Analysis of histopathologic and molecular pathologic findings in Czech LGMD2A patients

Abstract

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 (p 94). 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 550 delA is the most frequent CAPN3 defect in Czech LGMD2A patients (9 alleles of 28). Furthermore, we established that, in a patient with the 550 delA/R490W genotype, mRNA carrying frameshift mutation 550 delA 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 p 94 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.

Transcriptional and translational effects of intronic CAPN3 gene mutations

“…This mutation was identified in 6 unrelated LGMD2A patients in our series, and also in several previous reports, where its pathogenetic effects have been suggested [Piluso et al, 2005; Hermanova et al, 2006; Leiden Database], but unsupported by experimental data [Stehlikova et al, 2007; Krahn et al, 2007], or even reported as a polymorphism [Groen et al, 2007; Leiden Database]. …”

“…Furthermore, because of the lack of a molecular proof, some intronic variants have been reported either as polymorphisms or as “possibly pathogenetic”, thus generating confusion and compromising a conclusive genetic counselling. This is the case of the variation c. 1746-20C>G in intron 13 [Hermanova et al, 2006; Stehlikova et al, 2007; Krahn et al, 2007; Groen et al, 2007], for which we have provided a definite demonstration of the pathogenetic effect.…”

Comparison of Dysferlin Expression in Human Skeletal Muscle with That in Monocytes for the Diagnosis of Dysferlin Myopathy

“…Disruption of proteins in the dysferlin complex can affect the subcellular localization of dysferlin in the absence of mutations in the gene DYSF and may explain the abnormal immunohistochemical results described in the above-mentioned studies [14], [22], [28], [29], [30]. For this reason it is important to quantify dysferlin expression in skeletal muscle WB as well as by IH.…”

Limb-girdle muscular dystrophy type 2A

“…[14] Among the genetically confirmed Czech LGMD-2A patients, a few have been found on histopathology to have neurogenic changes in the muscle biopsy. [15] In one of the Japanese studies, pelvic and shoulder girdle muscle involvement was found to be the most prominent feature. [16] The article by Pathak et al [12] in this issue would be the first authentic report on phenotypic presentation of this Indian LGMD-2A cohort.…”