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.

Circulating miR-206 as a Biomarker for Patients Affected by Severe Limb Girdle Muscle Dystrophies.

Limb-girdle muscular dystrophies (LGMD) are clinically and genetically heterogeneous conditions, presenting with a wide clinical spectrum, leading to progressive proximal weakness caused by loss of muscle fibers. MiR-206 is a member of myomiRNAs, a group of miRNAs with important function in skeletal muscle. Our aim is to determine the value of miR-206 in detecting muscle disease evolution in patients affected by LGMD. We describe clinical features, disease history and progression of eleven patients affected by various types of LGMD: transportinopathy, sarcoglycanopathy and calpainopathy. We analyzed the patients' mutations and we studied the circulating miR-206 in serum by qRT-PCR; muscle MRI was done with a 1.5 Tesla apparatus. The severe evolution of disease type is associated with the expression levels of miR-206, which was significantly elevated in our LGMD patient cohort in comparison with a control group. In particular, we observed an over-expression of miR-206 that was 50-80 folds elevated in two patients with a severe and early disease course in the transportinopathy and calpainopathy sub-types. The functional impairment was observed clinically and muscle loss and atrophy documented by muscle MRI. This study provides the first evidence that miR-206 is associated with phenotypic expression and it could be used as a prognostic indicator of LGMD disease progression.

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.

A simple and rapid immunoassay predicts dysferlinopathies in peripheral blood film.

The assessment of dysferlin expression is useful to indicate or confirm the diagnosis of dysferlinopathies, a class of muscular diseases caused by mutations in the DYSF gene. Immunoblot analysis of skeletal muscle or monocytes is a specific and reliable diagnostic indicator of the disease, but the technique is specialized and laborious. We have developed a novel, robust immunoassay for detection of dysferlin in neutrophils requiring as little as one drop of blood. Our assay overcomes the issues of storage and handling of samples suggesting great promise as an inexpensive and rapid first screening for DYSF mutations. This relatively simple non-quantitative assay has the potential to benefit centers with limited resources, contributing to current diagnostic investigations into dysferlinopathies.

Clinical spectrum and gene mutations in a Chinese cohort with anoctaminopathy.

Recessive mutations in anoctamin-5 (ANO5) are causative for limb-girdle muscular dystrophy (LGMD) 2 L and non-dysferlin Miyoshi-like distal myopathy (MMD3). ANO5 mutations are highly prevalent in European countries; however it is not common in patients of Asian origin, and there is no data regarding the Chinese population. We retrospectively reviewed the clinical manifestations and gene mutations of Chinese patients with anoctaminopathy. A total of five ANO5 mutations including four novel mutations and one reported mutation were found in four patients from three families. No hotspot mutation was found. Three patients presented with presymptomatic hyperCKemia and one patient had limb muscle weakness. Muscle imaging of lower limbs showed preferential adductor magnus and medial gastrocnemius involvement. No hotspot mutation has been identified in Chinese patients to date.

Screening for late-onset Pompe disease in Poland.

OBJECTIVES: We aimed to screen for late-onset Pompe disease using the dried blood spot (DBS) test in a cohort of patients with limb-girdle muscle weakness or persistent hyperCKemia. MATERIALS AND METHODS: Patients with limb-girdle muscle weakness, persistently elevated CK, rigid spine syndrome, dyspnoea, myalgia or sibling of the patient diagnosed with LOPD were included in the study. Acid α-glucosidase (GAA) activity was measured on DBS by tandem mass spectrometry and followed by genetic testing when required. Study was conducted between June 2014 and May 2017. RESULTS: A total of 337 patients aged 32.2 years (range 2-80) were included in the study. Late-onset Pompe disease was diagnosed in 10 patients (3.0% of tested cohort). All were compound heterozygotes with common c.32-13T>G mutation on one allele and missense or frameshift mutation on the other. Two of the mutations (c.1951delG and c.397T>G) were not reported previously. Seven of the patients started enzyme replacement therapy. CONCLUSIONS: DBS test is a reliable method for screening for late-onset Pompe disease.

AL amyloidosis presenting with limb girdle myopathy.

An elderly Caucasian man presented with a 10-month history of proximal myopathy and dysphagia. His serum creatine kinase (CK) was elevated at 877 U/L (normal 40-320) and electromyography confirmed a myopathic process. Blood and urine tests suggested myeloma; bone marrow examination showed 30% plasma cells and stained positive for amyloid. The muscle biopsy was initially reported as normal but in the light of the bone marrow report, the biopsy specimen was stained for amyloid, which was positive. We diagnosed systemic amyloidosis causing a myopathy and have started treatment for myeloma.

Secondary Bone Defect in Neuromuscular Diseases in Childhood: A Longitudinal "Muscle-Bone Unit" Analysis.

To evaluate the potential bone defect in neuromuscular diseases, we conducted a longitudinal study including three groups of patients: 14 Duchenne muscular dystrophies (DMD) and 2 limb-girdle muscular dystrophies (LGMD); 3 Becker muscular dystrophies (BeMD) and 7 spinal muscular atrophies (SMA). Yearly osteodensitometries assessed body composition and bone mineral density (BMD) associated with bone markers and leptin. Along the 7-year study, 107 osteodensitometries showed that bone status evolved to osteopenia in most patients except BeMD. When analyzing the crude values, BMD improved with age in BeMD and SMA but not in DMD/LGMD. The correlation using the Z-scores displayed a decrease in BMD with age in DMD/LGMD for all regions, in SMA at total body less head, whereas BMD increased in BeMD at lumbar spine. As observed in healthy persons, muscular mass and bone tissue were significantly correlated. Glucocorticoids were deleterious on trabecular and cortical bone. Leptin was high in most patients and correlated to fat mass and bone parameters. This study confirms a secondary bone defect in neuromuscular diseases, further confirming the functional relationship between bone and muscle and arguing for regular bone follow-up in patients to prevent fracture risk. Adipose tissue seems to interfere with bone remodeling in neuromuscular diseases.

Late-onset Pompe disease: what is the prevalence of limb-girdle muscular weakness presentation?

Pompe disease is an inherited disease caused by acid alpha-glucosidase (GAA) deficiency. A single center observational study aimed at assessing the prevalence of late-onset Pompe disease in a high-risk Brazilian population, using the dried blood spot test to detect GAA deficiency as a main screening tool. Dried blood spots were collected for GAA activity assay from 24 patients with "unexplained" limb-girdle muscular weakness without vacuolar myopathy in their muscle biopsy. Samples with reduced enzyme activity were also investigated for GAA gene mutations. Of the 24 patients with dried blood spots, one patient (4.2%) showed low GAA enzyme activity (NaG/AaGIA: 40.42; %INH: 87.22%). In this patient, genetic analysis confirmed two heterozygous mutations in the GAA gene (c.-32-13T>G/p.Arg854Ter). Our data confirm that clinicians should look for late-onset Pompe disease in patients whose clinical manifestation is an "unexplained" limb-girdle weakness even without vacuolar myopathy in muscle biopsy.

Late-onset Pompe disease: what is the prevalence of limb-girdle muscular weakness presentation? / Doença de Pompe de início tardio: qual é a prevalência na apresentação de fraqueza muscular de cinturas?

ABSTRACT Pompe disease is an inherited disease caused by acid alpha-glucosidase (GAA) deficiency. A single center observational study aimed at assessing the prevalence of late-onset Pompe disease in a high-risk Brazilian population, using the dried blood spot test to detect GAA deficiency as a main screening tool. Dried blood spots were collected for GAA activity assay from 24 patients with "unexplained" limb-girdle muscular weakness without vacuolar myopathy in their muscle biopsy. Samples with reduced enzyme activity were also investigated for GAA gene mutations. Of the 24 patients with dried blood spots, one patient (4.2%) showed low GAA enzyme activity (NaG/AaGIA: 40.42; %INH: 87.22%). In this patient, genetic analysis confirmed two heterozygous mutations in the GAA gene (c.-32-13T>G/p.Arg854Ter). Our data confirm that clinicians should look for late-onset Pompe disease in patients whose clinical manifestation is an "unexplained" limb-girdle weakness even without vacuolar myopathy in muscle biopsy.

RESUMO A doença de Pompe é uma doença hereditária causada pela deficiência da enzima alfa-glicosidase ácida (GAA). Estudo observacional foi realizado, em um único centro, para determinar a prevalência da doença de Pompe de início tardio (LOPD) em uma população brasileira de alto risco, usando teste em gota seca (DBS) como ferramenta principal de triagem para detectar a deficiência da GAA. DBS foi coletado para avaliar a atividade da GAA em 24 pacientes com fraqueza muscular de cinturas "não explicada" sem miopatia vacuolar na biópsia muscular. As amostras com atividade enzimática reduzida foram também submetidas a análise de mutações no gene GAA. Dos 24 pacientes com DBS, baixa atividade da enzima GAA (NaG/AaGIA: 40.42; %INH: 87.22%) foi encontrada em um paciente (4.2%). Nessa paciente, a análise genética confirmou duas mutações em heterozigose composta no gene GAA (c.-32-13T > G/p.Arg854Ter). Nossos resultados confirmam que LOPD deve ser investigada quando a manifestação clínica é uma fraqueza muscular de cinturas "não explicada", mesmo na ausência de miopatia vacuolar na biópsia muscular.

Serum exosomes can restore cellular function in vitro and be used for diagnosis in dysferlinopathy.

Purpose: It is challenging to deliver the full-length dysferlin gene or protein to restore cellular functions of dysferlin-deficient (DYSF-/-) myofibres in dysferlinopathy, a disease caused by the absence of dysferlin, which is currently without effective treatment. Exosomes, efficient membranous nanoscale carriers of biological cargoes, could be useful. Experimental design: Myotube- and human serum-derived exosomes were investigated for their capabilities of restoring dysferlin protein and cellular functions in murine and human DYSF-/- cells. Moreover, dysferlinopathic patient serum- and urine-derived exosomes were assessed for their abilities as diagnostic tools for dysferlinopathy. Results: Here we show that exosomes from dysferlin-expressing myotubes carry abundant dysferlin and enable transfer of full-length dysferlin protein to DYSF-/- myotubes. Exogenous dysferlin correctly localizes on DYSF-/- myotube membranes, enabling membrane resealing in response to injury. Human serum exosomes also carry dysferlin protein and improve membrane repair capabilities of human DYSF-/- myotubes irrespective of mutations. Lack of dysferlin in dysferlinopathic patient serum and urine exosomes enables differentiation between healthy controls and dysferlinopathic patients. Conclusions: Our findings provide evidence that exosomes are efficient carriers of dysferlin and can be employed for the treatment and non-invasive diagnosis of dysferlinopathy.

Cross-sectional serum metabolomic study of multiple forms of muscular dystrophy.

Muscular dystrophies are characterized by a progressive loss of muscle tissue and/or muscle function. While metabolic alterations have been described in patients'-derived muscle biopsies, non-invasive readouts able to describe these alterations are needed in order to objectively monitor muscle condition and response to treatment targeting metabolic abnormalities. We used a metabolomic approach to study metabolites concentration in serum of patients affected by multiple forms of muscular dystrophy such as Duchenne and Becker muscular dystrophies, limb-girdle muscular dystrophies type 2A and 2B, myotonic dystrophy type 1 and facioscapulohumeral muscular dystrophy. We show that 15 metabolites involved in energy production, amino acid metabolism, testosterone metabolism and response to treatment with glucocorticoids were differentially expressed between healthy controls and Duchenne patients. Five metabolites were also able to discriminate other forms of muscular dystrophy. In particular, creatinine and the creatine/creatinine ratio were significantly associated with Duchenne patients performance as assessed by the 6-minute walk test and north star ambulatory assessment. The obtained results provide evidence that metabolomics analysis of serum samples can provide useful information regarding muscle condition and response to treatment, such as to glucocorticoids treatment.

Thrombospondin-1 and disease progression in dysferlinopathy.

The purpose of this study was to determine whether thrombospondin (TSP)-1 promotes macrophage activity and disease progression in dysferlinopathy. First, we found that levels of TSP-1 are elevated in blood of non-ambulant dysferlinopathy patients compared with ambulant patients and healthy controls, supporting the idea that TSP-1 levels are correlated with disease progression. We then crossed dysferlinopathic BlaJ mice with TSP-1 knockout mice and assessed disease progression longitudinally with magnetic resonance imaging (MRI). In these mice, deletion of TSP-1 ameliorated loss in volume and mass of the moderately affected gluteal muscle but not of the severely affected psoas muscle. T2 MRI parameters revealed that loss of TSP-1 modestly inhibited inflammation only in gluteal muscle of male mice. Histological assessment indicated that deletion of TSP-1 reduced inflammatory cell infiltration of muscle fibers, but only early in disease progression. In addition, flow cytometry analysis revealed that, in males, TSP-1 knockout reduced macrophage infiltration and phagocytic activity, which is consistent with TSP-1-enhanced phagocytosis and pro-inflammatory cytokine induction in cultured macrophages. In summary, TSP-1 appears to play an accessory role in modulating Mp activity in BlaJ mice in a gender, age and muscle-dependent manner, but is unlikely a primary driver of disease progression of dysferlinopathy.

[Clinical/genetic characteristics of patients with congenital muscular dystrophy caused by mutations in the LMNA gene].

OBJECTIVE: To study clinical/genetic characteristics of congenital muscular dystrophy caused by mutations in the LMNA gene in 5 patients from the Russian population. MATERIAL AND METHODS: DNA samples of 42 probands, aged from 2 months to 9 years, with characteristic signs of congenital muscular dystrophy from nonrelated families were studied. The diagnosis was based on the results of genealogical analysis, neurological examination, serum creatine phosphokinase activity, results of electroneuromyography. RESULTS AND CONCLUSION: In the Russian population, the frequency of congenital muscular dystrophy caused by mutations in the LMNA gene is not less than 12% of all cases of this group of diseases. The results indicate the presence of major mutation c.94_96delAAC in the LMNA gene. Specific phenotypic features of this form of congenital muscular dystrophy with symptoms of progressive flaccid paralysis with predominant lesions of axial muscles and plantar flexor muscles of the foot are described.

Toward an objective measure of functional disability in dysferlinopathy.

INTRODUCTION: Understanding the natural history of dysferlinopathy is essential to design and quantify novel therapeutic protocols. Our aim in this study was to assess, clinically and functionally, a cohort of patients with dysferlinopathy, using validated scales. METHODS: Thirty-one patients with genetically confirmed dysferlinopathy were assessed using the motor function measure (MFM), Modified Rankin Scale (MRS), Muscle Research Council (MRC) scale, serum creatine kinase (CK) assessment, baseline spirometry data, and echocardiographic and electrophysiologic studies. RESULTS: MFM and MRC scores showed a significant negative correlation with disease duration and inverse correlation with MRS, but not with onset age, clinical phenotype, or CK levels. Percent forced vital capacity (%FVC) correlated negatively with disease duration and onset age. Eight known pathogenic mutations were identified recurrently, 4 of which accounted for 79% of the total. CONCLUSIONS: The results suggest that MFM is a reliable outcome measure that may be useful for longitudinal follow-up in dysferlinopathy. Recurrent mutations suggest a founder effect in the Chilean population.

Respiratory and cardiac function in japanese patients with dysferlinopathy.

INTRODUCTION: We retrospectively reviewed respiratory and cardiac function in patients with dysferlinopathy, including 2 autopsy cases with respiratory dysfunction. METHODS: Subjects included 48 patients who underwent respiratory evaluation (n = 47), electrocardiography (n = 46), and echocardiography (n = 23). RESULTS: Of the 47 patients, 10 had reduced percent forced vital capacity (%FVC), and 4 required non-invasive positive pressure ventilation. %FVC was significantly correlated with disease duration, and mean %FVC was significantly lower in non-ambulatory patients, as well as in those aged ≥65 years with normal creatine kinase levels. On electrocardiography, QRS complex duration was prolonged in 19 patients, although no significant association with age, disease duration, or respiratory function was found. Echocardiography indicated no left ventricular dysfunction in any patient. Histopathology of autopsied cases revealed mild cardiomyopathy and moderate diaphragm involvement. CONCLUSION: Patients with dysferlinopathy may develop severe respiratory failure and latent cardiac dysfunction. Both respiratory and cardiac function should be monitored diligently.

Targeted screening for the detection of Pompe disease in patients with unclassified limb-girdle muscular dystrophy or asymptomatic hyperCKemia using dried blood: A Spanish cohort.

We aimed to screen for Pompe disease in patients with unclassified limb-girdle muscular dystrophy (LGMD) or asymptomatic hyperCKemia using dried blood spot (DBS) assays. Subsequently, we aimed to calculate the diagnostic delay between initial symptom presentation and the diagnosis. A prospective, multicenter, observational study was conducted in 348 patients: 146 with unclassified LGMD and 202 with asymptomatic or paucisymptomatic hyperCKemia. We quantified levels of acid alpha-glucosidase (GAA) from dried blood spots analyzed fluorometrically. The test was positive in 20 patients, and Pompe disease was confirmed by genetic testing in 16. Undiagnosed Pompe disease was detected in 7.5% of patients with LGMD and in 2.5% of patients with persistent, idiopathic elevation of serum creatine kinase. The c.-32-13 T > G mutation was found most commonly. The diagnostic delay was 15 years on average. In conclusion, DBS tests are useful and reliable screening tools for Pompe disease. We recommend the dried blood spot test to be included in the diagnostic work-up of patients with unclassified myopathies with proximal weakness and/or hyperCKemia of unknown cause and, when positive, to define the diagnosis, it will have to be confirmed by biochemical and/or molecular genetic analysis.

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-1ß 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-1ß 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.

Three novel serum biomarkers, miR-1, miR-133a, and miR-206 for Limb-girdle muscular dystrophy, Facioscapulohumeral muscular dystrophy, and Becker muscular dystrophy.

OBJECTIVES: Muscular dystrophies are a clinically and genetically heterogeneous group of inherited myogenic disorders. In clinical tests for these diseases, creatine kinase (CK) is generally used as diagnostic blood-based biomarker. However, because CK levels can be altered by various other factors, such as vigorous exercise, etc., false positive is observed. Therefore, three microRNAs (miRNAs), miR-1, miR-133a, and miR-206, were previously reported as alternative biomarkers for duchenne muscular dystrophy (DMD). However, no alternative biomarkers have been established for the other muscular dystrophies. METHODS: We, therefore, evaluated whether these miR-1, miR-133a, and miR-206 can be used as powerful biomarkers using the serum from muscular dystrophy patients including DMD, myotonic dystrophy 1 (DM1), limb-girdle muscular dystrophy (LGMD), facioscapulohumeral muscular dystrophy (FSHD), becker muscular dystrophy (BMD), and distal myopathy with rimmed vacuoles (DMRV) by qualitative polymerase chain reaction (PCR) amplification assay. RESULTS: Statistical analysis indicated that all these miRNA levels in serum represented no significant differences between all muscle disorders examined in this study and controls by Bonferroni correction. However, some of these indicated significant differences without correction for testing multiple diseases (P < 0.05). The median values of miR-1 levels in the serum of patients with LGMD, FSHD, and BMD were approximately 5.5, 3.3 and 1.7 compared to that in controls, 0.68, respectively. Similarly, those of miR-133a and miR-206 levels in the serum of BMD patients were about 2.5 and 2.1 compared to those in controls, 1.03 and 1.32, respectively. CONCLUSIONS: Taken together, our data demonstrate that levels of miR-1, miR-133a, and miR-206 in serum of BMD and miR-1 in sera of LGMD and FSHD patients showed no significant differences compared with those of controls by Bonferroni correction. However, the results might need increase in sample sizes to evaluate these three miRNAs as variable biomarkers.