Eva Zapletalová

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

Analysis of point mutations in the SMN1 gene in SMA patients bearing a single SMN1 copy

Spinal muscular atrophy (SMA) is caused by homozygous deletion of the SMN1 gene in approximately 96% of cases. Four percent of SMA patients have a combination of the deletion or conversion on one allele and an intragenic mutation on the second one. We performed analysis of point mutations in a set of our patients with suspicion of SMA and without homozygous deletion of the SMN1 gene. A quantitative test determining SMN1 copy number (using real-time PCR and/or MLPA analysis) was performed in 301 patients and only 1 SMN1 copy was detected in 14 of them. When these 14 patients were screened for the presence of point mutations we identified 6 mutations, p.Y272C (in three patients) and p.T274I, p.I33IfsX6, and p.A188S (each in one case). The mutations p.I33IfsX6 and p.A188S were found in two SMAI patients and were not detected previously. Further, evaluation of the relationship between mutation type, copy number of the SMN2 gene and clinical findings was performed. Among our SMA patients with a SMN1 homozygous deletion, we found a family with two patients: the son with SMAII possesses 3 SMN2 copies and the nearly asymptomatic father has a homozygous deletion of SMN1 exon 7 and carries 4 SMN2 copies. Generally, our results illustrate that an increased SMN2 gene copy number is associated with a milder SMA phenotype.

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.

Autosomal recessive congenital ichthyoses in the Czech Republic

Autosomal recessive congenital ichthyosis (ARCI) represents a heterogeneous group of disorders of epidermal cornification. Nine genes have been identified to be causative of ARCI, including TGM1 1,2 , ABCA12 3 , NIPAL4 4 , CYP4F22 5 , ALOX12B, ALOXE3 6 , PNPLA1 7 , LIPN 8 , and CERS3 9 . ARCI is rare, with a reported prevalence 1 in 200,000 in European and northern American populations 10 . We started DNA analysis of ARCI in 2012 when PCR-direct sequencing of the TGM1, ALOX12B, ALOXE3, NIPAL4, and CYP4F22 genes was introduced and patients were analysed step by step for mutations in these genes.

N.P.2 03 Correlation between SMN2 copy number variations in expression of SMN2 mRNA and clinical outcome in SMA patients treated with phenylbutyrate and valproic acid

The work is concerned with results of determination of SMN2 gene copy-number; correlation of SMN2 copy-number/clinical stage; corelation of SMN2 copy number/ full-length SMN2 mRNA level; and monitoring PBA and VA treatment in case of 25 SMA patients.