

A new evidence for the maintenance of the sarcoglycan complex in muscle sarcolemma in spite of the primary absence of delta-SG protein.

delta-Sarcoglycan (delta-SG) is one of the first proteins of the sarcoglycan complex (SGC) to be expressed during muscle development, and it has been considered fundamental for the assembling and insertion of the SGC in the sarcolemma. Studies using heterologous cell systems and co-precipitation have demonstrated that SGC assembly was dependent on the simultaneous synthesis of all four sarcoglycan proteins. Mutations in any one of sarcoglycan genes, including the common disease causing mutation c.656delC in the delta-SG gene, block complex formation and its insertion in the plasma membrane. Failure in complex assembly in patients with this mutation would be therefore expected. In this study, we provide evidence for the possibility of preservation of part of the SG complex in the sarcolemma, even in the absence of delta-SG. This is based on the study of one mildly affected patient with limb-girdle muscular dystrophy type 2F (LGMD2F) due to the homozygous c.656delC mutation in the delta-SG gene. Protein analysis in his muscle biopsy presented a significant deficiency of only delta-SG with retention of the other three SG proteins in the sarcolemma. RNA expression analysis showed that zeta-SG, a functionally homologous to delta-SG, is not atypically upregulated in his muscle and would not replace the absent delta-SG, retaining the complex alpha-beta-gamma-zeta. The patient started clinical manifestation at age 25, with frequent falls, but he is currently able to walk unassisted at age 42. His clinical course is significantly milder when compared to several other affected patients carrying the same mutation associated with a total deficiency of the four SG proteins in the muscle studied by our group and confirmed in other patients. Therefore, our results add a new in vivo evidence that alpha-, beta-, and gamma-SG proteins can be maintained in the sarcolemma without delta-SG. Additionally, LGMD2F, with retention of the part of the SGC, might be associated to a milder clinical course, which has important implications for clinical prognosis and genetic counseling of the family.

AHNAK, a novel component of the dysferlin protein complex, redistributes to the cytoplasm with dysferlin during skeletal muscle regeneration.

Mutations in dysferlin cause limb girdle muscular dystrophy 2B, Miyoshi myopathy and distal anterior compartment myopathy. Dysferlin is proposed to play a role in muscle membrane repair. To gain functional insight into the molecular mechanisms of dysferlin, we have searched for dysferlin-interacting proteins in skeletal muscle. By coimmunoprecipitation coupled with mass spectrometry, we demonstrate that AHNAK interacts with dysferlin. We defined the binding sites in dysferlin and AHNAK as the C2A domain in dysferlin and the carboxyterminal domain of AHNAK by glutathione S-transferase (GST)-pull down assays. As expected, the N-terminal domain of myoferlin also interacts with the carboxyterminal domain of AHNAK. In normal skeletal muscle, dysferlin and AHNAK colocalize at the sarcolemmal membrane and T-tubules. In dysferlinopathies, reduction or absence of dysferlin correlates with a secondary muscle-specific loss of AHNAK. Moreover, in regenerating rat muscle, dysferlin and AHNAK showed a marked increase and cytoplasmic localization, consistent with the direct interaction between them. Our data suggest that dysferlin participates in the recruitment and stabilization of AHNAK to the sarcolemma and that AHNAK plays a role in dysferlin membrane repair process. It may also have significant implications for understanding the biology of AHNAK-containing exocytotic vesicles, "enlargosomes," in plasma membrane remodeling and repair.

Patients with a non-dysferlin Miyoshi myopathy have a novel membrane repair defect.

Two autosomal recessive muscle diseases, limb girdle muscular dystrophy type 2B (LGMD2B) and Miyoshi myopathy (MM), are caused by mutations in the dysferlin gene. These mutations result in poor ability to repair cell membrane damage, which is suggested to be the cause for this disease. However, many patients who share clinical features with MM-type muscular dystrophy do not carry mutations in dysferlin gene. To understand the basis of MM that is not due to mutations in dysferlin gene, we analyzed cells from patients in one such family. In these patients, we found no defects in several potential candidates - annexin A2, caveolin-3, myoferlin and the MMD2 locus on chromosome 10p. Similar to dysferlinopathy, these cells also exhibit membrane repair defects and the severity of the defect correlated with severity of their disease. However, unlike dysferlinopathy, none of the conventional membrane repair pathways are defective in these patient cells. These results add to the existing evidence that cell membrane repair defect may be responsible for MM-type muscular dystrophy and indicate that a previously unsuspected genetic lesion that affects cell membrane repair pathway is responsible for the disease in the non-dysferlin MM patients.

Identification of putative in vivo substrates of calpain 3 by comparative proteomics of overexpressing transgenic and nontransgenic mice.

Calpain 3 (CAPN3) is a calcium-dependent protease, mutations in which cause limb girdle muscular dystrophy type 2A. To explore the physiological function of CAPN3, we compared the proteomes of transgenic mice that overexpress CAPN3 (CAPN3 Tg) and their nontransgenic (non-Tg) counterparts. We first examined known muscular dystrophy-related proteins to determine if overexpression of CAPN3 results in a change in their distribution or concentration. This analysis did not identify any known muscular dystrophy proteins as substrates of CAPN3. Next, we used a proteomic approach to compare and identify differentially represented proteins in 2-DE of CAPN3 Tg and non-Tg mice. LC-MS/MS analysis led to the identification of ten possible substrates for CAPN3, classified into two major functional categories: metabolic and myofibrillar. Myosin light chain 1 (MLC1) was focused upon because our previous studies suggested a role for CAPN3 in sarcomere remodeling. In this study, CAPN3 was shown to proteolyze MLC1 in vitro. These studies are the first to identify possible substrates for CAPN3 in an in vivo system and support a role for CAPN3 in sarcomere remodeling by cleavage of myofibrillar proteins such as MLC1. In addition, these data also suggest a role for CAPN3 in mitochondrial protein turnover.

The differential gene expression profiles of proximal and distal muscle groups are altered in pre-pathological dysferlin-deficient mice.

The selective pattern of muscle involvement is a key feature of muscular dystrophies. Dysferlinopathy is a good model for studying this process since it shows variable muscle involvement that can be highly selective even in individual patients. The transcriptomes of proximal and distal muscles from wildtype C57BL/10 and dysferlin deficient C57BL/10.SJL-Dysf mice at a prepathological stage were assessed using the Affymetrix oligonucleotide-microarray system. We detected significant variation in gene expression between proximal and distal muscle in wildtype mice. Dysferlin defiency, even in the absence of pathological changes, altered this proximal distal difference but with little specific overlap with previous microarray analyses of dysferlinopathy. In conclusion, proximal and distal muscle groups show distinct patterns of gene expression and respond differently to dysferlin deficiency. This has implications for the selection of muscles for future microarray analyses, and also offers new routes for investigating the selectivity of muscle involvement in muscular dystrophies.

Protein studies in dysferlinopathy patients using llama-derived antibody fragments selected by phage display.

Mutations in dysferlin, a member of the fer1-like protein family that plays a role in membrane integrity and repair, can give rise to a spectrum of neuromuscular disorders with phenotypic variability including limb-girdle muscular dystrophy 2B, Myoshi myopathy and distal anterior compartment myopathy. To improve the tools available for understanding the pathogenesis of the dysferlinopathies, we have established a large source of highly specific antibody reagents against dysferlin by selection of heavy-chain antibody fragments originating from a nonimmune llama-derived phage-display library. By utilizing different truncated forms of recombinant dysferlin for selection and diverse selection methodologies, antibody fragments with specificity for two different dysferlin domains could be identified. The selected llama antibody fragments are functional in Western blotting, immunofluorescence microscopy and immunoprecipitation applications. Using these antibody fragments, we found that calpain 3, which shows a secondary reduction in the dysferlinopathies, interacts with dysferlin.

Refined mapping of the Welander distal myopathy region on chromosome 2p13 positions the new candidate region telomeric of the DYSF locus.

Welander distal myopathy (WDM) is a late adult-onset autosomal dominant disorder, characterized by a slow progression and distal limb weakness of the extremity muscles. The WDM locus has been mapped to chromosome 2p13. Within this region a common shared haplotype co-segregates in all affected patients, indicating a founder effect. By undertaking an extended linkage analysis we have significantly reduced the WDM locus to a critical interval of approximately 1.2 Mb flanked by markers D2S358 and PAC3-H52. The dysferlin gene, a strong candidate gene responsible for two other distal myopathies in the same region, is located centromeric to PAC3-H52 and can thereby formally be excluded as cause for WDM.

Sarcoglycans of the zebrafish: orthology and localization to the sarcolemma and myosepta of muscle.

The zebrafish is an established model of vertebrate development and is also receiving increasing attention in terms of human disease modelling. In order to provide experimental support to realize this modelling potential, we report here the identification of apparent orthologues of many critical members of the dystrophin-associated glycoprotein complex (DGC) that have been implicated in a diverse range of neuromuscular disorders. In addition, immunohistochemical studies show the localization of the DGC to the sarcolemma of adult zebrafish muscle and in particular the myosepta. Together, these data suggest that the DGC in adult zebrafish may play a highly conserved functional role in muscle architecture that, when disrupted, could offer insight into human neuromuscular disease processes.