Redox state and mitochondrial respiratory chain function in skeletal muscle of LGMD2A patients.

BACKGROUND: Calpain-3 deficiency causes oxidative and nitrosative stress-induced damage in skeletal muscle of LGMD2A patients, but mitochondrial respiratory chain function and anti-oxidant levels have not been systematically assessed in this clinical population previously. METHODS: We identified 14 patients with phenotypes consistent with LGMD2A and performed CAPN3 gene sequencing, CAPN3 expression/autolysis measurements, and in silico predictions of pathogenicity. Oxidative damage, anti-oxidant capacity, and mitochondrial enzyme activities were determined in a subset of muscle biopsies. RESULTS: Twenty-one disease-causing variants were detected along the entire CAPN3 gene, five of which were novel (c.338 T>C, c.500 T>C, c.1525-1 G>T, c.2115+4 T>G, c.2366 T>A). Protein- and mRNA-based tests confirmed in silico predictions and the clinical diagnosis in 75% of patients. Reductions in antioxidant defense mechanisms (SOD-1 and NRF-2, but not SOD-2), coupled with increased lipid peroxidation and protein ubiquitination, were observed in calpain-3 deficient muscle, indicating a redox imbalance primarily affecting non-mitochondrial compartments. Although ATP synthase levels were significantly lower in LGMD2A patients, citrate synthase, cytochrome c oxidase, and complex I+III activities were not different from controls. CONCLUSIONS: Despite significant oxidative damage and redox imbalance in cytosolic/myofibrillar compartments, mitochondrial respiratory chain function is largely maintained in skeletal muscle of LGMD2A patients.

Dysferlin aggregation in limb-girdle muscular dystrophy type 2B/Miyoshi Myopathy necessitates mutational screen for diagnosis [corrected].

INTRODUCTION: Diagnosis of the limb-girdle muscular dystrophies (LGMDs) has been facilitated by the use of immunofluorescence microscopy, Western blot analysis, and rapid genetic testing. METHODS: We identified 7 patients with LGMD2B or Miyoshi myopathy (MM) phenotypes and performed detailed history, physical examination, and mutation analyses of genomic DNA. RESULTS: Ten disease-causing variants of the dysferlin gene (DYSF) were detected, 4 of which were novel and predicted to be pathogenic (IVS33+9G>T, c.1343T>C, c.4747T>G, and c.5066dupC). Two of these mutations (c.1343T>C and IVS33+9G>T) were associated with a reduction in sarcolemmal dysferlin expression, despite increased total mRNA and protein in mixed muscle homogenates, due to a pathological retention of the mutated polypeptide in the cytoplasm. CONCLUSIONS: Considering that protein-based assays may yield false negative test results and that dysferlin aggregation may be present in other LGMDs, mutational screening is necessary for specific diagnosis in primary dysferlinopathy patients exhibiting this phenotype.