M. Goicoechea

CAPN3 mutations in patients with idiopathic eosinophilic myositis

Eosinophilic myositis (EM) constitutes a rare pathological entity characterized by eosinophilic infiltration of skeletal muscles, usually associated with parasite infections, systemic disorders, or the intake of drugs or L‐tryptophan. The exclusion of such causes defines the spectrum of idiopathic EM. Based on a protein analysis performed in one affected patient, we identified the gene encoding calpain‐3, CAPN3, as a candidate for a subset of idiopathic EM.

CD24 V/V is an allele associated with the risk of developing multiple sclerosis in the Spanish population.

The allele C in the CD24 gene has been related to multiple sclerosis (MS). In this work we check this single nucleotide polymorphism (SNP) in a population of 135 patients and 285 controls. Our results confirm the association between the V/V genotype at aa 57 of this gene and MS and highlight the importance of taking into account the origin of the subjects to avoid a population bias.

Mis-splicing of Tau exon 10 in myotonic dystrophy type 1 is reproduced by overexpression of CELF2 but not by MBNL1 silencing

Tau is the proteinaceous component of intraneuronal aggregates common to neurodegenerative diseases called Tauopathies, including myotonic dystrophy type 1. In myotonic dystrophy type 1, the presence of microtubule-associated protein Tau aggregates is associated with a mis-splicing of Tau. A toxic gain-of-function at the ribonucleic acid level is a major etiological factor responsible for the mis-splicing of several transcripts in myotonic dystrophy type 1. These are probably the consequence of a loss of muscleblind-like 1 (MBNL1) function or gain of CUGBP1 and ETR3-like factor 1 (CELF1) splicing function. Whether these two dysfunctions occur together or separately and whether all mis-splicing events in myotonic dystrophy type 1 brain result from one or both of these dysfunctions remains unknown. Here, we analyzed the splicing of Tau exons 2 and 10 in the brain of myotonic dystrophy type 1 patients. Two myotonic dystrophy type 1 patients showed a mis-splicing of exon 10 whereas exon 2-inclusion was reduced in all myotonic dystrophy type 1 patients. In order to determine the potential factors responsible for exon 10 mis-splicing, we studied the effect of the splicing factors muscleblind-like 1 (MBNL1), CUGBP1 and ETR3-like factor 1 (CELF1), CUGBP1 and ETR3-like factor 2 (CELF2), and CUGBP1 and ETR3-like factor 4 (CELF4) or a dominant-negative CUGBP1 and ETR-3 like factor (CELF) factor on Tau exon 10 splicing by ectopic expression or siRNA. Interestingly, the inclusion of Tau exon 10 is reduced by CUGBP1 and ETR3-like factor 2 (CELF2) whereas it is insensitive to the loss-of-function of muscleblind-like 1 (MBNL1), CUGBP1 and ETR3-like factor 1 (CELF1) gain-of-function, or a dominant-negative of CUGBP1 and ETR-3 like factor (CELF) factor. Moreover, we observed an increased expression of CUGBP1 and ETR3-like factor 2 (CELF2) only in the brain of myotonic dystrophy type 1 patients with a mis-splicing of exon 10. Taken together, our results indicate the occurrence of a mis-splicing event in myotonic dystrophy type 1 that is induced neither by a loss of muscleblind-like 1 (MBNL1) function nor by a gain of CUGBP1 and ETR3-like factor 1 (CELF1) function but is rather associated to CUGBP1 and ETR3-like factor 2 (CELF2) gain-of-function.

Muscle wasting in myotonic dystrophies: a model of premature aging

Myotonic dystrophy type 1 (DM1 or Steinert’s disease) and type 2 (DM2) are multisystem disorders of genetic origin. Progressive muscular weakness, atrophy and myotonia are the most prominent neuromuscular features of these diseases, while other clinical manifestations such as cardiomyopathy, insulin resistance and cataracts are also common. From a clinical perspective, most DM symptoms are interpreted as a result of an accelerated aging (cataracts, muscular weakness and atrophy, cognitive decline, metabolic dysfunction, etc.), including an increased risk of developing tumors. From this point of view, DM1 could be described as a progeroid syndrome since a notable age-dependent dysfunction of all systems occurs. The underlying molecular disorder in DM1 consists of the existence of a pathological (CTG) triplet expansion in the 3′ untranslated region (UTR) of the Dystrophia Myotonica Protein Kinase (DMPK) gene, whereas (CCTG)n repeats in the first intron of the Cellular Nucleic acid Binding Protein/Zinc Finger Protein 9 (CNBP/ZNF9) gene cause DM2. The expansions are transcribed into (CUG)n and (CCUG)n-containing RNA, respectively, which form secondary structures and sequester RNA-binding proteins, such as the splicing factor muscleblind-like protein (MBNL), forming nuclear aggregates known as foci. Other splicing factors, such as CUGBP, are also disrupted, leading to a spliceopathy of a large number of downstream genes linked to the clinical features of these diseases. Skeletal muscle regeneration relies on muscle progenitor cells, known as satellite cells, which are activated after muscle damage, and which proliferate and differentiate to muscle cells, thus regenerating the damaged tissue. Satellite cell dysfunction seems to be a common feature of both age-dependent muscle degeneration (sarcopenia) and muscle wasting in DM and other muscle degenerative diseases. This review aims to describe the cellular, molecular and macrostructural processes involved in the muscular degeneration seen in DM patients, highlighting the similarities found with muscle aging.

Characterization of novel CAPN3 isoforms in white blood cells: an alternative approach for limb-girdle muscular dystrophy 2A diagnosis.

Limb-girdle muscular dystrophy type 2A (LGMD2A) is an autosomal recessive disorder caused by mutations in the CAPN3 gene. Its definitive diagnosis is laborious, since the clinical phenotype is often similar to other types of muscular dystrophy and since the CAPN3 gene encompasses a large genomic region with more than 300 pathogenic mutations described to date. In fact, it is estimated that nearly 25% of the cases with a phenotype suggestive of LGMD2A do not have mutations in the CAPN3 gene and that, in up to 22% of the cases, only one mutation is identified. In the present work, we have characterised CAPN3 messenger RNA (mRNA) expression in peripheral blood, and we have performed a retrospective diagnostic study with 26 LGMD2A patients, sequencing a transcript of CAPN3 present in white blood cells (WBCs). The 25% of the mutations presented in this paper (7/28) act modifying pre-mRNA splicing of the CAPN3 transcript, including the first deep-intronic mutation described to date in the CAPN3 gene. Our results determine that the sequencing of CAPN3 transcripts present in WBCs could be applied as a new approach for LGMD2A diagnosis. This method improves and simplifies diagnosis, since it combines the advantages of mRNA analysis in a more accessible and rapidly regenerated tissue. However, the lack of exon 15 in the CAPN3 isoforms present in blood, and the presence of mRNA degradation make it necessary to combine mRNA and DNA analyses in some specific cases.

Minimizing creatine kinase variability in rats for neuromuscular research purposes

Summary Rat serum or plasma creatine kinase (CK) activity is widely used to evaluate myopathic processes, to test the myotoxicity of different drugs, or to analyse the benefits of emerging gene therapies in some neuromuscular disorders. However, great variability is found in this determination. The aim of this study has been to control some factors of variation in order to reduce variability and increase the reproducibility of analytical data. 8–10-week-old Wistar-Han rats were used. The study consisted of four sequential phases. Phase I aimed to analyse the effect of ether and isoflurane as anaesthetic drugs. The objective of Phase II was to evaluate bleeding rats via retro-orbital sinus vs. tail vein. Phases III and IV were designed as two separate, repeated measure experiments on two factors: habituation to laboratory handling procedures in Phase III and gender in Phase IV. The repeated factor was the storage temperature of blood sample prior to centrifugation. Ether did not significantly increased the CK value. Using isoflurane, getting rats accustomed to laboratory handling procedures and whole blood refrigeration prior to centrifugation and serum separation resulted in statistically significant reduction in CK value and variability. Male rats showed significantly higher values than female rats. In the light of our findings, CK value and variability in rats may be minimized by choosing tail vein as site of bleeding, getting rats accustomed to laboratory handling procedures and maintaining whole blood refrigerated until centrifugation and serum separation.

Eosinophilic infiltration related to CAPN3 mutations: a pathophysiological component of primary calpainopathy?

To the Editor : Mutations in the gene encoding calpain-3 (CAPN3 ) cause the most prevalent form of autosomalrecessive limb girdle muscular dystrophy (LGMD), which is type 2A (LGMD2A OMIM#253600) (1), also referred to as calpainopathy. We previously reported six children with CAPN3 mutations that constitute a genetic cause of idiopathic eosinophilic myositis (EM) (2). This was subsequently confirmed in two adults (3), whereas eosinophilic infiltration is not known as a typical feature of LGMD2A. To further evaluate the relationship between calpain-3 mutations and EM/infiltration, we retrospectively studied five additional unrelated patients with idiopathic EM (one adult: PEM7 and four children: PEM8, PEM9, PEM10 and PEM11). Moreover, we evaluated eosinophil infiltration in muscle biopsies in 17 LGMD2A patients and report a case of lymphocytic myositis associated with CAPN3 mutations. Patient PEM7 presented with progressive proximal weakness and patients PEM8, PEM9, PEM10 and PEM11 presented with initial isolated increase in creatinine phosphokinase (CPK) levels in plasma. Circulating eosinophilia was identified at diagnosis in all children, but not in the adult. Repeated eosinophil counts, available for PEM10 and PEM11, showed intermittent hypereosinophilia between 9 and 12 years of age (oscillating between 601 and 1400/μl, with intermittent normal levels) and between 4 and 9 years of age (highest value 819/μl, with intermittent normal levels), respectively. For PEM10, mainly normal eosinophil counts were seen after he had developed muscle weakness at 12 years of age. After informed consent, we screened for CAPN3 mutations as described (2), based on the particular histopathological presentation. Retrospectively, a frozen muscle sample could only be obtained for PEM10, revealing complete absence of calpain-3. CAPN3 disease-causing mutations were identified either as a homozygote (PEM7 and PEM10) or compound heterozygote (PEM8, PEM9 and PEM11). Additionally, we identified two CAPN3 mutations in a 5-year-old boy (PEM12) who presented with proximal weakness of the lower limbs and a discrete focal lymphocytic infiltrate mainly composed of CD8+ T-lymphocytes, without dystrophic features, on muscle biopsy. Complete calpain-3 deficiency was found on routine immunoblot. All clinical and mutational data are compiled in Table 1. To further assess our previous hypothesis of EM being a possible histopathological manifestation of LGMD2A, we retrospectively analysed haematoxylin–eosin-stained cryosections from muscle samples of 17 patients with genetically confirmed, ‘typical’ LGMD2A (mean age of biopsy 29 years). This showed inflammatory changes, associated sparse presence of eosinophils, in 5 of the 17 patients (data not shown). The average disease duration at biopsy for patients presenting without or with eosinophils in the infiltrates was 13.9 years and 6 years, respectively. Our findings further confirm mutations in CAPN3 as a genetic cause of EM and highlight eosinophilic infiltration as an ‘early’ component (or event) of primary calpainopathy (4). Importantly, as in our previous report, inclusion criteria were based only on the particular histopathological presentation, without any identified aetiological factor (even after two muscle biopsies in patients PEM7 and PEM8). T-lymphocytes may be a key component in the eosinophilic infiltrative process (5) which together with macrophages are the main component of inflammatory lesions in the vicinity of damaged muscle fibres; they play a central role in the chemotaxis of eosinophils; and they express calpain-3. Interestingly, Baumeister et al. recently reported a case of EM caused by a homozygous gamma-sarcoglycan mutation (6),

Identification and Characterization of the Dermal Panniculus Carnosus Muscle Stem Cells

Summary The dermal Panniculus carnosus (PC) muscle is important for wound contraction in lower mammals and represents an interesting model of muscle regeneration due to its high cell turnover. The resident satellite cells (the bona fide muscle stem cells) remain poorly characterized. Here we analyzed PC satellite cells with regard to developmental origin and purported function. Lineage tracing shows that they originate in Myf5+, Pax3/Pax7+ cell populations. Skin and muscle wounding increased PC myofiber turnover, with the satellite cell progeny being involved in muscle regeneration but with no detectable contribution to the wound-bed myofibroblasts. Since hematopoietic stem cells fuse to PC myofibers in the absence of injury, we also studied the contribution of bone marrow-derived cells to the PC satellite cell compartment, demonstrating that cells of donor origin are capable of repopulating the PC muscle stem cell niche after irradiation and bone marrow transplantation but may not fully acquire the relevant myogenic commitment.

Does the severity of the LGMD2A phenotype in compound heterozygotes depend on the combination of mutations

Introduction: Limb‐girdle muscular dystrophy type 2A (LGMD2A) is caused by a deficiency of calpain‐3/p94. Although the symptoms in most LGMD2A patients are generally homogeneous, some variation in the severity and progression of the disease has been reported. Methods: We describe 2 patients who carry the same combination of compound heterozygous mutations (pG222R/pR748Q) and whose symptoms are exceptionally benign compared to homozygotes with each missense mutation. Results: The benign phenotype observed in association with the combined pG222R and pR748Q mutations suggested that it may result from a compensatory effect of compound heterozygosity rather than the individual mutations themselves. Our analyses revealed that these two mutations exert different effects on the protease activity of calpain‐3, suggesting “molecular complementation” in these patients. Conclusion: We propose several hypotheses to explain how this specific combination of mutations may rescue the normal proteolytic activity of calpain‐3, resulting in an exceptionally benign phenotype. Muscle Nerve, 2011

Negative evidence for stachydrine or Galeopsis ladanum L. seeds as the causal agents of coturnism after quail meat ingestion.

Quail poisoning is known to produce an acute myoglobinuric syndrome called coturnism. The cause of this syndrome is still unknown, although it has been postulated that Galeopsis ladanum L. seeds, in particular lipidic compounds or stachydrine, are responsible for this toxicity. Thus, we aimed to study the implication of this plant in coturnism in order to explore the physiopathology of the disease, especially with regard to stachydrine and lipidic compounds extracted from seeds. For this purpose, Wistar rats were fed with G. ladanum seed extracts or with quail meat. However, the rhabdomyolysis outbreak could not be reproduced in any case. Therefore, in view of our results and experimental conditions, seeds of G. ladanum and stachydrine do not appear to be the responsible agents of the myopathic outbreak. This conclusion is supported by the following facts: direct administration of extracts of seeds of G. ladanum or stachydrine produces no myotoxicity in rats; G. ladanum seeds are not toxic to quails and meat from quails fed G. ladanum seeds is not toxic to rats.