Limb-girdle Muscular Dystrophy Type 2A Research Articles

A knock down strategy for rapid, generic, and versatile modelling of muscular dystrophies in 3D-tissue-engineered-skeletal muscle

BackgroundHuman iPSC-derived 3D-tissue-engineered-skeletal muscles (3D-TESMs) offer advanced technology for disease modelling. However, due to the inherent genetic heterogeneity among human individuals, it is often difficult to distinguish disease-related readouts from random variability. The generation of genetically matched isogenic controls using gene editing can reduce variability, but the generation of isogenic hiPSC-derived 3D-TESMs can take up to 6 months, thereby reducing throughput.MethodsHere, by combining 3D-TESM and shRNA technologies, we developed a disease modelling strategy to induce distinct genetic deficiencies in a single hiPSC-derived myogenic progenitor cell line within 1 week.ResultsAs proof of principle, we recapitulated disease-associated pathology of Duchenne muscular dystrophy and limb-girdle muscular dystrophy type 2A caused by loss of function of DMD and CAPN3, respectively. shRNA-mediated knock down of DMD or CAPN3 induced a loss of contractile function, disruption of tissue architecture, and disease-specific proteomes. Pathology in DMD-deficient 3D-TESMs was partially rescued by a candidate gene therapy treatment using micro-dystrophin, with similar efficacy compared to animal models.ConclusionsThese results show that isogenic shRNA-based humanized 3D-TESM models provide a fast, cheap, and efficient tool to model muscular dystrophies and are useful for the preclinical evaluation of novel therapies.

Comorbidity of bathing suit ichthyosis and limb-girdle muscular dystrophy type 2 A in a Tunisian patient revealed by Whole Exome Sequencing

Elevated rates of consanguinity and inbreeding are responsible for the high prevalence of recessively inherited diseases among inbred populations including Tunisia. In addition, the co-occurrence of two of these conditions, called also comorbidity, within the same individual or in members of the same family are often described in Tunisia which is challenging for diagnosis. The high throughput sequencing has improved the diagnosis of inherited diseases. We report here on a 32-year-old woman born to consanguineous parents. She presented with congenital ichthyosis and muscular dystrophy. She was primarily suspected as suffering from Chanarin-Dorfman syndrome (CDS) with unusual form. Screening of founder mutations allowed only the elucidation of the molecular etiology of Ichthyosis. As the result was inconclusive, Whole Exome Sequencing (WES) was conducted. WES data analysis led to the identification of a mutation in the CAPN3 gene underlying limb-girdle muscular dystrophy type 2A (LGMD2A). Sanger sequencing confirmed the familial segregation of mutations. This work presents the first case worldwide of individual comorbidity of bathing suit ichthyosis and LGMD2A. The co-occurrence of two diseases should be systematically considered when establishing a diagnosis especially in consanguineous populations. WES is a powerful tool for molecular diagnosis in particular for revealing comorbidities and rectifying the diagnosis.

Ryanodine receptor type 1 content decrease-induced endoplasmic reticulum stress is a hallmark of myopathies.

Decreased ryanodine receptor type 1 (RyR1) protein levels are a well-described feature of recessive RYR1-related myopathies. The aim of the present study was twofold: (1) to determine whether RyR1 content is also decreased in other myopathies and (2) to investigate the mechanisms by which decreased RyR1 protein triggers muscular disorders. We used publicly available datasets, muscles from human inflammatory and mitochondrial myopathies, an inducible muscle-specific RYR1 recessive mouse model and RyR1 knockdown in C2C12 muscle cells to measure RyR1 content and endoplasmic reticulum (ER) stress markers. Proteomics, lipidomics, molecular biology and transmission electron microscopy approaches were used to decipher the alterations associated with the reduction of RyR1 protein levels. RYR1 transcripts were reduced in muscle samples of patients suffering from necrotizing myopathy (P=0.026), inclusion body myopathy (P=0.003), polymyositis (P<0.001) and juvenile dermatomyositis (P<0.001) and in muscle samples of myotonic dystrophy type 2 (P<0.001), presymptomatic (P<0.001) and symptomatic (P<0.001) Duchenne muscular dystrophy, Becker muscular dystrophy (P=0.004) and limb-girdle muscular dystrophy type 2A (P=0.004). RyR1 protein content was also significantly decreased in inflammatory myopathy (-75%, P<0.001) and mitochondrial myopathy (-71%, P<0.001) muscles. Proteomics data showed that depletion of RyR1 protein in C2C12 myoblasts leads to myotubes recapitulating the common molecular alterations observed in myopathies. Mechanistically, RyR1 protein depletion reduces ER-mitochondria contact length (-26%, P<0.001), Ca2+ transfer to mitochondria (-48%, P=0.002) and the mitophagy gene Parkinson protein 2 transcripts (P=0.037) and induces mitochondrial accumulation (+99%, P=0.005) and dysfunction (P<0.001). This was associated to the accumulation of deleterious sphingolipid species. Our data showed increased levels of the ER stress marker chaperone-binding protein/glucose regulated protein 78, GRP78-Bip, in RyR1 knockdown myotubes (+45%, P=0.046), in mouse RyR1 recessive muscles (+58%, P=0.001) and in human inflammatory (+96%, P=0.006) and mitochondrial (+64%, P=0.049) myopathy muscles. This was accompanied by increased protein levels of the pro-apoptotic protein CCAAT-enhancer-binding protein homologous protein, CHOP-DDIT3, in RyR1 knockdown myotubes (+27%, P<0.001), mouse RyR1 recessive muscles (+63%, P=0.009), human inflammatory (+50%, P=0.038) and mitochondrial (+51%, P=0.035) myopathy muscles. In publicly available datasets, the decrease in RYR1 content in myopathies was also associated to increased ER stress markers and RYR1 transcript levels are inversely correlated with ER stress markers in the control population. Decreased RyR1 is commonly observed in myopathies and associated to ER stress in vitro, in mouse muscle and in human myopathy muscles, suggesting a potent role of RyR1 depletion-induced ER stress in the pathogenesis of myopathies.

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.

Serum miRNAs as biomarkers for the rare types of muscular dystrophy

Muscular dystrophies are a group of disorders that cause progressive muscle weakness. There is an increasing interest for the development of biomarkers for these disorders and specifically for Duchene Muscular Dystrophy. Limited research however, has been performed on the biomarkers’ development for the most rare muscular dystrophies, like the Facioscapulohumeral Muscular Dystrophy, Limb-Girdle Muscular Dystrophy and Myotonic Dystrophy type 2. Here, we aimed to identify novel serum-based miRNA biomarkers for these rare muscular dystrophies, through high-throughput next-generation RNA sequencing. We identified many miRNAs that associate with muscular dystrophy patients compared to controls. Based on a series of selection criteria, the two best candidate miRNAs for each of these disorders were chosen and validated in a larger number of patients. Our results showed that miR-223-3p and miR-206 are promising serum-based biomarkers for Facioscapulohumeral Muscular Dystrophy type 1, miR-143-3p and miR-486-3p for Limb-Girdle Muscular Dystrophy type 2A whereas miR-363-3p and miR-25-3p associate with Myotonic Dystrophy type 2. Some of the identified miRNAs were significantly elevated in the serum of the patients compared to controls, whereas some others were lower. In conclusion, we provide new evidence that certain circulating miRNAs may be used as biomarkers for three types of rare muscular dystrophies.

Аutosomal recessive limb-girdle muscular dystrophy type 2A: two cases in Ukraine with different age of onset

The paper reports on two cases of young women from Ukraine with autosomal recessive limb-girdle muscular dystrophy type 2A with different age of symptoms onset and an absence of any family history presented with gradual onset of proximal muscle weakness in four limbs and thinning of shoulders, arms and thighs. Both patients had elevated creatine phosphokinase level and c.550delA mutations in CAPN3 gene. Sequence analysis and deletion/duplication testing of the 159 genes from skeletal muscles disease testing panel of 5-year-old girl identified deletion of exon 8 (heterozygous) and c.550delA (p.Thr184Argfs*36) mutation (heterozygous), were in CAPN3 gene. Magnetic Resonance Imaging of soft tissue of the proximal lower extremities was performed which showed signs of symmetrical atrophic changes in the major adductor muscle, the long and short adductor muscles, the semitendinosus muscle of the thigh, as a manifestations of autosomal recessive limb-girdle muscular dystrophy type 2A. Homozygous, pathogenic variant of the defect in the CAPN3 gene c.550del (p.Thr184Argfs * 36) was identified in a 25-year-old woman. Type 2A is the most common form of limb-girdle muscular dystrophy, accounting for about 30% of cases. The autosomal recessive limb-girdle muscular dystrophy type 2A is on caused by mutations in the CAPN3 gene, and it is characterized by selective atrophy and weakness of proximal limb and girdle muscles. The age of onset of muscle weakness is extremely variable; the most common being between 8 and 15 years, although it can range between 2 and 50 years. The diagnosis can be suspected by findings on a muscle biopsy or when a doctor experienced in muscular dystrophy examines you. A serum creatine kinase blood test may also show raised levels which indicate a problem in the muscles. The diagnosis has to be confirmed by means of identifying a mutation in the CAPN3 gene which is done on a deoxyribonucleic acid sample from a blood test. To date there are no specific treatments for limb-girdle muscular dystrophy, however careful management of the symptoms of the condition can improve a person’s quality of life. Joint contractures (tightening) can occur in limb-girdle muscular dystrophy and therefore regular physiotherapy is recommended.

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.

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.

Regional variation of thigh muscle fat infiltration in patients with neuromuscular diseases compared to healthy controls.

Chemical shift encoding-based water-fat magnetic resonance imaging (CSE-MRI) measures a quantitative biomarker: the proton density fat fraction (PDFF). The aim was to assess regional and proximo-distal PDFF variations at the thigh in patients with myotonic dystrophy type 2 (DM2), limb-girdle muscular dystrophy type 2A (LGMD2A), and late-onset Pompe disease (LOPD) as compared to healthy controls. Seven patients (n=2 DM2, n=2 LGMD2A, n=3 LOPD) and 20 controls were recruited. A 3D-spoiled gradient echo sequence was used to scan the thigh musculature. Muscles were manually segmented to generate mean muscle PDFF. In all three disease entities, there was an increase in muscle fat replacement compared to healthy controls. However, within each disease group, there were patients with a shorter time since symptom onset that only showed mild PDFF elevation (range, 10% to 20%) compared to controls (P≤0.05), whereas patients with a longer period since symptom onset showed a more severe grade of fat replacement with a range of 50% to 70% (P<0.01). Increased PDFF of around 5% was observed for vastus medialis, semimembranosus and gracilis muscles in advanced compared to early DM2. LGMD2A_1 showed an early disease stage with predominantly mild PDFF elevations over all muscles and levels (10.9%±7.1%) compared to controls. The quadriceps, gracilis and biceps femoris muscles showed the highest difference between LGMD2A_1 with 5 years since symptom onset (average PDFF 11.1%±6.9%) compared to LGMD2A_2 with 32 years since symptom onset (average PDFF 66.3%±6.3%). For LOPD patients, overall PDFF elevations were observed in all major hip flexors and extensors (range, 25.8% to 30.8%) compared to controls (range, 1.7% to 2.3%, P<0.05). Proximal-to-distal PDFF highly varied within and between diseases and within controls. The intra-reader reliability was high (reproducibility coefficient ≤2.19%). By quantitatively measuring muscle fat infiltration at the thigh, we identified candidate muscles for disease monitoring due to their gradual PDFF elevation with longer disease duration. Regional variation between proximal, central, and distal muscle PDFF was high and is important to consider when performing longitudinal MRI follow-ups in the clinical setting or in longitudinal studies.

Mitochondrial dysfunction and consequences in calpain-3-deficient muscle

BackgroundNonsense or loss-of-function mutations in the non-lysosomal cysteine protease calpain-3 result in limb-girdle muscular dystrophy type 2A (LGMD2A). While calpain-3 is implicated in muscle cell differentiation, sarcomere formation, and muscle cytoskeletal remodeling, the physiological basis for LGMD2A has remained elusive.MethodsCell growth, gene expression profiling, and mitochondrial content and function were analyzed using muscle and muscle cell cultures established from healthy and calpain-3-deficient mice. Calpain-3-deficient mice were also treated with PPAR-delta agonist (GW501516) to assess mitochondrial function and membrane repair. The unpaired t test was used to assess the significance of the differences observed between the two groups or treatments. ANOVAs were used to assess significance over time.ResultsWe find that calpain-3 deficiency causes mitochondrial dysfunction in the muscles and myoblasts. Calpain-3-deficient myoblasts showed increased proliferation, and their gene expression profile showed aberrant mitochondrial biogenesis. Myotube gene expression analysis further revealed altered lipid metabolism in calpain-3-deficient muscle. Mitochondrial defects were validated in vitro and in vivo. We used GW501516 to improve mitochondrial biogenesis in vivo in 7-month-old calpain-3-deficient mice. This treatment improved satellite cell activity as indicated by increased MyoD and Pax7 mRNA expression. It also decreased muscle fatigability and reduced serum creatine kinase levels. The decreased mitochondrial function also impaired sarcolemmal repair in the calpain-3-deficient skeletal muscle. Improving mitochondrial activity by acute pyruvate treatment improved sarcolemmal repair.ConclusionOur results provide evidence that calpain-3 deficiency in the skeletal muscle is associated with poor mitochondrial biogenesis and function resulting in poor sarcolemmal repair. Addressing this deficit by drugs that improve mitochondrial activity offers new therapeutic avenues for LGMD2A.

Variants of CAPN3 cause limb-girdle muscular dystrophy type 2A in two Chinese families.

Limb-girdle muscular dystrophies (LGMDs) are a group of neuromuscular diseases that are characterized by progressive muscle weakness. LGMD type 2A (LGMD2A), caused by variants in the calpain-3 (CAPN3) gene, is the most prevalent type. The present study aimed to analyze pathogenic CAPN3 gene variants in two pedigrees affected by LGMD2A. Each family contains three patients who are siblings and sought genetic counseling. Genomic DNA was extracted from the peripheral blood samples collected from the probands and family members and whole-exome sequencing (WES) was used to detect the pathogenic genes in the probands. Suspected variants were subsequently validated by Sanger sequencing. In family 1, WES revealed that the proband carried the compound heterogeneous variants c.1194-9A>G and c.1437C>T (p.Ser479=) in CAPN3 (NM_000070.2). In family 2, WES identified that the proband carried the compound heterogeneous variants c.632+4A>G and c.1468C>T (p.Arg490Trp) in CAPN3 (NM_000070.2). In conclusion, the present study indicated that the compound heterogeneous variants of the CAPN3 gene were most likely responsible for LGMD2A in the two Chinese families.

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.

Titin splicing regulates cardiotoxicity associated with calpain 3 gene therapy for limb-girdle muscular dystrophy type 2A.

Limb-girdle muscular dystrophy type 2A (LGMD2A or LGMDR1) is a neuromuscular disorder caused by mutations in the calpain 3 gene (CAPN3). Previous experiments using adeno-associated viral (AAV) vector-mediated calpain 3 gene transfer in mice indicated cardiac toxicity associated with the ectopic expression of the calpain 3 transgene. Here, we performed a preliminary dose study in a severe double-knockout mouse model deficient in calpain 3 and dysferlin. We evaluated safety and biodistribution of AAV9-desmin-hCAPN3 vector administration to nonhuman primates (NHPs) with a dose of 3 × 1013 viral genomes/kg. Vector administration did not lead to observable adverse effects or to detectable toxicity in NHP. Of note, the transgene expression did not produce any abnormal changes in cardiac morphology or function of injected animals while reaching therapeutic expression in skeletal muscle. Additional investigation on the underlying causes of cardiac toxicity observed after gene transfer in mice and the role of titin in this phenomenon suggest species-specific titin splicing. Mice have a reduced capacity for buffering calpain 3 activity compared to NHPs and humans. Our studies highlight a complex interplay between calpain 3 and titin binding sites and demonstrate an effective and safe profile for systemic calpain 3 vector delivery in NHP, providing critical support for the clinical potential of calpain 3 gene therapy in humans.

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.

Novel Missense CAPN3 Mutation Responsible for Adult-Onset Limb Girdle Muscular Dystrophy with Calves Hypertrophy.

CAPN3 gene encodes for calpain-3; this protein is a calcium-dependent intracellular protease. Deficiency of this enzyme leads to weakness of the proximal limb muscles and pelvic and shoulder girdles, the so-called limb-girdle muscular dystrophy type 2A (LGMD2A). Here, we reported the case of a Tunisian patient with LGMD2A associated with a novel missense mutation (c.T1681C/p.Y561H). A 61-year-old man, with consanguineous parents, was referred for gait difficulties and slowly progressive proximal weakness of the four limbs associated with moderate hypertrophy of the calves but his facial muscles were unaffected. Electromyography showed that the profile was myopathic pattern and creatine kinase (CK) level was high. Muscle biopsy processing included routine histological, immunohistochemical, and Western Blot reactions, using a panel of antibodies directed against dystrophin, dysferlin, calpain-3, sarcoglycan α, β, γ, and δ. For mutation analysis, we designed an NGS-based screening. Immunological analyses demonstrated a total deficiency in calpain-3 and δ-sarcoglycan, and a reduced expression of dysferlin. The genetic study yielded a homozygous missense mutation (c.T1681C) of the 13th exon of the CAPN3 gene. The mutation found in our patient (c.T1681C/p.Y561H) has not been previously reported. It is responsible for complete calpain-3 and δ-sarcoglycan deficiency and reduced dysferlin expression. The genetic study is mandatory in such cases with multiple-protein deficiency and ambiguous results of immune-histology and Western Blot studies.

Insertion sequence 1 from calpain-3 is functional in calpain-2 as an internal propeptide

Calpains are intracellular, calcium-activated cysteine proteases. Calpain-3 is abundant in skeletal muscle, where its mutation-induced loss of function causes limb-girdle muscular dystrophy type 2A. Unlike the small subunit-containing calpain-1 and -2, the calpain-3 isoform homodimerizes through pairing of its C-terminal penta-EF-hand domain. It also has two unique insertion sequences (ISs) not found in the other calpains: IS1 within calpain-3's protease core and IS2 just prior to the penta-EF-hand domain. Production of either native or recombinant full-length calpain-3 to characterize the function of these ISs is challenging. Therefore, here we used recombinant rat calpain-2 as a stable surrogate and inserted IS1 into its equivalent position in the protease core. As it does in calpain-3, IS1 occupied the catalytic cleft and restricted the enzyme's access to substrate and inhibitors. Following activation by Ca2+, IS1 was rapidly cleaved by intramolecular autolysis, permitting the enzyme to freely accept substrate and inhibitors. The surrogate remained functional until extensive intermolecular autoproteolysis inactivated the enzyme, as is typical of calpain-2. Although the small-molecule inhibitors E-64 and leupeptin limited intermolecular autolysis of the surrogate, they did not block the initial intramolecular cleavage of IS1, establishing its role as a propeptide. Surprisingly, the large-molecule calpain inhibitor, calpastatin, completely blocked enzyme activity, even with IS1 intact. We suggest that calpastatin is large enough to oust IS1 from the catalytic cleft and take its place. We propose an explanation for why calpastatin can inhibit calpain-2 bearing the IS1 insertion but cannot inhibit WT calpain-3.

Hippo signaling pathway is altered in Duchenne muscular dystrophy.

Hippo signaling pathway is considered a key regulator of tissue homeostasis, cell proliferation, apoptosis and it is involved in cancer development. In skeletal muscle, YAP, a downstream target of the Hippo pathway, is an important player in myoblast proliferation, atrophy/hypertrophy regulation, and in mechano-trasduction, transferring mechanical signals into transcriptional responses. We studied components of Hippo pathway in muscle specimens from patients with Duchenne muscular dystrophy (DMD), Becker muscular dystrophy, limb-girdle muscular dystrophy type 2A and type 2B and healthy subjects. Only DMD muscles had decreased YAP1 protein expression, increased LATS1/2 kinase activity, low Survivin mRNA expression and high miR-21 expression. In light of our novel results, a schematic model is postulated: low levels of YOD1 caused by increased inhibition by miR-21 lead to an increase of LATS1/2 activity which in turn augments phosphorylation of YAP. Reduced amount of active YAP, which is also a target of increased miR-21, causes decreased nuclear expression of YAP-mediated target genes. Since it is known that YAP has beneficial roles in promoting tissue repair and regeneration after injury so that its activation may be therapeutically useful, our results suggest that some components of Hippo pathway could become novel therapeutic targets for DMD treatment.

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.

F42. EMG findings in patients with limb-girdle muscular dystrophy due to heterozygous CAPN3 mutations

Introduction Limb-girdle muscular dystrophy type 2A (LGMD2A) is the most common autosomal recessive (AR) LGMD subtype worldwide. The disease is caused by homozygous pathogenic mutations in the calpain 3 gene (CAPN3). Although strict AR inheritance is assumed for CAPN3-related myopathies, recent reports described symptomatic patients carrying a single CAPN3 mutation. The LGMDs share common clinical and laboratory findings such as weakness and atrophy of proximal muscles, often affecting lower extremities first; elevated creatine kinase (CK) levels; and dystrophic findings on muscle biopsy. Some clinical and imaging findings help to differentiate LGMD2A, such as asymmetric weakness and atrophy and early scapular winging. On muscle MRI, calpain deficiency characteristically reveals preferential involvement of posterior thigh muscles and the adductor magnus muscle with relative sparing of the anterior thigh muscles. There are few reports on EMG findings in patients with single CAPN3 mutations. Methods Two unrelated patients with LGMD and harboring single CAPN3 mutations were identified. They underwent detailed neurological evaluation, muscle biopsy, muscle MRI and electroneuromyography (ENMG). Results Patient one: man, 28 years old, born from nonconsanguineous parents, presenting progressive calves atrophy since 24 years old. Genetic analysis showed the variant c.258dupT;p. (Leu87Serfs*4) at CAPN3 in heterozygosity. ENMG showed myopathic motor unit action potential (MUAP) on left gastrocnemius and pectoralis major, and on right biceps brachii, without fibs or positive sharp waves. Right gastrocnemius muscle biopsy showed moderate myopathic changes. Patient two: woman, 77 years old, born from nonconsanguineous parents, presenting progressive proximal lower limb weakness in the last 2 years. Genetic analysis revealed the heterozygous frameshift variant c.550delA; p. (Thr184Argfs*36) in the CAPN3 gene. ENMG showed moderate diffuse myopathy, with preferential involvement of posterior tigh muscles and gastrocnemius with fibs and positive sharp waves. MRI showed severe atrophy of posterior tight muscles. Right quadriceps femoris biopsy revealed minimal myopathic changes. Conclusion The typical clinical, imaging and electrophysiological findings from LGMD2A can be found in patients presenting heterozygous pathogenic mutations in CAPN3 gene, indicating that a dominantly inherited form of calpainopathy exists, but with a milder fenotype. EMG reveals a predominantly posterior leg pattern of involvement.

Coincidence of hereditary motor and sensory neuropathy type 1A and limb lumbo-muscular dystrophy type 2A

A rare case of two neuromuscular disorders in a 29-year-old female is presented: autosomal dominant hereditary motor and sensory neuropathy type 1A (HMSN1A) due to PMP22 duplication and autosomal recessive limb girdle muscular dystrophy type 2A (LGMD2A) produced by CAPN3 common mutation c.550delA and novel c.575C>G (p.Thr192Ser).Walking difficulties appeared in 27 years, the patient had signs of both disorders, more of LGMD, but was not disabled. HMSN1A was inherited from her father whose disease was not recognized earlier.