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Dysferlin is a large transmembrane protein involved in critical cellular processes including membrane repair and vesicle fusion. Mutations in the dysferlin gene (DYSF) can result in rare forms of muscular dystrophy; Miyoshi myopathy; limb girdle muscular dystrophy type 2B (LGMD2B); and distal myopathy. These conditions are collectively known as dysferlinopathies and are caused by more than 600 mutations that have been identified across the DYSF gene to date. In this review, we discuss the key molecular and clinical features of LGMD2B, the causative gene DYSF, and the associated dysferlin protein structure. We also provide an update on current approaches to LGMD2B diagnosis and advances in drug development, including splice switching antisense oligonucleotides. We give a brief update on clinical trials involving adeno-associated viral gene therapy and the current progress on CRISPR/Cas9 mediated therapy for LGMD2B, and then conclude by discussing the prospects of antisense oligomer-based intervention to treat selected mutations causing dysferlinopathies. [ABSTRACT FROM AUTHOR]

Dysferlin is a large transmembrane protein involved in critical cellular processes including membrane repair and vesicle fusion. Mutations in the dysferlin gene (DYSF) can result in rare forms of muscular dystrophy; Miyoshi myopathy; limb girdle muscular dystrophy type 2B (LGMD2B); and distal myopathy. These conditions are collectively known as dysferlinopathies and are caused by more than 600 mutations that have been identified across the DYSF gene to date. In this review, we discuss the key molecular and clinical features of LGMD2B, the causative gene DYSF, and the associated dysferlin protein structure. We also provide an update on current approaches to LGMD2B diagnosis and advances in drug development, including splice switching antisense oligonucleotides. We give a brief update on clinical trials involving adeno-associated viral gene therapy and the current progress on CRISPR/Cas9 mediated therapy for LGMD2B, and then conclude by discussing the prospects of antisense oligomer-based intervention to treat selected mutations causing dysferlinopathies.

Dysferlinopathy is a disease caused by a dysferlin deficiency due to mutations in the DYSF gene. Dysferlin is a membrane protein in the sarcolemma and is involved in different functions, such as membrane repair and vesicle fusion, T-tubule development and maintenance, Ca2+ signalling, and the regulation of various molecules. Miyoshi Myopathy type 1 (MMD1) and Limb–Girdle Muscular Dystrophy 2B/R2 (LGMD2B/LGMDR2) are two possible clinical presentations, yet the same mutations can cause both presentations in the same family. They are therefore grouped under the name dysferlinopathy. Onset is typically during the teenage years or young adulthood and is characterized by a loss of Achilles tendon reflexes and difficulty in standing on tiptoes or climbing stairs, followed by a slow progressive loss of strength in limb muscles. The MRI pattern of patient muscles and their biopsies show various fibre sizes, necrotic and regenerative fibres, and fat and connective tissue accumulation. Recent tools were developed for diagnosis and research, especially to evaluate the evolution of the patient condition and to prevent misdiagnosis caused by similarities with polymyositis and Charcot–Marie–Tooth disease. The specific characteristic of dysferlinopathy is dysferlin deficiency. Recently, mouse models with patient mutations were developed to study genetic approaches to treat dysferlinopathy. The research fields for dysferlinopathy therapy include symptomatic treatments, as well as antisense-mediated exon skipping, myoblast transplantation, and gene editing. [ABSTRACT FROM AUTHOR]

For inherited diseases, obtaining a definitive diagnosis is critical for proper disease management, family planning, and participation in clinical trials. This can be challenging for dysferlinopathy due to the significant clinical overlap between the 30+ subtypes of limb–girdle muscular dystrophy (LGMD) and the large number of variants of unknown significance (VUSs) that are identified in the dysferlin gene, DYSF. We performed targeted RNA-Seq using a custom gene-panel in 77 individuals with a clinical/genetic suspicion of dysferlinopathy and evaluated all 111 identified DYSF variants according to the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP) guidelines. This evaluation identified 11 novel DYSF variants and allowed for the classification of 87 DYSF variants as pathogenic/likely pathogenic, 8 likely benign, while 16 variants remained VUSs. By the end of the study, 60 of the 77 cases had a definitive diagnosis of dysferlinopathy, which was a 47% increase in diagnostic yield over the rate at study onset. This data shows the ability of RNA-Seq to assist in variant pathogenicity classification and diagnosis of dysferlinopathy and is, therefore, a type of analysis that should be considered when DNA-based genetic analysis is not sufficient to provide a definitive diagnosis. [ABSTRACT FROM AUTHOR]

Background: To characterize the phenotypic, neurophysiological, radiological, pathological, and genetic profile of 33 Saudi Arabian families with dysferlinopathy. Methods: A descriptive observational study was done on a cohort of 112 Saudi Arabian families with LGMD. Screening for the Dysferlin (DYSF) gene was done in a tertiary care referral hospital in Saudi Arabia. Clinical, Neurophysiological, Radiological, Pathological, and Genetic findings in subjects with dysferlin mutation were the primary outcome variables. Statistical analysis was done by Epi-info. Results: 33 out of 112 families (29.46%) registered in the LGMD cohort had Dysferlinopathy. 53 subjects (28 males, 52.83%) from 33 families were followed up for various periods ranging from 1 to 28 years. The mean age of onset was 17.79 ± 3.48 years (Range 10 to 25 years). Miyoshi Myopathy phenotype was observed in 50.94% (27 out of 53), LGMDR2 phenotype in 30.19% (16 out of 53), and proximodistal phenotype in 15.09% (8 out of 53) of the subjects. Loss of ambulation was observed in 39.62% (21 out of 53 subjects). Electrophysiological, Radiological, and histopathological changes were compatible with the diagnosis. Mean serum Creatinine Kinase was 6,464.45 ± 4,149.24 with a range from 302 to 21,483 IU/L. In addition, 13 dysferlin mutations were identified two of them were compound heterozygous. One founder mutation was observed c.164_165insA in 19 unrelated families. Conclusion: The prevalence of Dysferlinopathy was 29.46% in the native Saudi LGMD cohort. It is the most prevalent subtype seconded by calpainopathy. The clinical course varied among the study subjects and was consistent with those reported from different ethnic groups. One founder mutation was identified. Initial screening of the founder mutations in new families is highly recommended. [ABSTRACT FROM AUTHOR]

BackgroundTo characterize the phenotypic, neurophysiological, radiological, pathological, and genetic profile of 33 Saudi Arabian families with dysferlinopathy.MethodsA descriptive observational study was done on a cohort of 112 Saudi Arabian families with LGMD. Screening for the Dysferlin (DYSF) gene was done in a tertiary care referral hospital in Saudi Arabia. Clinical, Neurophysiological, Radiological, Pathological, and Genetic findings in subjects with dysferlin mutation were the primary outcome variables. Statistical analysis was done by Epi-info.Results33 out of 112 families (29.46%) registered in the LGMD cohort had Dysferlinopathy. 53 subjects (28 males, 52.83%) from 33 families were followed up for various periods ranging from 1 to 28 years. The mean age of onset was 17.79 ± 3.48 years (Range 10 to 25 years). Miyoshi Myopathy phenotype was observed in 50.94% (27 out of 53), LGMDR2 phenotype in 30.19% (16 out of 53), and proximodistal phenotype in 15.09% (8 out of 53) of the subjects. Loss of ambulation was observed in 39.62% (21 out of 53 subjects). Electrophysiological, Radiological, and histopathological changes were compatible with the diagnosis. Mean serum Creatinine Kinase was 6,464.45 ± 4,149.24 with a range from 302 to 21,483 IU/L. In addition, 13 dysferlin mutations were identified two of them were compound heterozygous. One founder mutation was observed c.164_165insA in 19 unrelated families.ConclusionThe prevalence of Dysferlinopathy was 29.46% in the native Saudi LGMD cohort. It is the most prevalent subtype seconded by calpainopathy. The clinical course varied among the study subjects and was consistent with those reported from different ethnic groups. One founder mutation was identified. Initial screening of the founder mutations in new families is highly recommended.

• Pelvic girdle weakness becomes clinically evident in LGMDR1/LGMD2A and LGMDR2/LGMD2B after 12–15 years from disease onset. • Upper limbs are involved later in LGMDR2/LGMD2B than LGMDR1/LGMD2A. • Discrepancy between lower limbs muscular strength impairment and the corresponding motor function occurs, presumably because of compensatory mechanisms. • The slow progression of the diseases does not make possible to identify specific milestones. • Mathematical models in medicine are fundamental tools that can expand the knowledge base from actual study data. We aimed to describe the natural history of Limb Girdle Muscular Dystrophy type 2A and 2B over more than three decades by considering muscular strength, motor, cardiac and respiratory function. 428 visits of nineteen 2A and twenty 2B patients were retrospectively analysed through a regression model to create the curves of evolution with disease duration of muscle strength (through Medical Research Council grading), motor function measure scale (D1, D2 and D3 domains) and cardio-pulmonary function tests. Clinically relevant muscular and motor function alterations occurred after the first decade of disease, while mild respiratory function alterations started after the second, with preserved cardiac function. Although type 2A showed relatively stronger distal lower limb muscles, while type 2B started with relatively stronger upper limb muscles, the corresponding motor functions were similar, becoming severely compromised after 25 years of disease. This was the longest retrospective study in types 2A and 2B. It defined curves of disease evolution not only from a neuromuscular, but also from functional, cardiac, and respiratory points of view, to be used to evaluate how the natural progression is changed by therapies. Due to slow disease progression, it was not possible to identify time sensitive endpoints. [ABSTRACT FROM AUTHOR]

Two of the main pathologies characterizing dysferlinopathies are disrupted muscle membrane repair and chronic inflammation, which lead to symptoms of muscle weakness and wasting. Here, we used recombinant human Galectin-1 (rHsGal-1) as a therapeutic for LGMD2B mouse and human models. Various redox and multimerization states of Gal-1 show that rHsGal-1 is the most effective form in both increasing muscle repair and decreasing inflammation, due to its monomer-dimer equilibrium. Dose-response testing shows an effective 25-fold safety profile between 0.54 and 13.5 mg/kg rHsGal-1 in Bla/J mice. Mice treated weekly with rHsGal-1 showed downregulation of canonical NF-κB inflammation markers, decreased muscle fat deposition, upregulated anti-inflammatory cytokines, increased membrane repair, and increased functional movement compared to non-treated mice. Gal-1 treatment also resulted in a positive self-upregulation loop of increased endogenous Gal-1 expression independent of NF-κB activation. A similar reduction in disease pathologies in patient-derived human cells demonstrates the therapeutic potential of Gal-1 in LGMD2B patients.

Introduction: 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.Methods: 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).Results: 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.Conclusions: qMRI measures may help to quantify muscular degeneration, but careful interpretation is needed when fat infiltration is massive. Muscle Nerve 58: 550-558, 2018. [ABSTRACT FROM AUTHOR]

Dysferlinopathy, a progressive muscular dystrophy, results from mutations in the Dysferlin gene (DYSF, MIM*603009). Traditional diagnosis relies on the reduction or absence of dysferlin. However, altered dysferlin has been observed in other myopathies, leading to a precise diagnosis through molecular genetics. In this study, we report a patient who was previously misdiagnosed as inflammatory myopathy based on routine clinicopathological examinations alone. However, muscle biopsy specimens were analyzed further by immunohistochemistry of muscular dystrophy‐related proteins, and gene‐targeted next generation sequencing (NGS) was used to correctly identify muscular dystrophy. DNA was sequenced with NGS and the detected mutation was verified by Sanger sequencing. Our targeted NGS found a novel missense mutation (c.5392G > A) in the DYSF gene, allowing correct diagnosis of LGMD2B in our patient. We discovered of a novel missense mutation in the DYSF gene and have broadened the DYSF mutation spectrum, which may be correlated in patients with presumed dysferlinopathy, especially when lymphocytic infiltration is observed. [ABSTRACT FROM AUTHOR]

Dysferlinopathies comprise a family of disorders caused by mutations in the dysferlin ( DYSF ) gene, leading to a progressive dystrophy characterized by chronic muscle fiber loss, fat replacement, and fibrosis. To correct the underlying histopathology and function, expression of full-length DYSF is required. Dual adeno-associated virus vectors have been developed, defined by a region of homology, to serve as a substrate for reconstitution of the full 6.5 kb dysferlin cDNA. Previous work studied the efficacy of this treatment through intramuscular and regional delivery routes. To maximize clinical efficacy, dysferlin-deficient mice were treated systemically to target all muscles through the vasculature for efficacy and safety studies. Mice were evaluated at multiple time points between 4 and 13 months post treatment for dysferlin expression and functional improvement using magnetic resonance imaging and magnetic resonance spectroscopy and membrane repair. A systemic dose of 6 × 1012 vector genomes resulted in widespread gene expression in the muscles. Treated muscles showed a significant decrease in central nucleation, collagen deposition, and improvement of membrane repair to wild-type levels. Treated gluteus muscles were significantly improved compared to placebo-treated muscles and were equivalent to wild type in volume, intra- and extramyocellular lipid accumulation, and fat percentage using magnetic resonance imaging and magnetic resonance spectroscopy. Dual-vector treatment allows for production of full-length functional dysferlin with no toxicity. This confirms previous safety data and validates translation of systemic gene delivery for dysferlinopathy patients. [ABSTRACT FROM AUTHOR]

B6.A-Dysfprmd/GeneJ (BLAJ) mice model human limb-girdle muscular dystrophy 2B (LGMD2B), which is linked to mutations in the dysferlin (DYSF) gene. We tested the hypothesis that, the calcium ion (Ca2+) channel blocker diltiazem (DTZ), reduces contraction-induced skeletal muscle damage, in BLAJ mice. We randomly assigned mice (N = 12; 3-4 month old males) to one of two groups - DTZ (N = 6) or vehicle (VEH, distilled water, N = 6). We conditioned mice with either DTZ or VEH for 1 week, after which, their tibialis anterior (TA) muscles were tested for contractile torque and susceptibility to injury from forced eccentric contractions. We continued dosing with DTZ or VEH for 3 days following eccentric contractions, and then studied torque recovery and muscle damage. We analyzed contractile torque before eccentric contractions, immediately after eccentric contractions, and at 3 days after eccentric contractions; and counted damaged fibers in the injured and uninjured TA muscles. We found that DTZ improved contractile torque before and immediately after forced eccentric contractions, but did not reduce delayed-onset muscle damage that was observed at 3 days after eccentric contractions. [ABSTRACT FROM AUTHOR]

To date, over 30 genes with mutations causing limb-girdle muscle dystrophy have been described. Dysferlinopathies are a form of limb-girdle muscle dystrophy type 2B with an incidence ranging from 1:1,300 to 1:200,000 in different populations. In 1996, Dr. S. N. Illarioshkin described a family from the Botlikhsky district of Dagestan, where limb-girdle muscle dystrophy type 2B and Miyoshi myopathy were diagnosed in 12 members from three generations of a large Avar family. In 2000, a previously undescribed mutation in the DYSF gene (c.TG573/574AT; p. Val67Asp) was detected in the affected members of this family. Twenty years later, in this work, we re-examine five known and seven newly affected family members previously diagnosed with dysferlinopathy. We observed disease progression in family members who were previously diagnosed and noted obvious clinical polymorphism of the disease. A typical clinical case is provided. [ABSTRACT FROM AUTHOR]

Dysferlinopathies are caused by mutations in the DYSF gene and patients may present with proximal or distal myopathy. Dysferlin is responsible for membrane resealing, and mutations may result in a defect in membrane repair following mechanical or chemical stress, causing an influx of Ca 2+ . Since mitochondria are involved in Ca 2+ buffering, we hypothesised that mitochondrial defects may be present in skeletal muscle biopsies from patients with mutations in this gene. The aim was to characterise mitochondrial defects in muscle from patients with dysferlinopathies. Here, we analysed skeletal muscle biopsies for eight patients by quadruple immunofluorescent assay to assess oxidative phosphorylation protein abundance. Long-range PCR in single muscle fibres was used to look for presence of clonally expanded large-scale mitochondrial DNA rearrangements in patients' skeletal muscle (n = 3). Immunofluorescence demonstrated that the percentage of complex I- and complex IV-deficient fibres was higher in patients with DYSF mutations than in age-matched controls. No clonally expanded mtDNA deletions were detected using long-range PCR in any of the analysed muscle fibres. We conclude that complex I and complex IV deficiency is higher in patients than age matched controls but patients do not have rearrangements of the mtDNA. We hypothesise that respiratory chain deficiency may be the results of an increased cytosolic Ca 2+ concentration (due to a membrane resealing defect) causing mitochondrial aberrations. [ABSTRACT FROM AUTHOR]

Dysferlin-deficient limb girdle muscular dystrophy type 2B, distal Miyoshi myopathy, and other less frequent phenotypes are a group of recessive disorders called dysferlinopathies. They are characterized by wide clinical heterogeneity. To diagnose dysferlinopathy, a clinical neuromuscular workup, including electrophysiological and muscle imaging investigations, is essential to support subsequent laboratory testing. Increased serum creatine kinase levels, distal or proximal muscle weakness, and myalgia with onset in the second or third decades are the main clinical features of the disease. In muscle biopsies, severe dysferlin deficiency by immunoblot or its abnormal localization by immunohistochemistry are the gold standard, as they have a high diagnostic value. Dysferlin testing on monocytes is a valuable alternative to muscle immunoblotting. Molecular techniques for gene mutation detection, such as next generation sequencing, have improved the genetic diagnosis, which is crucial for treatment and genetic counselling. Muscle Nerve 54: 821-835, 2016. [ABSTRACT FROM AUTHOR]

Summary Dysferlin is a member of the evolutionarily conserved ferlin gene family. Mutations in Dysferlin lead to Limb Girdle Muscular Dystrophy 2B (LGMD2B), an inherited, progressive and incurable muscle disorder. However, the molecular mechanisms underlying disease pathogenesis are not fully understood. We found that both loss-of-function mutations and muscle-specific overexpression of C. elegans fer-1, the founding member of the Dysferlin gene family, caused defects in muscle cholinergic signaling. To determine if Dysferlin-dependent regulation of cholinergic signaling is evolutionarily conserved, we examined the in vivo physiological properties of skeletal muscle synaptic signaling in a mouse model of Dysferlin-deficiency. In addition to a loss in muscle strength, Dysferlin −/− mice also exhibited a cholinergic deficit manifested by a progressive, frequency-dependent decrement in their compound muscle action potentials following repetitive nerve stimulation, which was observed in another Dysferlin mouse model but not in a Dysferlin-independent mouse model of muscular dystrophy. Oral administration of Pyridostigmine bromide, a clinically used acetylcholinesterase inhibitor (AchE.I) known to increase synaptic efficacy, reversed the action potential defect and restored in vivo muscle strength to Dysferlin −/− mice without altering muscle pathophysiology. Our data demonstrate a previously unappreciated role for Dysferlin in the regulation of cholinergic signaling and suggest that such regulation may play a significant pathophysiological role in LGMD2B disease.

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. [ABSTRACT FROM AUTHOR]

Dysferlinopathies are a group of disabling muscular dystrophies  that includes limb girdle muscular dystrophy type 2B (LGMD2B), Miyoshi myopathy, and distal myopathy with anterior tibial onset (DMAT) as the main phenotypes. They are associated with molecular defects in DYSF, which encodes dysferlin, a key player in sarcolemmal homeostasis. Previous investigations have suggested that exon skipping may be a promising therapy for many patients with dysferlinopathies. It was reported that exons 28-29 of DYSF are dispensable for dysferlin functions. Here, we present a method for multiexon skipping of DYSF exons 28-29 using a cocktail of two phosphorodiamidate morpholino oligomers (PMOs) on cells derived from a dystrophinopathy patient. Also, we describe assays to characterize the multiexon skipped dysferlin at several levels by using one-step RT-PCR, immunoblotting, and a membrane wounding assay., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Loss-of-function mutations in the dysferlin gene (DYSF) result in a family of muscle disorders known collectively as the dysferlinopathies. Dysferlin-deficient muscle is characterized by inflammatory foci and macrophage infiltration with subsequent decline in muscle function. Whereas macrophages function to remove necrotic tissue in acute injury, their prevalence in chronic myopathy is thought to inhibit resolution of muscle regeneration. Two major classes of macrophages, classical (M1) and alternative (M2a), play distinct roles during the acute injury process. However, their individual roles in chronic myopathy remain unclear and were explored in this study. Methods: To test the roles of the two macrophage phenotypes on regeneration in dysferlin-deficient muscle, we developed an in vitro co-culture model of macrophages and muscle cells. We assayed the co-cultures using ELISA and cytokine arrays to identify secreted factors and performed transcriptome analysis of molecular networks induced in the myoblasts. Results: Dysferlin-deficient muscle contained an excess of M1 macrophage markers, compared with WT, and regenerated poorly in response to toxin injury. Co-culturing macrophages with muscle cells showed that M1 macrophages inhibit muscle regeneration whereas M2a macrophages promote it, especially in dysferlin-deficient muscle cells. Examination of soluble factors released in the co-cultures and transcriptome analysis implicated two soluble factors in mediating the effects: IL-1ß and IL-4, which during acute injury are secreted from M1 and M2a macrophages, respectively. To test the roles of these two factors in dysferlin-deficient muscle, myoblasts were treated with IL-4, which improved muscle differentiation, or IL-1ß, which inhibited it. Importantly, blockade of IL-1ß signaling significantly improved differentiation of dysferlin-deficient cells. Conclusions: We propose that the inhibitory effects of M1 macrophages on myogenesis are mediated by IL-1ß signals and suppression of the M1-mediated immune response may improve muscle regeneration in dysferlin deficiency. Our studies identify a potential therapeutic approach to promote muscle regeneration in dystrophic muscle. [ABSTRACT FROM AUTHOR]

Introduction: Dysferlin deficiency causes dysferlinopathies. Among peripheral blood mononuclear cells (PBMCs), the dysferlin protein is expressed specifically in CD14+ monocytes. Methods: We quantified dysferlin protein levels in PBMC lysates of 77 individuals suspected clinically of having a dysferlinopathy to screen for true positives. Subsequent molecular confirmation was done by Sanger sequencing and comparative genomic hybridization arrays to establish diagnosis. Results: Of the 44 individuals who had significantly reduced dysferlin levels (⩽10%), 41 underwent molecular testing. We identified at least 1 mutation in 85% (35 of 41), and 2 mutations, establishing a dysferlinopathy diagnosis, in 61% (25 of 41) of these individuals. Among those with dysferlin protein levels of >10% (33 of 77), only 1 individual (of 14 who underwent molecular testing) had a detectable mutation. Conclusions: Our results suggest that dysferlin protein levels of ⩽10% in PBMCs, are highly indicative of primary dysferlinopathies. However, this assay may not distinguish carriers from those with secondary dysferlin reduction. [ABSTRACT FROM AUTHOR]

Introduction: Muscle fiber atrophy and the molecular pathways underlying this process have not been investigated in dysferlinopathy patients. Methods: In 22 muscles from dysferlinopathy patients we investigated fiber atrophy by morphometry and ubiquitin-proteasome and autophagic pathways using protein and/or transcriptional analysis of atrophy- and autophagyrelated genes (MuRF1, atrogin1, LC3, p62, Bnip3). Results: Dysferlinopathy showed significant fiber atrophy and higher MuRF-1 protein and mRNA levels, which correlated with fiber size, suggesting activation of the atrophy program by proteasome induction. Conclusions: Some of the MuRF-1 upregulation and proteasome induction may be attributed to the prominent regeneration found. A potential role of impaired autophagy was suggested by p62-positive protein aggregates in atrophic fibers and significantly higher levels of LC3-II and p62 proteins and overexpression of p62 and Bnip3 mRNA. Damaged muscle fibers and prominent inflammatory changes may also enhance autophagy due to the insufficient level of proteasomal degradation of mutant dysferlin. [ABSTRACT FROM AUTHOR]

Mutations in human dysferlin (DYSF) gene cause both limb girdle muscular dystrophy type 2B (LGMD2B) and Miyoshi myopathy (MM), also named dysferlinopathy. They are autosomal recessive muscular dystrophies characterized by degeneration and weakness of proximal and/or distal limb girdle muscles caused by partial or complete absence of a sarcolemmal protein dysferlin. The size and large mutational spectrum of DYSF impose a multistep diagnosis strategy before gene analysis. Here we report the first three patients from two unrelated Croatian families in which diagnosis of dysferlinopathy was suggested on the basis of clinical picture, family history and linkage analysis. In order to confirm the presumed diagnosis, we performed a blood-based assay in which dysferlin expression is screened in blood monocytes. All three tested patients showed complete absence of dysferlin expression, giving strong evidence of dysferlinopathy that was recently confirmed by mutation analysis. In conclusion, we would suggest the presented diagnostic strategy as a reliable and non-invasive method to be used as an alternative to muscle tissue protein analysis in routine diagnostics of dysferlinopathies, prior to the more complex and demanding search for causative DYSF mutations. This non-aggressive approach seems especially useful in situation in which multiplex Western blot (WB) analysis of different muscular dystrophy proteins on muscle sample is not available. [ABSTRACT FROM AUTHOR]

Abstract: Primary dysferlinopathies are a group of recessive heterogeneous muscular dystrophies. The most common clinical presentations are Miyoshi myopathy and LGMD2B. Additional presentations range from isolated hyperCKemia to severe functional disability. Symptomatology begins in the posterior muscle compartment of the calf and its clinical course progresses slowly in Miyoshi myopathy whereas LGMD2B involves predominantly the proximal muscles of the lower limbs. The age of onset ranges from 13 to 60years in Caucasians. We present five patients that carry a novel mutation in the exon12/intron12 boundary: c.1180_1180+7delAGTGCGTG (r.1054_1284del). We provide evidence of a founder effect due to a common ancestral origin of this mutation, detected in heterozygosity in four patients and in homozygosity in one patient. [Copyright &y& Elsevier]

Abstract: Dysferlinopathy refers to a variety of autosomal recessive, skeletal muscle disorders due to the mutations of dysferlin-encoding gene, DYSF. It encompasses limb-girdle muscular dystrophy type 2B (LGMD2B), Miyoshi myopathy (MM), distal myopathy with anterior tibial onset (DMAT), isolated hyperCKemia, rigid spine syndrome and congenital muscular dystrophy. Herein, we report five Thai patients with distal myopathy due to dysferlinopathy including four MM and one DMAT patients. Muscle biopsy from one MM patient depicted numerous ring fibers which is an atypical finding in dysferlinopathy. Mutation analysis of DYSF revealed novel compound heterozygous mutations of p.Tyr309X and c.236+1G>T in two related MM patients, known homozygous mutations, p.Arg89X and p.Gln176X, in two MM patients and a heterozygous missense mutation, p.Arg555Trp, in a DMAT patient. Most of the previously reported DMAT patients were Hispanic. To the best of our knowledge, this is the first report of genetically confirmed patients with dysferlinopathy in Thailand. [Copyright &y& Elsevier]

Abstract: We report two patients with a new phenotype of dysferlinopathy presenting as congenital muscular disease. Both patients showed weakness in proximal lower limbs and neck flexor muscles at birth. The presence of normal CK levels during the first years should be noted. Initial MRI showed no abnormalities but short-time-inversion-recovery (STIR) sequences revealed a striking myoedema in gastrocnemius and hamstring muscles at the age of 5. Muscle biopsy showed mild dystrophic features and the absence of dysferlin. Dysferlin gene (DYSF) analysis revealed a p.Ala927LeufsX21 mutation in a homozygous state in both siblings. This new phenotype widens the clinical spectrum of dysferlin myopathies. [Copyright &y& Elsevier]

Two of the main pathologies characterizing dysferlinopathies are disrupted muscle membrane repair and chronic inflammation, which lead to symptoms of muscle weakness and wasting. Here, we used recombinant human Galectin-1 (rHsGal-1) as a therapeutic for LGMD2B mouse and human models. Various redox and multimerization states of Gal-1 show that rHsGal-1 is the most effective form in both increasing muscle repair and decreasing inflammation, due to its monomer-dimer equilibrium. Dose-response testing shows an effective 25-fold safety profile between 0.54 and 13.5 mg/kg rHsGal-1 in Bla/J mice. Mice treated weekly with rHsGal-1 showed downregulation of canonical NF-κB inflammation markers, decreased muscle fat deposition, upregulated anti-inflammatory cytokines, increased membrane repair, and increased functional movement compared to non-treated mice. Gal-1 treatment also resulted in a positive self-upregulation loop of increased endogenous Gal-1 expression independent of NF-κB activation. A similar reduction in disease pathologies in patient-derived human cells demonstrates the therapeutic potential of Gal-1 in LGMD2B patients. [ABSTRACT FROM AUTHOR]

Background: Miyoshi myopathy (MM) and limb girdle muscular dystrophy (LGMD2B) are distinct clinical entities because different muscle groups are involved at the onset. We describe the clinical features in 28 patients with dysferlin deficiency confirmed by muscle immunohistochemistry (IHC). Settings and Design: A case series from a tertiary national referral center for neurological disorders. Materials and Methods: Patients with classical phenotype of MM and LGMD2B underwent a thorough phenotypic characterization followed by muscle histopathological study including IHC for dysferlin deficiency. Results: There were 28 patients (20 men and eight women) presenting with manifestations of distal myopathy or LGMD2B and had absence of dysferlin staining on IHC. Patients presented predominantly with distal myopathy of Miyoshi type (MM) or proximal LGMD type and were diagnosed to have dysferlinopathy on IHC. Two patients had the proximodistal form and two had onset as tibial muscular dystrophy. The main clinical features in these patients were onset in late adolescence or early adulthood (mean age of onset for MM was 22.0 ± 6.7 years and for LGMD2B 19.4 ± 5.1 years). There was early and predominant involvement of the posterior compartment muscles of the leg or proximal pelvic girdle muscles, dystrophic features with necrotic regeneration pattern without vacuoles on muscle biopsy and markedly elevated serum creatine kinase values with mean of 10033.8 ± 9283 IU/l (range 402-27460). Consanguinity was reported in 46.4%. The mean duration of illness was 6.4 ± 4.2 years. Dysferlinopathies formed nearly one-fourth of our patients with LGMD. Conclusion: In our experience dysferlinopathies was not an uncommon form of LGMD. [ABSTRACT FROM AUTHOR]

Abstract: Dysferlin encoding gene (DYS) is mutated in the autosomal recessive disorders Miyoshi myopathy, Limb Girdle Muscular Dystrophy type 2B (LGMD2B) and distal anterior compartment myopathy, causing dysferlin deficiency in muscle biopsy. Three ethnic clusters have previously been described in Dysferlinopathy: the Libyan Jewish population originating in the area of Tripoli, Italian and Spanish populations. We report another cluster of this muscular dystrophy in Israel among Jews of the Caucasus region. A genomic analysis of the dysferlin coding sequence performed in patients from this ethnic group, who demonstrated an absence of dysferlin expression in muscle biopsy, revealed a homozygous frameshift mutation of G deletion at codon 927 (2779delG) predicting a truncated protein and a complete loss of functional protein. The possible existence of a founder effect is strengthened by our finding of a 4% carrier frequency in this community. These findings are important for genetic counseling and also enable a molecular diagnosis of LGMD2B in Jews of the Caucasus region. [Copyright &y& Elsevier]

Mutations in dysferlin cause limb girdle muscular dystrophy 2B, Miyoshi myopathy and distal anterior compartment myopathy. Dysferlin is proposed to play a role in muscle membrane repair. To gain functional insight into the molecular mechanisms of dysferlin, we have searched for dysferlin-interacting proteins in skeletal muscle. By coimmunoprecipitation coupled with mass spectrometry, we demonstrate that AHNAK interacts with dysferlin. We defined the binding sites in dysferlin and AHNAK as the C2A domain in dysferlin and the carboxyterminal domain of AHNAK by glutathione S-transferase (GST)-pull down assays. As expected, the N-terminal domain of myoferlin also interacts with the carboxyterminal domain of AHNAK. In normal skeletal muscle, dysferlin and AHNAK colocalize at the sarcolemmal membrane and T-tubules. In dysferlinopathies, reduction or absence of dysferlin correlates with a secondary muscle-specific loss of AHNAIL Moreover, in regenerating rat muscle, dysferlin and AHNAK showed a marked increase and cytoplasmic localization, consistent with the direct interaction between them. Our data suggest that dysferlin participates in the recruitment and stabilization of AHNAK to the sarcolemma and that AHNAK plays a role in dysferlin membrane repair process. It may also have significant implications for understanding the biology of AHNAK-containing exocytotic vesicles, "enlargosomes," in plasma membrane remodeling and repair. [ABSTRACT FROM AUTHOR]

Mutations in the human dysferlin gene (DYSF) cause autosomal recessive muscular dystrophies characterized by degeneration and weakness of proximal and/or distal muscles: limb girdle muscular dystrophy type 2B (LGMD2B) and Miyoshi myopathy (MM). Recently, an interaction between caveolin-3 and dysferlin in normal and dystrophic muscle (primary caveolin-3 deficiency; LGMD1C) was shown. In this study, clinical,morphological and genetic analysis was carried out in four independent LGMD2B/MM patients. All patients presented with an adult-onset, slowly progressive muscular dystrophy with variable involvement of proximal and distal muscles. We found complete lack of dysferlin in the four LGMD2B/MM patients. Secondary reduction of caveolin-3 was detected in three out of the four patients. Regular caveolae were detected along the basal lamina in two patients by electron microscopy. We provide further evidence that dysferlin and caveolin-3 interact in human skeletal muscle. It remains to be elucidated whether the loss of this interaction contributes to pathogenic events in muscular dystrophy. [ABSTRACT FROM AUTHOR]

Dysferlin is the protein product of the DYSF gene mapped at 2p31, which mutations cause limb-girdle muscular dystrophy type 2B (LGMD2B) and Miyoshi myopathy. To date, nine autosomal recessive forms (AR-LGMD) have been identified: four genes, which code for the sarcoglycan glycoproteins, are associated with both mild and severe forms, the sarcoglycanopathies (LGMD2C, 2D, 2E and 2F). The other five forms, usually causing a milder phenotype are LGMD2A (calpain 3), LGMD2B (dysferlin), LGMD2G (telethonin), LGMD2H (9q31-11), and LGMD2I (19q13.3). We studied dysferlin expression in a total of 176 patients, from 166 LGMD families: 12 LGMD2B patients, 70 with other known forms of muscular dystrophies (LGMD2A, sarcoglycanopathies, LGMD2G), in an attempt to assess the effect of the primary gene-product deficiency on dysferlin. In addition, 94 still unclassified LGMD families were screened for dysferlin deficiency. In eight LGMD2B patients from five families, no dysferlin was observed in muscle biopsies, both through immunofluorescence (IF) and Western blot methodologies, while in two families, a very faint band was detected. Both patterns, negative or very faint bands, were concordant in patients belonging to the same families, suggesting that dysferlin deficiency is specific to LGMD2B. Myoferlin, the newly identified homologue of dysferlin was studied for the first time in LGMD2B patients. Since no difference was observed between patients mildly and severely affected, this protein do not seem to modify the phenotype in the present dysferlin-deficient patients. Dystrophin, sarcoglycans, and telethonin were normal in all LGMD2B patients, while patients with sarcoglycanopathies (2C, 2D, and 2E), LGMD2A, LGMD2G, and DMD showed the presence of a normal dysferlin band by Western blot and a positive pattern on IF. These data suggest that there is no interaction between dysferlin and these proteins. However, calpain analysis showed a weaker band in four patients from two families with intra-familial concordance. Therefore, this secondary deficiency of calpain in LGMD2B families, may indicate an interaction between dysferlin and calpain in muscle. Dysferlin was also present in cultured myotubes, in chorionic villus, and in the skin. Dysferlin deficiency was found in 24 out of a total of 166 Brazilian AR-LGMD families screened for muscle proteins (∼14%), thus representing the second most frequent known LGMD form, after calpainopathy, in our population. [ABSTRACT FROM AUTHOR]

Galectin-1 is a small (14.5 kDa) multifunctional protein with cell-cell and cell-ECM adhesion due to interactions with the carbohydrate recognition domain (CRD). In two types of muscular dystrophies, this lectin protein has shown therapeutic properties, including positive regulation of skeletal muscle differentiation and regeneration. Both Duchenne and limb-girdle muscular dystrophy 2B (LGMD2B) are subtypes of muscular dystrophies characterized by deficient membrane repair, muscle weakness, and eventual loss of ambulation. This chapter explains confocal techniques such as laser injury, calcium imaging, and galectin-1 localization to examine the effects of galectin-1 on membrane repair in injured LGMD2B models., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

The widespread use of magnetic resonance imaging (MRI) in the diagnosis of myopathies has made it possible to clarify the typical MRI pattern of dysferlinopathy. However, sufficient attention has not been given to the variability of MRI patterns in dysferlinopathy., Materials and Methods: Twenty-five patients with the clinical manifestations of dysferlinopathy were examined. For all patients, creatine phosphokinase levels were measured and molecular genetics were examined. In two patients, immunohistochemical examinations of muscle biopsies were performed. MRI scanning was included T2 multi-slice multi-echo, T1 weighted, T2 weighted and Short Tau Inversion Recovery T2 weighted sequences. Quantitative and semi-quantitative evaluations of fatty replacement and swelling of the muscles were undertaken., Results: Variability in the MRI patterns was lowest in the pelvis and leg muscles and highest in the thigh muscles. Three main types of MRI patterns were distinguished: posterior-dominant (80%), anterior-dominant (16%), and diffuse (4%). Among patients with the anterior-dominant pattern, the collagen-like variant (4%), proximal variant (4%) and pseudo-myositis (8%) were separately distinguished., Conclusions: Awareness of atypical MRI patterns in dysferlinopathy is important for increasing the efficiency of routine diagnostics and optimizing the search for causative gene mutations., (©2021 Gaetano Conte Academy - Mediterranean Society of Myology, Naples, Italy.)