Kristýna Stehlíková

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 limb-girdle muscular dystrophies in the Czech Republic

BackgroundAutosomal recessive limb-girdle muscular dystrophies (LGMD2) include a number of disorders with heterogeneous etiology that cause predominantly weakness and wasting of the shoulder and pelvic girdle muscles. In this study, we determined the frequency of LGMD subtypes within a cohort of Czech LGMD2 patients using mutational analysis of the CAPN3, FKRP, SGCA, and ANO5 genes.MethodsPCR-sequencing analysis; sequence capture and targeted resequencing.ResultsMutations of the CAPN3 gene are the most common cause of LGMD2, and mutations in this gene were identified in 71 patients in a set of 218 Czech probands with a suspicion of LGMD2. Totally, we detected 37 different mutations of which 12 have been described only in Czech LGMD2A patients. The mutation c.550delA is the most frequent among our LGMD2A probands and was detected in 47.1% of CAPN3 mutant alleles. The frequency of particular forms of LGMD2 was 32.6% for LGMD2A (71 probands), 4.1% for LGMD2I (9 probands), 2.8% for LGMD2D (6 probands), and 1.4% for LGMD2L (3 probands).Further, we present the first results of a new approach established in the Czech Republic for diagnosis of neuromuscular diseases: sequence capture and targeted resequencing. Using this approach, we identified patients with mutations in the DYSF and SGCB genes.ConclusionsWe characterised a cohort of Czech LGMD2 patients on the basis of mutation analysis of genes associated with the most common forms of LGMD2 in the European population and subsequently compared the occurrence of particular forms of LGMD2 among countries on the basis of our results and published studies.

Ten years of DNA diagnostics of epidermolysis bullosa in the Czech Republic.

Epidermolysis bullosa (EB) is a heterogeneous group of inherited skin disorders characterized by blister formation. Classification of patients with EB begins with their separation into one of the four major EB groups, based on the level to which blisters develop: EB simplex (EBS), junctional EB (JEB), dystrophic EB (DEB) and Kindler syndrome. The next level of subclassification takes into account the clinical features present in a given patient, most notably the distribution and severity of cutaneous and extracutaneous disease involvement.

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.

Muscular dystrophies and myopathies: the spectrum of mutated genes in the Czech Republic

Inherited neuromuscular disorder (NMD) is a wide term covering different genetic disorders affecting muscles, nerves, and neuromuscular junctions. Genetic and clinical heterogeneity is the main drawback in a routine gene‐by‐gene diagnostics. We present Czech NMD patients with a genetic cause identified using targeted next‐generation sequencing (NGS) and the spectrum of these causes. Overall 167 unrelated patients presenting NMD falling into categories of muscular dystrophies, congenital muscular dystrophies, congenital myopathies, distal myopathies, and other myopathies were tested by targeted NGS of 42 known NMD‐related genes. Pathogenic or probably pathogenic sequence changes were identified in 79 patients (47.3%). In total, 37 novel and 51 known disease‐causing variants were detected in 23 genes. In addition, variants of uncertain significance were suspected in 7 cases (4.2%), and in 81 cases (48.5%) sequence changes associated with NMD were not found. Our results strongly indicate that for molecular diagnostics of heterogeneous disorders such as NMDs, targeted panel testing has a high‐clinical yield and should therefore be the preferred first‐tier approach. Further, we show that in the genetic diagnostic practice of NMDs, it is necessary to take into account different types of inheritance including the occurrence of an autosomal recessive disorder in two generations of one family.

G.P.222

Limb-girdle muscular dystrophy (LGMD) is defined as a muscular dystrophy with predominantly proximal distribution of muscle weakness. It includes a number of disorders with heterogeneous etiology. We determined the frequency of recessive LGMD subtypes (LGMD2A, LGMD2D, LGMD2I and LGMD2L) within a cohort of Czech LGMD2 patients using mutation analysis of the calpain3 (CAPN3), fukutin-related protein (FKRP), alpha-sarcoglycan (SGCA), and anoctamin5 (ANO5) genes. Last year we introduced next generation sequencing to accelerate patient diagnosis and to widen spectrum of analysed genes. We designed capture library to target the coding exons of genes responsible for all known types of LGMD and genes responsible for muscular dystrophy with similar phenotype to LGMD. We observed that mutations of the CAPN3 gene are the most common cause of LGMD2. The frequency of particular forms of LGMD2 was 32.6% for LGMD2A, 4.1% for LGMD2I, 2.8% for LGMD2D, and 1.4% for LGMD2L. Using next-generation sequencing, we identified two patients with mutations in the gene encoding dysferlin (DYSF) – LGMD2B and a patient with mutations in the gene encoding beta-sarcoglycan (SGCB) – LGMD2E. In total, we determined mutations in 41% of Czech LGMD2 patients. This work was funded by the project of the Internal Grant Agency of the Czech Ministry of Health (NT/14574-3); the projects of the Czech Ministry of Education “CEITEC – Central European Institute of Technology” (CZ.1.05/1.1.00/02.0068) and SuPReMMe (CZ.1.07/2.3.00/20.0045).

G.P.44 Spectrum of mutations identified in the cohort of Czech LGMD patients

Abstract Limb girdle muscular dystrophies (LGMDs) represent a group of clinically and genetically heterogeneous disorders predominantly affecting shoulder and pelvic girdles. To date, 15 forms (2A-N) of autosomal recessive (AR) and eight forms (1A-H) of autosomal dominant LGMDs have been described. LGMD2A is the most frequent form of LGMD in many European countries, and is caused by mutations in the CAPN3 gene that encodes a muscle specific protease, calpain-3. Besides LGMD2A, we perform molecular genetic diagnostics of LGMD2I, LGMD2D, and LGMD2L caused by mutations in genes encoding fukutin-related protein (FKRP), alpha-sarcoglycan (SGCA) and anoctamin-5 (ANO5). Based on the results of clinical assessment and histopathological examination of muscle biopsies (including protein analysis using immunohistochemistry and immunoblotting), the mutational analysis of the CAPN3 , FKRP , SGCA , and ANO5 genes was performed at the mRNA level (using reverse transcription-PCR-direct sequencing) and/or at the DNA level (using PCR-direct sequencing) in a cohort of Czech patients with a preliminary diagnosis of LGMD. In total, we screened 230 unrelated patients and mutations associated with the disease were determined in 70 of them (30.4%). Mutations in the CAPN3 gene were found in 55 patients, and LGMD2A was confirmed to represent the most frequent AR LGMD in the Czech Republic. The homozygous occurence of the most common mutation in the FKRP gene (p.Leu276Ile) associated with LGMD2I was determined in eight patients. Mutations in the SGCA and ANO5 genes were detected in 5 LGMD2D and 2 LGMD2L patients, respectively. This work was funded by the project “CEITEC – Central European Institute of Technology” (CZ.1.05/1.1.00/02.0068).

P1.17 Limb-girdle muscular dystrophies in Czech Republic

able affected family members and at least one unaffected sibling in the first 6 families were genotyped at 10,204 SNP markers and linkage analysis was performed in MERLIN. Three families showed linkage to overlapping intervals on chromosome 19q13 and sequence analysis of FKRP identified a novel missense mutation, T314M, in two of these families that shared a haplotype across a portion of the linkage peak containing FKRP, indicating they likely inherited a founder mutation from a common ancestor. In the third family we discovered a second novel FKRP missense mutation, V338L. Two additional families mapped to chromosome 17q21, and one of these carried the previously-reported R34H mutation in SGCA (Adhalin), while in the other we found a novel 50 splice site mutation IVS5 + 5G > A, which was absent from >300 control DNAs. A sixth family showed linkage to chromosome 5q33-34 and a novel nonsense mutation R33X in SGCD. FKRP and SGCA (Adhalin) were then screened in the remaining 7 families, yielding FKRP T314M in one and SGCA R34H in another. Genotyping the remaining 5 families, followed by homozygosity mapping, yielded mutations in SGCB in 3 families and SGCG in 1 family. The final family maps to SEPN1 with two potential mutations identified, and the phenotype upon review is suggestive of multiminicore disease. Our study shows linkage mapping in consanguineous pedigrees is a productive and efficient method for discovering novel mutations and loci.

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.