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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]

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]

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]

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]

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]

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]

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]

ABSTRACT 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 autophagy-related 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. Muscle Nerve 50: 340-347, 2014 [ABSTRACT FROM AUTHOR]

ABSTRACT 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. Muscle Nerve 50: 333-339, 2014 [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]

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]

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]