Analysis of histopathologic and molecular pathologic findings in Czech LGMD2A patients

Limb-girdle muscular dystrophy type 2A (LGMD2A) is an autosomal-recessive disorder characterized by selective atrophy and progressive weakness of proximal girdle muscles. LGMD2A, the most prevalent form of LGMD, is caused by mutations in the CAPN3 gene that encodes the skeletal muscle-specific member of the calpain family, calpain-3 (p94). We examined the histopathologic and molecular pathologic findings in 14 Czech LGMD2A patients. Analysis of the CAPN3 gene was performed at the mRNA level, using reverse transcription-polymerase chain reaction (RT-PCR) and sequencing, and/or DNA level, using PCR and denaturing high-performance liquid chromatography (DHPLC). Our results confirm that mutation 550delA is the most frequent CAPN3 defect in Czech LGMD2A patients (9 alleles of 28). Furthermore, we established that, in a patient with the 550delA/R490W genotype, mRNA carrying frameshift mutation 550delA was not detected, probably due to its degradation by nonsense-mediated mRNA decay. In muscle biopsies of two LGMD2A patients, a neurogenic pattern simulating a neurogenic lesion was observed. Immunoblot analysis revealed the deficiency of p94 in all genetically confirmed cases of LGMD2A, and secondary dysferlin deficiency was demonstrated on muscle membranes in 6 patients using immunofluorescence. Thus, we find a combination of DNA and mRNA mutational analysis to be useful in the diagnosis of LGMD2A. Moreover, our study expands the spectrum of calpainopathies to cases that simulate a neurogenic lesion in muscle biopsies, and the knowledge of possible secondary deficiencies of muscular proteins also contributes to a diagnosis of LGMD2A.