Limb-girdle Muscular Dystrophy Type 2A Patients Research Articles

Quantitative Muscle MRI in Patients with Neuromuscular Diseases-Association of Muscle Proton Density Fat Fraction with Semi-Quantitative Grading of Fatty Infiltration and Muscle Strength at the Thigh Region.

(1) Background and Purpose: The skeletal muscles of patients suffering from neuromuscular diseases (NMD) are affected by atrophy, hypertrophy, fatty infiltration, and edematous changes. Magnetic resonance imaging (MRI) is an important tool for diagnosis and monitoring. Concerning fatty infiltration, T1-weighted or T2-weighted DIXON turbo spin echo (TSE) sequences enable a qualitative assessment of muscle involvement. To achieve higher comparability, semi-quantitative grading scales, such as the four-point Mercuri scale, are commonly applied. However, the evaluation remains investigator-dependent. Therefore, effort is being invested to develop quantitative MRI techniques for determination of imaging markers such as the proton density fat fraction (PDFF). The present work aims to assess the diagnostic value of PDFF in correlation to Mercuri grading and clinically determined muscle strength in patients with myotonic dystrophy type 2 (DM2), limb girdle muscular dystrophy type 2A (LGMD2A), and adult Pompe disease. (2) Methods: T2-weighted two-dimensional (2D) DIXON TSE and chemical shift encoding-based water-fat MRI were acquired in 13 patients (DM2: n = 5; LGMD2A: n = 5; Pompe disease: n = 3). Nine different thigh muscles were rated in all patients according to the Mercuri grading and segmented to extract PDFF values. Muscle strength was assessed according to the British Medical Research Council (BMRC) scale. For correlation analyses between Mercuri grading, muscle strength, and PDFF, the Spearman correlation coefficient (rs) was computed. (3) Results: Mean PDFF values ranged from 7% to 37% in adults with Pompe disease and DM2 and up to 79% in LGMD2A patients. In all three groups, a strong correlation of the Mercuri grading and PDFF values was observed for almost all muscles (rs > 0.70, p < 0.05). PDFF values correlated significantly to muscle strength for muscle groups responsible for knee flexion (rs = −0.80, p < 0.01). (4) Conclusion: In the small, investigated patient cohort, PDFF offers similar diagnostic precision as the clinically established Mercuri grading. Based on these preliminary data, PDFF could be further considered as an MRI-based biomarker in the assessment of fatty infiltration of muscle tissue in NMD. Further studies with larger patient cohorts are needed to advance PDFF as an MRI-based biomarker in NMD, with advantages such as its greater dynamic range, enabling the assessment of subtler changes, the amplified objectivity, and the potential of direct correlation to muscle function for selected muscles.

LIMB GIRDLE MUSCULAR DYSTROPHIES: P.145 Prevalence of CAPN3 mutations in Limb-girdle muscular dystrophy type 2A: From a multi-center analysis in China to a worldwide perspective

Limb-girdle muscular dystrophy type 2A (LGMD2A) is a rare autosomal recessive limb-girdle muscular dystrophy caused by mutations in CAPN3 gene. To date, no systemic evaluation was performed to investigate the mutational distribution and the hot spot regions. We retrospectively reviewed CAPN3 gene mutations from 124 Chinese patients recruited from four diagnostic centers (2012 January to 2019 September). Then we integrated the multicenter analysis with open-source databases (Clinvar, LOVD, gnomAD) and annotated the combined dataset (Ref: NM_000070.3). Functional evaluating tools (SIFT, PolyPhen, dbscSNV and MaxEntScan) were used to analyze the pathogenicity of the mutations. We evaluated the frequency and distribution of allelic CAPN3 mutation in LGMD2A patients. In the allelic mutational dataset from Chinese cohort (n=112), 62 novel mutations (55.4%) were identified according to ClinVar. The most prevalent mutation in China was c.2120A>G (18.9%) while the previous reported hotspot in LOVD was c.550del. Functional analysis with Polyphen and dbscSNV revealed a higher deleterious effect in Chinese cohort in comparison to the reported data (PolyPhen: g= 0.52, p < 0.0001; dbscSNV: g=0.49, p = 0.0036). After integrating the Chinese data with reported alleles (n= 467 in total), mutations in exon 1 (8.9%) and exon 10 (8.0%) of CAPN3 had the highest frequencies, while missense and frameshift mutations accounted for the majority of mutations. CAPN3 mutations had a high frequency in Exon 1 and 10 among LGMD2A patients. Exon 1 encodes the muscle-specific NS region of Calpain-3 protein while Exon 10 corresponds to Protease Core subdomain 2 (PC2). The frequent mutational regions revealed in the LGMD2A patients.

Gene Correction of LGMD2A Patient-Specific iPSCs for the Development of Targeted Autologous Cell Therapy.

Limb girdle muscular dystrophy type 2A (LGMD2A), caused by mutations in the Calpain 3 (CAPN3) gene, is an incurable autosomal recessive disorder that results in muscle wasting and loss of ambulation. To test the feasibility of an autologous induced pluripotent stem cell (iPSC)-based therapy for LGMD2A, here we applied CRISPR-Cas9-mediated genome editing to iPSCs from three LGMD2A patients to enable correction of mutations in the CAPN3 gene. Using a gene knockin approach, we genome edited iPSCs carrying three different CAPN3 mutations, and we demonstrated the rescue of CAPN3 protein in myotube derivatives invitro. Transplantation of gene-corrected LGMD2A myogenic progenitors in a novel mouse model combining immunodeficiency and a lack of CAPN3 resulted in muscle engraftment and rescue of the CAPN3 mRNA. Thus, we provide here proof of concept for the integration of genome editing and iPSC technologies to develop a novel autologous cell therapy for LGMD2A.

Fatty infiltration evaluation and selective pattern characterization of lower limbs in limb‐girdle muscular dystrophy type 2A by muscle magnetic resonance imaging

Limb-girdle muscular dystrophy type 2A (LGMD2A) is characterized by progressive wasting of muscles for which the disease-monitoring tools are still deficient. We performed muscle MRI of the lower limbs in 32 LGMD2A patients and 21 controls. The modified Mercuri scale was employed to evaluate the degree of fatty infiltration. Severe fatty infiltration in the long head of biceps femoris (modified Mercuri scale 3.99) and sparing of extensor digitorum longus (modified Mercuri scale 0.17) were observed. The sensitivity and specificity of this pattern in diagnostic testing was 76.00% and 90.48%, respectively. A comprehensive clinical and MRI evaluation revealed that progressive fatty infiltration in the upper leg correlated well with disease progression, but neither calf involvement nor muscle strength deterioration showed a good correlation. The selective involvement pattern is potentially useful for LGMD2A diagnosis. Upper leg muscle MRI is a sensitive evaluation method for monitoring disease progression. Muscle Nerve 58: 536-541, 2018.

FRZB and melusin, overexpressed in LGMD2A, regulate integrin β1D isoform replacement altering myoblast fusion and the integrin-signalling pathway.

Limb-girdle muscular dystrophy type 2A (LGMD2A) is characterised by muscle wasting and progressive degeneration of proximal muscles because of mutations in the CAPN3 gene. However, the underlying pathophysiological mechanisms of muscle degeneration are still not well understood. The objective of this study was to assess the relevance of genes with differential expression in the muscle of LGMD2A patients. For this purpose, we analysed their in vitro expression in primary cultures of human myoblasts and myotubes. Abnormal fusion was observed in the myotubes of these patients, which may be explained by the lack of physiological replacement of integrin β1D. Owing to this observation, we focused on deregulated genes coding proteins that directly interact with integrin, ITGB1BP2 and CD9, as well as FRZB gene, because of its in vitro upregulation in myotubes. Silencing studies established that these genes are closely regulated, CD9 and FRZB being positive regulators of the expression of ITGB1BP2, and in turn, this gene being a negative regulator of the expression of FRZB. Interestingly, we observed that FRZB regulates integrin β1D expression, its silencing increasing integrin β1D expression to levels similar to those in controls. Finally, the administration of LiCl, an enhancer of the Wnt-signalling pathway showed similar experimentally beneficial effects, suggesting FRZB silencing or LiCl administration as potential therapeutic targets, though further studies are required.

312. Gene Correction of LGMD2A Patient-Specific iPS Cells for Targeted Autologous Cell Therapy

Limb girdle muscular dystrophy type 2A (LGMD2A), an incurable autosomal recessive disorder and the most common form of LGMD, is characterized by symmetric and progressive weakness of limb-girdle muscles leading to loss of ambulation. LGMD2A is caused by mutations in the Calpain 3 (CAPN3) gene which encodes a muscle-specific intracellular calcium-sensitive cysteine protease, hypothesized to act as a gate keeper for proper assembly and maintenance of the sarcomere. A cell therapy based on induced pluripotent stem (iPS) cells has the potential for treating muscular dystrophy. We have demonstrated that transplantation of skeletal muscle progenitors obtained from disease-free human iPS cells can restore muscle contractility in a mouse model for Duchenne Muscular Dystrophy. The goal of this study is to apply gene editing tools to correct the CAPN3 mutation in LGMD2A iPS cells, and to determine the feasibility of an iPS-cell based autologous cell therapy for LGMD2A. Here we investigated two approaches to genetically correct a LGMD2A patient-specific iPS cell line with homozygous deletion of exon 17 to 24 in the CAPN3 gene. The first approach consisted of knocking in the coding region of CAPN3 cDNA with SV40 polyadenylation signal sequence downstream in exon 1 of the endogenous CAPN3 gene by CRISPR-CAS9 induced double stranded break (DSB) mediated homology directed DNA repair from exogenous donor template, allowing for universal correction of all types of CAPN3 mutations. PCR, sequencing and Southern blot analysis on iPS colonies post- genome editing confirmed the integration of the cDNA in exon 1 of the CAPN3 gene. The second approach involved inserting the missing exons 17-24 with SV40 polyadenylation signal sequence downstream contiguous with the exon 16 of the endogenous CAPN3 gene. PCR and sequencing analysis on iPS colonies post-genome editing confirmed proper integration in this approach as well. LGMD2A iPS cell clones, genetically corrected using these two gene editing methods will be differentiated into skeletal muscle progenitors using conditional expression of Pax7, as previously described by our group, and evaluated in vitro and in vivo for restoration of full length CAPN3. For the latter, we have generated an immunodeficient LGMD2A mouse model by combining the CAPN3-KO mouse model with NOD/SCID and IL2Rg mutations. This mouse is ideal for studies on the transplantation of human cells. In summary, we have corrected the CAPN3 mutation in LGMD2A iPS cells for the first time, and ongoing transplantation studies with corrected iPS cells will provide proof-of-principal for a potential new cell therapy that can permanently restore CAPN3 function in all LGMD2A patients.

Calpain 3 deficiency affects SERCA expression and function in the skeletal muscle.

Limb-girdle muscular dystrophy type 2A (LGMD2A) is a form of muscular dystrophy caused by mutations in calpain 3 (CAPN3). Several studies have implicated Ca2+ dysregulation as an underlying event in several muscular dystrophies, including LGMD2A. In this study we used mouse and human myotube cultures, and muscle biopsies in order to determine whether dysfunction of sarco/endoplasmatic Ca2+-ATPase (SERCA) is involved in the pathology of this disease. In CAPN3-deficient myotubes, we found decreased levels of SERCA 1 and 2 proteins, while mRNA levels remained comparable with control myotubes. Also, we found a significant reduction in SERCA function that resulted in impairment of Ca2+ homeostasis, and elevated basal intracellular [Ca2+] in human myotubes. Furthermore, small Ankyrin 1 (sAnk1), a SERCA1-binding protein that is involved in sarcoplasmic reticulum integrity, was also diminished in CAPN3-deficient fibres. Interestingly, SERCA2 protein was patently reduced in muscles from LGMD2A patients, while it was normally expressed in other forms of muscular dystrophy. Thus, analysis of SERCA2 expression may prove useful for diagnostic purposes as a potential indicator of CAPN3 deficiency in muscle biopsies. Altogether, our results indicate that CAPN3 deficiency leads to degradation of SERCA proteins and Ca2+ dysregulation in the skeletal muscle. While further studies are needed in order to elucidate the specific contribution of SERCA towards muscle degeneration in LGMD2A, this study constitutes a reasonable foundation for the development of therapeutic approaches targeting SERCA1, SERCA2 or sAnk1.

Neurotrophins, cytokines, oxidative parameters and funcionality in Progressive Muscular Dystrophies.

We investigated the levels of brain derived-neurotrophic factor (BDNF), cytokines and oxidative parameters in serum and tried to correlate them with the age and functionality of patients with Progressive Muscle Dystrophies (PMD). The patients were separated into six groups (case and controls pared by age and gender), as follows: Duchenne Muscular Dystrophy (DMD); Steinert Myotonic Dystrophy (SMD); and Limb-girdle Muscular Dystrophy type-2A (LGMD2A). DMD patients (± 17.9 years old) had a decrease of functionality, an increase in the IL-1β and TNF-α levels and a decrease of IL-10 levels and superoxide dismutase activity in serum. SMD patients (± 25.8 years old) had a decrease of BDNF and IL-10 levels and superoxide dismutase activity and an increase of IL-1β levels in serum. LGMD2A patients (± 27.7 years old) had an decrease only in serum levels of IL-10. This research showed the first evidence of BDNF involvement in the SMD patients and a possible unbalance between pro-inflammatory and anti-inflammatory cytokine levels, along with decreased superoxide dismutase activity in serum of DMD and SMD patients.

Genetic variability and clinical spectrum of Chinese patients with limb‐girdle muscular dystrophy type 2A

Previous studies of limb-girdle muscular dystrophy type 2A (LGMD2A) patients in many countries have suggested a heterogeneous genetic and clinical spectrum, but the genotypes and phenotypes of Chinese LGMD2A patients remain unclear. We directly screened calpain-3 (CAPN3) in 18 Chinese Han subjects who exhibited severely reduced or completely absent calpain-3 expression, as determined by Western blot analysis. We subsequently analyzed genotype/phenotype correlations. Seventeen patients (94.4%) were identified who had at least 1 causative mutation. All 18 mutations were distributed along the entire gene, and 11 of the mutations were novel, including 4 missense mutations, 5 deletions, and 2 splicing mutations. The phenotypes of these Chinese LGMD2A patients varied from severe LGMD to distal myopathy, and even asymptomatic hyper-CK-emia. No evidential correlation was found between the genotypes and phenotypes of the patients assessed in this study. Western blot analysis is still a useful diagnostic method when genetic analysis is unavailable.

C3KO mouse expression analysis: downregulation of the muscular dystrophy Ky protein and alterations in muscle aging

Mutations in CAPN3 gene cause limb-girdle muscular dystrophy type 2A (LGMD2A) characterized by muscle wasting and progressive degeneration of scapular and pelvic musculature. Since CAPN3 knockout mice (C3KO) display features of muscle pathology similar to those features observed in the earliest-stage or preclinical LGMD2A patients, gene expression profiling analysis in C3KO mice was performed to gain insight into mechanisms of disease. Two different comparisons were carried out in order to determine, first, the differential gene expression between wild-type (WT) and C3KO soleus and, second, to identify the transcripts differentially expressed in aging muscles of WT and C3KO mice. The up/downregulation of two genes, important for normal muscle function, was identified in C3KO mice: the Ky gene, encoding a protease implicated in muscle development, and Park2 gene encoding an E3 ubiquitin ligase (parkin). The Ky gene was downregulated in C3KO muscles suggesting that Ky protease may play a complementary role in regulating muscle cytoskeleton homeostasis in response to changes in muscle activity. Park2 was upregulated in the aged WT muscles but not in C3KO muscles. Taking into account the known functions of parkin E3 ligase, it is possible that it plays a role in ubiquitination and degradation of atrophy-specific and damaged proteins that are necessary to avoid cellular toxicity and a cellular stress response in aging muscles.

Impaired calcium calmodulin kinase signaling and muscle adaptation response in the absence of calpain 3

Mutations in the non-lysosomal, cysteine protease calpain 3 (CAPN3) result in the disease limb girdle muscular dystrophy type 2A (LGMD2A). CAPN3 is localized to several subcellular compartments, including triads, where it plays a structural, rather than a proteolytic, role. In the absence of CAPN3, several triad components are reduced, including the major Ca(2+) release channel, ryanodine receptor (RyR). Furthermore, Ca(2+) release upon excitation is impaired in the absence of CAPN3. In the present study, we show that Ca-calmodulin protein kinase II (CaMKII) signaling is compromised in CAPN3 knockout (C3KO) mice. The CaMK pathway has been previously implicated in promoting the slow skeletal muscle phenotype. As expected, the decrease in CaMKII signaling that was observed in the absence of CAPN3 is associated with a reduction in the slow versus fast muscle fiber phenotype. We show that muscles of WT mice subjected to exercise training activate the CaMKII signaling pathway and increase expression of the slow form of myosin; however, muscles of C3KO mice do not exhibit these adaptive changes to exercise. These data strongly suggest that skeletal muscle's adaptive response to functional demand is compromised in the absence of CAPN3. In agreement with our mouse studies, RyR levels were also decreased in biopsies from LGMD2A patients. Moreover, we observed a preferential pathological involvement of slow fibers in LGMD2A biopsies. Thus, impaired CaMKII signaling and, as a result, a weakened muscle adaptation response identify a novel mechanism that may underlie LGMD2A and suggest a pharmacological target that should be explored for therapy.

CAPN3 mRNA processing alteration caused by splicing mutation associated with novel genomic rearrangement of Alu elements

Recessive mutations of CAPN3 gene are reported to be responsible for limb girdle muscular dystrophy type 2A (LGMD2A). In all, 15-25% of intronic nucleotide changes identified in this gene were investigated by in silico analysis, but occasionally supported by experimental data or reported in some cases as a polymorphism. We report here genetic and transcriptional analyses in three Tunisian patients belonging to the same consanguineous family sharing the same mutation c.1194-9 A>G and Alu repeats insertion in intron 7 of CAPN3 gene. Reverse transcriptase-PCR experiments performed on total RNA from the patient's muscle biopsy showed retention of the eight last nucleotides of intron 9 in the CAPN3 transcript lacking the first seven exons. Our results provide evidence regarding the potential involvement of Alu elements in aberrant processing of pre-mRNA owing to the disruption of pre-existing intronic splicing regulatory elements. We also demonstrated variable mRNA alternative splicing among tissues and between LGMD2A patients. A deep intronic variation and rearrangement have been reported in the literature as causing genetic diseases in humans. However, this is the first report on a potential pathogenic CAPN3 gene mutation resulting from an Alu insertion.

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

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. 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. 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. 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.

Gene Expression Profiling in Limb-Girdle Muscular Dystrophy 2A

Limb-girdle muscular dystrophy type 2A (LGMD2A) is a recessive genetic disorder caused by mutations in calpain 3 (CAPN3). Calpain 3 plays different roles in muscular cells, but little is known about its functions or in vivo substrates. The aim of this study was to identify the genes showing an altered expression in LGMD2A patients and the possible pathways they are implicated in. Ten muscle samples from LGMD2A patients with in which molecular diagnosis was ascertained were investigated using array technology to analyze gene expression profiling as compared to ten normal muscle samples. Upregulated genes were mostly those related to extracellular matrix (different collagens), cell adhesion (fibronectin), muscle development (myosins and melusin) and signal transduction. It is therefore suggested that different proteins located or participating in the costameric region are implicated in processes regulated by calpain 3 during skeletal muscle development. Genes participating in the ubiquitin proteasome degradation pathway were found to be deregulated in LGMD2A patients, suggesting that regulation of this pathway may be under the control of calpain 3 activity. As frizzled-related protein (FRZB) is upregulated in LGMD2A muscle samples, it could be hypothesized that β-catenin regulation is also altered at the Wnt signaling pathway, leading to an incorrect myogenesis. Conversely, expression of most transcription factor genes was downregulated (MYC, FOS and EGR1). Finally, the upregulation of IL-32 and immunoglobulin genes may induce the eosinophil chemoattraction explaining the inflammatory findings observed in presymptomatic stages. The obtained results try to shed some light on identification of novel therapeutic targets for limb-girdle muscular dystrophies.

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.

CDNA analyses of CAPN3 enhance mutation detection and reveal a low prevalence of LGMD2A patients in Denmark

Calpainopathy or limb-girdle muscular dystrophy type 2A (LGMD2A) is generally recognized as the most prevalent form of recessive LGMD and is caused by mutations in the CAPN3 gene. Out of a cohort of 119 patients fulfilling clinical criteria for LGMD2, referred to our neuromuscular clinic, 46 were suspected to have LGMD2A, based on western blot results. Four of these patients were shown to have LGMD2I upon molecular analysis, whereas 16 of the remaining 42 patients harbored mutations in CAPN3 by both direct genomic sequencing and cDNA analyses. In 10 patients, we identified both mutant alleles. In three other, only one heterozygous mutation could be identified on the genomic level; however, CAPN3 cDNA analyses demonstrated homozygosity for the mutant allele, indicating the presence of an unidentified allele that somehow compromise correct CAPN3 RNA processing. In the three remaining patients, only a single heterozygous mutation could be identified both at the genomic level and on full-length CAPN3 cDNA. All three patients exhibited a highly abnormal western blot for calpain-3 and clinical characteristics of LGMD2A. Only three of the genetically confirmed LGMD2A patients were of Danish origin, indicating a five- to sixfold lower prevalence in Denmark compared to other European countries. A total of 16 different CAPN3 mutations were identified, of which 5 were novel. The present study demonstrates the value of cDNA analysis for CAPN3 in LGMD2A patients and indicates that calpainopathy is an uncommon cause of LGMD in the Denmark.

NF‐NF‐κBB‐dependent expression of the antiapoptotic factor c‐FLIP is regulated by calpain 3, the protein involved in limb‐girdle muscular dystrophy type 2A

Limb-girdle muscular dystrophy type 2A (LGMD2A) is a recessive genetic disorder caused by mutations in the cysteine protease calpain 3 (CAPN3) that leads to selective muscle wasting. We previously showed that CAPN3 deficiency is associated with a profound perturbation of the NF-kappaB/IkappaB alpha survival pathway. In this study, the consequences of altered NF-kappaB/IkappaB alpha pathway were investigated using biological materials from LGMD2A patients. We first show that the antiapoptotic factor cellular-FLICE inhibitory protein (c-FLIP), which is dependent on the NF-kappaB pathway in normal muscle cells, is down-regulated in LGMD2A biopsies. In muscle cells isolated from LGMD2A patients, NF-kappaB is readily activated on cytokine induction as shown by an increase in its DNA binding activity. However, we observed discrepant transcriptional responses depending on the NF-kappaB target genes. IkappaB alpha is expressed following NF-kappaB activation independent of the CAPN3 status, whereas expression of c-FLIP is obtained only when CAPN3 is present. These data lead us to postulate that CAPN3 intervenes in the regulation of the expression of NF-kappaB-dependent survival genes to prevent apoptosis in skeletal muscle. Deregulations in the NF-kappaB pathway could be part of the mechanism responsible for the muscle wasting resulting from CAPN3 deficiency.

G.P.4.15 CAPN3 mutations in patients with idiopathic eosinophilic myositis: A predystrophic stage of LGMD2A?

Objective and Background: Mutations in the gene encoding calpain-3 (CAPN3) cause autosomal-recessive limb girdle muscular dystrophy type 2A (LGMD2A). In six children, we recently identified CAPN3 mutations as a genetic cause of idiopathic eosinophilic myositis (EM), a rare pathological entity characterised by eosinophilic infiltration of skeletal muscles. Here, we further evaluated the hypothesis of EM being a possible histopathological pre-dystrophic developmental stage of LGMD2A. Methods: We screened CAPN3 for mutations using DHPLC and direct sequencing, in five unrelated patients (four children and one young adult), recruited on the basis of EM diagnosed after histological examination of muscle biopsy samples, without any identified aetiological factor. In addition, information on the presence of eosinophils was retrospectively assessed in 17 genetically confirmed LGMD2A patients and inflammatory changes by analysing haematoxylin-eosin stained cryosections from muscle samples (mean age of biopsy 28 years). Results: Two disease-causing CAPN3 mutations were identified in the five patients affected with idiopathic EM. In one additional patient, only one disease-causing CAPN3 mutation was identified. Additionally, we found a sparse presence of eosinophils in muscle biopsies from 5/17 patients with inflammatory (n = 3 mild focal, n = 2 moderate focal) and myopathic changes (n = 2 mild, n = 2 moderate, n = 1 severe). Conclusion: Our findings confirm mutations in CAPN3 as a genetic cause of eosinophilic myositis. The presence of eosinophils has been discussed before as a component of muscular dystrophy (Cai et al. 2000). The presence of marked eosinophilic infiltrates in EM patients, together with the findings of our retrospective analysis of biopsies from LGMD2A patients might suggest the following possibilities (1) EM associated with CAPN3 mutations could be a distinct phenotype, (2) eosinophilic infiltration might represent an early or transient developmental stage in the inflammation associated with primary calpainopathy.

Correlations between clinical severity, genotype and muscle pathology in limb girdle muscular dystrophy type 2A

Background: Limb girdle muscular dystrophy type 2A (LGMD2A) is characterised by wide variability in clinical features and rate of progression. Patients with two null mutations usually have a rapid course,...

A large deletion and novel point mutations in the calpain 3 gene (CAPN3) in Bulgarian LGMD2A patients

Limb-girdle muscular dystrophy type 2A (LGMD2A) is caused by mutations in the calpain 3 (CAPN3) gene. The clinical diagnoses of these cases in Bulgaria are very complicated, no protein analysis on muscular biopsy is available in our country, and genetic tests are the only possibility to clarify the diagnoses in clinically ambiguous cases. We screened 48 unrelated Bulgarian cases with preliminary diagnoses of different types of muscular dystrophy for mutations in the CAPN3 gene. Altogether, 20 families (42%) were found to carry mutations in the CAPN3 gene. Several misdiagnosed cases were clarified. Three novel and six recurrent mutations were identified. In total, 40% of the patients are homozygous for c.550delA, and 70% carry it at least on one allele. The affected group of women in our sample shows later onset, milder clinical manifestation, slower progression, and later invalidization.