Limb Girdle Muscular Dystrophy 2A Research Articles

Evaluation of cardiomyopathy with two-dimensional speckle tracking echocardiography in limb-girdle muscular dystrophy type 2A and 2B.

Cardiac involvement in limb-girdle muscular dystrophy (LGMD)2A and LGMD2B, the most common subgroups of LGMD, is controversial. Our study aims to determine whether myocardial dysfunction develops in LGMD2A and LGMD2B patients. The study included 16 LGMD2A, 12 LGMD2B patients, and 48 healthy individuals. Comparisons included demographic, clinical, and laboratory parameters of LGMD2A and LGMD2B subgroups and traditional echocardiography and two-dimensional speckle tracking echocardiography (2D-STE) parameters with the normal population. The median age was 33 (22-39 interquartile range [IQR]) in the LGMD2A group, 33 (27-38 IQR) in the LGMD2B group, and 28 (25-35 IQR) in the control group. The left ventricular (LV) ejection fraction of both LGMD2A and LGMD2B groups was similar to the control group (p=0.296 and p=0.918). Apical 4-chamber longitudinal strain (LS), Apical 2-chamber LS, Apical 3-chamber LS, left ventricular global longitudinal strain (LVGLS)-mid-myocardial, LVGLS-endocardium, and LVGLS-epicardium were lower (less negative) in the LGMD2B group compared to the control group (p=0.006, p=0.001, p < 0.001, p < 0.001, p < 0.001, and p < 0.001, respectively). LV 2D-STE parameters of LGMD2A patients were similar to the control group, while they decreased significantly (less negative) in LGMD2B patients, indicating that LV subclinical myocardial dysfunction may develop in LGMD2B patients.

Systemic delivery of AAVrh74.tMCK.hCAPN3 rescues the phenotype in a mouse model for LGMD2A/R1

Limb girdle muscular dystrophy (LGMD) 2A/R1, caused by mutations in the CAPN3 gene and CAPN3 loss of function, is known to play a role in disease pathogenicity. In this study, AAVrh74.tMCK.CAPN3 was delivered systemically to two different age groups of CAPN3 knockout (KO) mice; each group included two treatment cohorts receiving low (1.17 × 1014 vg/kg) and high (2.35 × 1014 vg/kg) doses of the vector and untreated controls. Treatment efficacy was tested 20 weeks after gene delivery using functional (treadmill), physiological (in vivo muscle contractility assay), and histopathological outcomes. AAV.CAPN3 gene therapy resulted in significant, robust improvements in functional outcomes and muscle physiology at low and high doses in both age groups. Histological analyses of skeletal muscle showed remodeling of muscle, a switch to fatigue-resistant oxidative fibers in females, and fiber size increases in both sexes. Safety studies revealed no organ tissue abnormalities; specifically, there was no histopathological evidence of cardiotoxicity. These results show that CAPN3 gene replacement therapy improved the phenotype in the CAPN3 KO mouse model at both doses independent of age at the time of vector administration. The improvements were supported by an absence of cardiotoxicity, showing the efficacy and safety of the AAV.CAPN3 vector as a potential gene therapy for LGMDR1.

OMICs AND AI APPROACHES FOR MUSCLE DISEASES: O.08 Deep convolutional-neural-network can differentiate twelve major muscular diseases better than human

Muscle pathology plays an important role in the diagnosis of muscle disease even in the molecular era, as most muscle diseases have been classified or even partly defined by pathological features. Nevertheless, availability of experts is limited especially in underserved areas of the world. This study aims to establish the algorithm to assist muscle pathology diagnosis using deep convolutional neural networks (CNNs), which can differentiate major muscle diseases only by hematoxylin and eosin (H&E)-stained pathological images. Target diseases were dystrophinopathy (DYST), limb-girdle muscular dystrophy (LGMD) 2A (LG2A), LGMD2B (LG2B), Ullrich congenital muscular dystrophy (CMD) (UCMD), Fukuyama CMD (FCMD), congenital myopathy (CM), GNE myopathy (GNEM), dermatomyositis (DM), inclusion body myositis (IBM), immune-mediated necrotizing myopathy (IMNM), antisynthetase syndrome (ASS), and neuropathic disease (NP). All images to train CNNs were taken from slides with CCD cameras via light microscopes. A total of 4041 images were obtained from 1400 slides. The algorithm consists of two steps: 1) to differentiate between idiopathic inflammatory myopathy (IIM) and other conditions, and 2) to subclassify each category. For the first step, images of DM, IBM, IMNM, and ASS were combined to create the IIM group. Meanwhile, those of DYST, FCMD, LG2A, LG2B, UCMD, CM, GNEM, and NP were combined to create another group. At the second step, four subtypes of IIM were analyzed. The first step achieved 0.996 area under the curve (AUC). The accuracy of the CNN and the best pathologist of all were 96.9% and 93.8%, respectively, indicating the CNN outperformed muscle specialists. In the second step, the CNNs classified four subtypes (DM, IBM, IMNM, and ASS) with high AUCs: 0.953 (IMNM), 0.969 (IBM), 0.965 (DM), and 0.944 (ASS). The algorithm used only a few H&E images obtained by CCD, which does not need a huge financial investment. This system should be helpful in clinical settings especially in underserved regions, where experts are not easily available.

LIMB GIRDLE MUSCULAR DYSTROPHIES: P.148 Semiquantitative CT assessment of muscle involvement in limb girdle muscular dystrophy 2A and 2B with the wide range of duration

Limb girdle muscular dystrophy (LGMD) 2A and 2B represent the most frequent forms of LGMDs in the Japanese population. We investigated muscle CT images of both LGMDs with the wide range of disease duration to characterize muscle involvement of this disease and to determine the rate of progression. We applied the Mercuri grading system to CT scans cross-sectional images of six cross section (shoulder, upper and lower trunk, pelvis, thigh and calf). This study included 15 patients with LGMD2A (8 male, 7 female, 41.2±21.6) and 27 patients with LGMD2B (13 male, 14 female, 50.5±17.4 years) registered in IBIC (Integrative Brain Imaging Center:the database of skeletal muscle images in Japan). The patients were classified into 3 groups according to disease duration: stage1 (<10years), stage 2 (11∼30years) and stage 3 (31<years). In stage1, both types showed early predominant degeneration of subscapularis, paraspinal, gluteus minimus and the medial head of the gastrocnemius. There was mild muscle involvement in 2A, but moderate involvement in 2B at shoulder level. The involvement of the pelvic muscles in 2A was equal to, or slightly more than those of 2B. The degeneration of the posterior segment of the thigh with sparing of the gracilis muscle was observed in both. In stage2, biceps brachii muscle was much more severely involved than triceps brachii muscle in 2A, but this finding was not true for 2B. Patchy involvement of the quadriceps was observed in 2B. In stage3, almost all muscles were replaced by fat in 2B whereas there were some residual muscles in 2A at the thigh and calf level. The rate of disease progression was much slower in LGMD2A than LGMD2B. This finding is concordant with our previous study using %MVI.

Plasmid-Mediated Gene Therapy in Mouse Models of Limb Girdle Muscular Dystrophy

We delivered plasmid DNA encoding therapeutic genes to the muscles of mouse models of limb girdle muscular dystrophy (LGMD) 2A, 2B, and 2D, deficient in calpain3, dysferlin, and alpha-sarcoglycan, respectively. We also delivered the human follistatin gene, which has the potential to increase therapeutic benefit. After intramuscular injection of DNA, electroporation was applied to enhance delivery to muscle fibers. When plasmids encoding the human calpain3 or dysferlin cDNA sequences were injected into quadriceps muscles of LGMD2A and LGMD2B mouse models, respectively, in 3-month studies, robust levels of calpain3 and dysferlin proteins were detected. We observed a statistically significant decrease in Evans blue dye penetration in LGMD2B mouse muscles after delivery of the dysferlin gene, consistent with repair of the muscle membrane defect in these mice. The therapeutic value of delivery of the genes for alpha-sarcoglycan and follistatin was documented by significant drops in Evans blue dye penetration in gastrocnemius muscles of LGMD2D mice. These results indicated for the first time that a combined gene therapy involving both alpha-sarcoglycan and follistatin would be valuable for LGMD2D patients. We suggest that this non-viral gene delivery method should be explored for its translational potential in patients.

Multiparametric quantitative MRI assessment of thigh muscles in limb-girdle muscular dystrophy 2A and 2B.

The aim of this study was to apply quantitative MRI (qMRI) to assess structural modifications in thigh muscles of subjects with limb girdle muscular dystrophy (LGMD) 2A and 2B with long disease duration. Eleven LGMD2A, 9 LGMD2B patients and 11 healthy controls underwent a multi-parametric 3T MRI examination of the thigh. The protocol included structural T1-weighted images, DIXON sequences for fat fraction calculation, T2 values quantification and diffusion MRI. Region of interest analysis was performed on 4 different compartments (anterior compartment, posterior compartment, gracilis, sartorius). Patients showed high levels of fat infiltration as measured by DIXON sequences. Sartorius and anterior compartment were more infiltrated in LGMD2B than LGMD2A patients. T2 values were mildly reduced in both disorders. Correlations between clinical scores and qMRI were found. qMRI measures may help to quantify muscular degeneration, but careful interpretation is needed when fat infiltration is massive. Muscle Nerve 58: 550-558, 2018.

Calpain 3 and CaMKIIβ signaling are required to induce HSP70 necessary for adaptive muscle growth after atrophy

Mutations in CAPN3 cause autosomal recessive limb girdle muscular dystrophy 2A. Calpain 3 (CAPN3) is a calcium dependent protease residing in the myofibrillar, cytosolic and triad fractions of skeletal muscle. At the triad, it colocalizes with calcium calmodulin kinase IIβ (CaMKIIβ). CAPN3 knock out mice (C3KO) show reduced triad integrity and blunted CaMKIIβ signaling, which correlates with impaired transcriptional activation of myofibrillar and oxidative metabolism genes in response to running exercise. These data suggest a role for CAPN3 and CaMKIIβ in gene regulation that takes place during adaptation to endurance exercise. To assess whether CAPN3- CaMKIIβ signaling influences skeletal muscle remodeling in other contexts, we subjected C3KO and wild type mice to hindlimb unloading and reloading and assessed CaMKIIβ signaling and gene expression by RNA-sequencing. After induced atrophy followed by 4 days of reloading, both CaMKIIβ activation and expression of inflammatory and cellular stress genes were increased. C3KO muscles failed to activate CaMKIIβ signaling, did not activate the same pattern of gene expression and demonstrated impaired growth at 4 days of reloading. Moreover, C3KO muscles failed to activate inducible HSP70, which was previously shown to be indispensible for the inflammatory response needed to promote muscle recovery. Likewise, C3KO showed diminished immune cell infiltration and decreased expression of pro-myogenic genes. These data support a role for CaMKIIβ signaling in induction of HSP70 and promotion of the inflammatory response during muscle growth and remodeling that occurs after atrophy, suggesting that CaMKIIβ regulates remodeling in multiple contexts: endurance exercise and growth after atrophy.

P.154 - Severe respiratory involvement in a young-onset limb-girdle muscular dystrophy 2A from Guinea-Bissau associated with a novel pathogenic mutation

P.154 - Severe respiratory involvement in a young-onset limb-girdle muscular dystrophy 2A from Guinea-Bissau associated with a novel pathogenic mutation

Failure to up-regulate transcription of genes necessary for muscle adaptation underlies limb girdle muscular dystrophy 2A (calpainopathy).

Limb girdle muscular dystrophy 2A is due to loss-of-function mutations in the Calpain 3 (CAPN3) gene. Our previous data suggest that CAPN3 helps to maintain the integrity of the triad complex in skeletal muscle. In Capn3 knock-out mice (C3KO), Ca2+ release and Ca2+/calmodulin kinase II (CaMKII) signaling are attenuated. We hypothesized that calpainopathy may result from a failure to transmit loading-induced Ca2+-mediated signals, necessary to up-regulate expression of muscle adaptation genes. To test this hypothesis, we compared transcriptomes of muscles from wild type (WT) and C3KO mice subjected to endurance exercise. In WT mice, exercise induces a gene signature that includes myofibrillar, mitochondrial and oxidative lipid metabolism genes, necessary for muscle adaptation. C3KO muscles fail to activate the same gene signature. Furthermore, in agreement with the aberrant transcriptional profile, we observe a commensurate functional defect in lipid metabolism whereby C3KO muscles fail to release fatty acids from stored triacylglycerol. In conjunction with the defects in oxidative metabolism, C3KO mice demonstrate reduced exercise endurance. Failure to up-regulate genes in C3KO muscles is due, in part, to decreased levels of PGC1α, a transcriptional co-regulator that orchestrates the muscle adaptation response. Destabilization of PGC1α is attributable to decreased p38 MAPK activation via diminished CaMKII signaling. Thus, we elucidate a pathway downstream of Ca2+-mediated CaMKII activation that is dysfunctional in C3KO mice, leading to reduced transcription of genes involved in muscle adaptation. These studies identify a novel mechanism of muscular dystrophy: a blunted transcriptional response to muscle loading resulting in chronic failure to adapt and remodel.

Attenuated Ca(2+) release in a mouse model of limb girdle muscular dystrophy 2A.

BackgroundMutations in CAPN3 cause limb girdle muscular dystrophy type 2A (LGMD2A), a progressive muscle wasting disease. CAPN3 is a non-lysosomal, Ca-dependent, muscle-specific proteinase. Ablation of CAPN3 (calpain-3 knockout (C3KO) mice) leads to reduced ryanodine receptor (RyR1) expression and abnormal Ca2+/calmodulin-dependent protein kinase II (Ca-CaMKII)-mediated signaling. We previously reported that Ca2+ release measured by fura2-FF imaging in response to single action potential stimulation was reduced in old C3KO mice; however, the use of field stimulation prevented investigation of the mechanisms underlying this impairment. Furthermore, our prior studies were conducted on older animals, whose muscles showed advanced muscular dystrophy, which prevented us from establishing whether impaired Ca2+ handling is an early feature of disease. In the current study, we sought to overcome these matters by studying single fibers isolated from young wild-type (WT) and C3KO mice using a low affinity calcium dye and high intracellular ethylene glycol-bis(2-aminoethylether)-n,n,n′,n′-tetraacetic acid (EGTA) to measure Ca2+ fluxes. Muscles were subjected to both current and voltage clamp conditions.MethodsStandard and confocal fluorescence microscopy was used to study Ca2+ release in single fibers enzymatically isolated from hind limb muscles of wild-type and C3KO mice. Two microelectrode amplifier and experiments were performed under current or voltage clamp conditions. Calcium concentration changes were detected with an impermeant low affinity dye in the presence of high EGTA intracellular concentrations, and fluxes were calculated with a single compartment model. Standard Western blotting analysis was used to measure the concentration of RyR1 and the α subunit of the dihydropyridine (αDHPR) receptors. Data are presented as mean ± SEM and compared with the Student’s test with significance set at p < 0.05.ResultsWe found that the peak value of Ca2+ fluxes elicited by single action potentials was significantly reduced by 15–20 % in C3KO fibers, but the kinetics was unaltered. Ca2+ release elicited by tetanic stimulation was also impaired in C3KO fibers. Confocal studies confirmed that Ca2+ release was similarly reduced in all triads of C3KO mice. Voltage clamp experiments revealed a normal voltage dependence of Ca2+ release in C3KO mice but reduced peak Ca2+ fluxes as with action potential stimulation. These findings concur with biochemical observations of reduced RyR1 and αDHPR levels in C3KO muscles and reduced mechanical output. Confocal studies revealed a similar decrease in Ca2+ release at all triads consistent with a homogenous reduction of functional voltage activated Ca2+ release sites.ConclusionsOverall, these results suggest that decreased Ca2+ release is an early defect in calpainopathy and may contribute to the observed reduction of CaMKII activation in C3KO mice.

Calpain-3-mediated regulation of the Na⁺-Ca²⁺ exchanger isoform 3.

Ca2+ disturbances are observed when Ca2+-dependent cysteine proteases malfunction, causing muscle weakness and wasting. For example, loss of calpain-3 (CAPN3) activity leads to limb-girdle muscular dystrophy 2A (LGMD2A). In neuronal excitotoxicity, the cleavage of the Na+-Ca2+ exchanger isoform 3 (NCX3) has been associated with an increase in activity and elevation of the Ca2+ content in the endoplasmic reticulum (ER). Since NCX3 is expressed in skeletal muscle, we evaluated the cleavage of different NCX3 splice variants by CAPN1 and CAPN3. Using Fura-2-based cellular Ca2+ imaging, we showed for the first time that CAPN3 increases NCX3 activity and that only NCX3-AC, the variant predominantly expressed in skeletal muscle, is sensitive to calpain. The silencing of the endogenous CAPN1 and the expression of the inactive form of CAPN3 (C129S CAPN3) confirmed the specificity for CAPN1 and CAPN3. Functional studies revealed that cellular Ca2+ uptake through the reverse mode of NCX3 was significantly increased independently of the mode of activation of the exchanger by either a rise in intracellular Ca2+ ([Ca2+]i) or Na+ ([Na+]i). Subsequently, the sensitivity to CAPN1 and CAPN3 could be abrogated by removal of the six residues coded in exon C of NCX3-AC. Additionally, mutation of the Leu-600 and Leu-601 suggested the presence of a cleavage site at Leu-602. The increased Ca2+ uptake of NCX3 might participate in the Ca2+ refilling of the sarcoplasmic reticulum (SR) after the excitation-contraction uncoupling following exercise and therefore be implicated in the impaired reticular Ca2+ storage observed in LGMD2A.

The Frequency of c.550delA Mutation of the CANP3 Gene in the Polish LGMD2A Population

Limb girdle muscular dystrophy 2A (LGMD2A) is the most frequent LGMD variant in the European population, representing about 40% of LGMD. The c.550delA mutation in the CANP3 (calcium activated neutral protease 3) gene is the most commonly reported mutation in LGMD2A. Prevalence of this mutation in the Polish population has not been previously investigated. The aim of this study was to identify and estimate the frequency of the c.550delA mutation in Polish LGMD2A patients. Polymerase chain reaction-sequencing analysis, restriction fragment length polymorphism polymerase chain reaction method. We analyzed 76 families affected with LGMD and identified 62 probands with mutations in the CANP3 gene. C.550delA was the most common mutation identified, being found in 78% of the LGMD2A families. The remaining mutations observed multiple times were as follows: c.598-612del15ntd; c.2242C>T; c.418dupC; c.1356insT, listed in terms of decreasing frequency. Two novel variants in the CANP3 gene, that is, c.700G>A Gly234Arg and c.661G>A Gly221Ser were also characterized. Overall, mutations in the LGMD2A gene were estimated to be present in 81% of patients with the LGMD phenotype who were without sarcoglycans and dysferlin deficiency on immunocytochemical analysis. The frequency of the heterozygous c.550delA mutation in the healthy Polish population was estimated at 1/124. The c.550delA is the most frequent CANP3 mutation in the Polish population, thus sequencing of exon 4 of this gene could identify the majority of LGMD2A patients in Poland.

G.P.158 - Description of muscle strength in a Brazilian sample of limb-girdle muscular dystrophy 2A (calpainopathy)

G.P.158 - Description of muscle strength in a Brazilian sample of limb-girdle muscular dystrophy 2A (calpainopathy)

CAPN3-mediated processing of C-terminal titin replaced by pathological cleavage in titinopathy.

Mutations in the extreme C-terminus of titin (TTN), situated in the sarcomeric M-band, cause tibial muscular dystrophy (TMD) and limb-girdle muscular dystrophy 2J (LGMD2J). The mutations ultimately cause a loss of C-terminal titin, including a binding site for the protease calpain 3 (CAPN3), and lead to a secondary CAPN3 deficiency in LGMD2J muscle. CAPN3 has been previously shown to bind C-terminal titin and to use it as a substrate in vitro. Interestingly, mutations in CAPN3 underlie limb-girdle muscular dystrophy 2A (LGMD2A). Here, we aimed to clarify the relationship of CAPN3 and M-band titin in normal and pathological muscle. In vitro analyses identified several CAPN3 cleavage sites in C-terminal titin that were defined by protein sequencing. Furthermore, cleavage products were detected in normal muscle extracts by western blotting and in situ by immunofluorescence microscopy. The TMD/LGMD2J mutation FINmaj proved to alter this processing in vitro, while binding of CAPN3 to mutant titin was preserved. Unexpectedly, the pathological loss of M-band titin due to TMD/LGMD2J mutations was found to be independent of CAPN3, whereas the involvement of ubiquitous calpains is likely. We conclude that proteolytic processing of C-terminal titin by CAPN3 may have an important role in normal muscle, and that this process is disrupted in LGMD2A and in TMD/LGMD2J due to CAPN3 deficiency and to the loss of C-terminal titin, respectively.

Differential Diagnosis between Duchenne Muscular Dystrophy and Limb Girdle Muscular Dystrophy 2a.

Limb Girdle Muscular Dystrophy 2A (LGMD 2A) is the most common form of limb girdle muscular dystrophies caused by mutations in the calpain-3 gene (CAPN-3). The pattern of LGMD 2A can be clinically indistinguishable from that of Duchenne Muscular Dystrophy (DMD). We report a case of a 14-year-old boy which has the initial diagnosed as DMD at 6 years old, based on clinical features and very elevated serum creatine kinase levels. A muscle biopsy at the age of 10 showed atypical features which suggested a histiocytosis or neural damage. An MRI conducted 2 years later revealed fatty degeneration predominantly in the posterior region of the thigh and led the diagnosis to LGMD 2A, as well as the necessity to repeat the biopsy. Immunohistochemical analysis was normal for dystrophin, but the Western Blott showed a normal/borderline amount of calpain-3 in the muscle. We also performed a molecular analysis that identified a compound heterozygous mutation of the calpain 3 gene (CAPN 3). LGMD 2A was often misdiagnosed as DMD due to the similarities in clinical manifestations and technique limitations; the immunohistochemical examination, the magnetic resonance imaging examination and the molecular analysis are an essential tool for establishing a right diagnosis.

Autolytic Activation of Calpain 3 Proteinase Is Facilitated by Calmodulin Protein

Calpains are broadly distributed, calcium-dependent enzymes that induce limited proteolysis in a wide range of substrates. Mutations in the gene encoding the muscle-specific family member calpain 3 (CAPN3) underlie limb-girdle muscular dystrophy 2A. We have shown previously that CAPN3 knockout muscles exhibit attenuated calcium release, reduced calmodulin kinase (CaMKII) signaling, and impaired muscle adaptation to exercise. However, neither the precise role of CAPN3 in these processes nor the mechanisms of CAPN3 activation in vivo have been fully elucidated. In this study, we identify calmodulin (CaM), a known transducer of the calcium signal, as the first positive regulator of CAPN3 autolytic activity. CaM was shown to bind CAPN3 at two sites located in the C2L domain. Biochemical studies using muscle extracts from transgenic mice overexpressing CAPN3 or its inactive mutant revealed that CaM binding enhanced CAPN3 autolytic activation. Furthermore, CaM facilitated CAPN3-mediated cleavage of its in vivo substrate titin in tissue extracts. Therefore, these studies reveal a novel interaction between CAPN3 and CaM and identify CaM as the first positive regulator of CAPN3 activity.

Clinical and molecular genetic study of limb-girdle muscular dystrophy type 2A in a Chinese family

Objective To analyze the clinical, muscle pathological features and molecular mutations in the 2 Chinese Han siblings with limb-girdle muscular dystrophy(LGMD) and conclude the phenotype/genotype correlations. Methods Clinical and muscle pathological data were collected.Genomic DNA of the two siblings and their parents were extracted using standard procedures from the peripheral leukocytes.A custom of targeted gene panel including 61 neuromuscular genes were designed by using the Agilent Sureselect Target Enrichment Kit.Targeted next generation sequencing(NGS) was performed in the proband, and CAPN3 gene mutation was verified with Sanger sequencing in the two siblings and their parents.The dbSNP138 and http: //www.dmd.nl were searched to determine the disease-causing mutations. Results The proband slowly showed muscle weakness profoundly with pelvic muscles, developed difficulty in squatting and standing and climbing stairs.She had a tight Achilles tendon, high CK level(1 908-9 241 IU/L), wi-thout winging scapula and hypertrophy calf.The affected brother was only diagnosed hyper CKemia. By using the targeted NGS, the two siblings possessed the same two compound heterozygous mutations(c.717delT and c. 2243G>A) in CAPN3 gene.The two mutations both were verified by Sanger sequencing and had been reported before. Conclusions LGMD is clinically and genetically heterogeneous, and targeted NGS is powerful in defining the causal mutation of LGMD and helpful in investigating the exact genotype/phenotype analysis. Key words: Limb-girdle muscular dystrophy 2A; CAPN3 gene; Next generation sequencing; Mutation

G.P.230: Clinical and genetic features in Korean patients with CAPN3 mutations

Recessive mutations in the CAPN3 gene are responsible for Limb-girdle muscular dystrophy (LGMD) 2A, one of the most common type of LGMD worldwide. However, the characteristics of LGMD2A in Koreans have not yet been evaluated. Therefore, the present study was designed to investigate the characteristics in Korean patients with CAPN3 mutations. Mutations were identified by direct or targeted sequencing using a primer set that covered all 24 exons and flanking intron regions of the CAPN3 gene. Then, we evaluated genetic, clinical, and pathological spectrum. Results: We identified eight distinct mutations in 12 patients from nine families. Four mutations were missense, two were frameshift, and two were aberrant splicing. Additionally, four mutations (c.1524+1G>T, c.1789_1790inA, c.2120A>G, c.2384C>T) were novel. We detected two mutations in eight patients from six families and only one mutation in four patients from three families. Patient ages at symptom onset ranged from eight to 33 years. Family history was positive in four of nine families. Three patients were initially diagnosed with Emery-Dreifuss muscular dystrophy, hyperCKemia, and polymyositis, respectively. This is because one patient exhibited prominent contracture of ankle and elbow joints, another patient presented hyperCKemia without definite muscle weakness, and the other patient showed minimal inflammatory cell infiltration on muscle pathology. Winged scapula, joint contracture and lordosis were found in four, six and three patients, respectively. In addition, lobulated fibers on muscle pathology were detected in four of eight tested patients. In the present study, we identify four novel CAPN3 mutations and confirm the usefulness of clinical and pathologic findings, especially lobulated fibers.

Muscle atrophy in Limb Girdle Muscular Dystrophy 2A: a morphometric and molecular study

The peculiar clinical features and the pathogenic mechanism related to calpain-3 deficiency (impaired sarcomere remodelling) suggest that the ubiquitin-proteasome degradation pathway may have a crucial role in Limb Girdle Muscular Dystrophy 2A (LGMD2A). We therefore investigated muscle atrophy and the role of the ubiquitin-proteasome and lysosomal-autophagic degradation pathways. We selected 25 adult male LGMD2A patients (and seven controls), classified them using clinical severity score, analysed muscle fibre size by morphometry and protein and/or transcriptional expression levels of the most important atrophy- and autophagy-related genes (MuRF1, atrogin1, LC3, p62, Bnip3). Muscle fibre size was significantly lower in LGMD2A than in controls and it was significantly correlated with patients' clinical disability score recorded at the time of biopsy, suggesting that functional and structural muscle impairment are dependent. The large majority of atrophic fibres originate from a mechanism different from regeneration, as assessed by neonatal myosin immunolabelling. As compared with controls, LGMD2A muscles have higher MuRF1 (but not atrogin1) protein and MuRF1 gene expression levels, and MuRF1 protein levels significantly correlated with both muscle fibre size and clinical disability score. LGMD2A muscles have slightly increased levels of LC3-II and p62 proteins and a significant up-regulation of p62 and Bnip3 gene expression. In LGMD2A muscles the activation of the atrophy programme appeared to depend mainly upon induction of the ubiquitin-proteasome system and, to a lesser extent, the autophagic-lysosomal degradation pathway.

MRI muscle fat quantification in ambulant patients with limb girdle muscular dystrophy 2A and correlation with clinical severity

Background: Muscle MRI provides information on the distribution and evolution of limb girdle muscular dystrophies (LGMD). In the last years quantitative sequences like 3-point Dixon technique have been developed to measure muscle fat fraction (MFF).