Jana Sedláčková

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. Muscle Nerve, 2006

Quantitative analysis of CAPN3 transcripts in LGMD2A patients: involvement of nonsense-mediated mRNA decay.

Limb girdle muscular dystrophy type 2A (LGMD2A) is caused by single or small nucleotide changes widespread along the CAPN3 gene, which encodes the muscle-specific proteolytic enzyme calpain-3. About 356 unique allelic variants of CAPN3 have been identified to date. We performed analysis of the CAPN3 gene in LGMD2A patients at both the mRNA level using reverse transcription-PCR, and at the DNA level using PCR and denaturing high performance liquid chromatography. In four patients, we detected homozygous occurrence of a missense mutation or an in-frame deletion at the mRNA level although the DNA was heterozygous for this mutation in conjunction with a frame-shift mutation. The relationship observed in 12 patients between the quantity of CAPN3 mRNA, determined using real-time PCR, and the genotype leads us to propose that CAPN3 mRNAs which contain frame-shift mutations are degraded by nonsense-mediated mRNA decay. Our results illustrate the importance of DNA analysis for reliable establishment of mutation status, and provide a new insight into the process of mRNA decay in cells of LGMD2A patients.

Charcot-Marie-Tooth neuropathy type 1A combined with Duchenne muscular dystrophy

We report a 24‐year‐old male with an unusual combination of two inherited neuromuscular disorders – Charcot‐Marie‐Tooth (CMT) disease type 1A and Duchenne muscular dystrophy (DMD). A phenotypic presentation of this patient included features of both these disorders. Nerve conduction studies revealed demyelinating peripheral neuropathy. Electromyography showed a profound myogenic pattern. The serum creatine kinase level was highly elevated. Muscle biopsy revealed a dystrophic picture with deficient dystrophin immunostaining. CMT1A duplication on chromosome 17p11.2 was found. The frame‐shift mutation c.3609–3612delTAAAinsCTT (p.K1204LfsX11) was detected in the dystrophin gene by analysing mRNA isolated from the muscle tissue. The patient inherited both these mutations from his mother. The combination of CMT1A and DMD has not been reported as yet.

G.P.14.03 Quantitative analysis of CAPN3 and DMD transcripts: Involvement of nonsense-mediated mRNA decay

Duchenne and Becker muscular dystrophy (DMD/BMD) are associated with mutations in the DMD gene (Xp21); limb girdle muscular dystrophy type 2A (LGMD2A) with mutations in the CAPN3 gene (15q15). Pathological mutations connected with these phenotypes frequently disrupt the translational reading frame, giving rise to truncated proteins. Nonsense-mediated mRNA decay (NMD) is a type of quality control mechanism that selectively degrades mRNAs with premature termination codons (PTCs). Using real-time PCR, we performed quantitative analysis of DMD and CAPN3 transcripts carrying various mutated alleles and examined mRNA degradation by reason of NMD.

P.P.2 07 Molecular genetic diagnostics of myotonic dystrophy and facioscapulohumeral muscular dystrophy in Czech patients

DNA diagnostics of MD2 was performed in 100 patients with suspicion of MD2. In case of 48 patients, MD2 was confirmed. DNA diagnostics of FSHD was performed in 32 patients with suspicion of FSHD and confirmed in case of 19 patients.