A Rare Case of Dysferlinopathy in an Adolescent from a Resource-Limited Setting

To describe the phenotypic spectrum of dysferlin (DYSF) gene mutations (which cause dysferlinopathies, autosomal recessive muscular dystrophies) in patients with a dysferlin protein deficiency. Clinical, biological, and pathological data from 40 patients were reviewed. The diagnosis of dysferlinopathy was based on the absence or strong reduction of dysferlin in muscle, and confirmed by mutational screening of the DYSF gene. Two French neuromuscular diseases centers (in Paris and Marseilles). Two main dysferlinopathy phenotypes are well recognized: Miyoshi myopathy and limb-girdle muscular dystrophy type 2B. Typical Miyoshi myopathy and limb-girdle muscular dystrophy type 2B were found in 20 (50%) patients only. Unusual phenotypes included a mixed phenotype, referred to as "proximodistal," combining distal and proximal onset in 14 (35%) patients, pseudometabolic myopathy in 4 (10%), and asymptomatic hyperCKemia (an increased serum creatine kinase level) in 2 (5%). The disease may worsen rapidly, and 10 (25%) patients were initially misdiagnosed as having polymyositis. We suggest a relationship between proximodistal phenotype, inflammation, and severity. In addition to typical Miyoshi myopathy and limb-girdle muscular dystrophy type 2B, dysferlinopathies are a clinically heterogeneous group of disorders ranging from asymptomatism to severe functional disability.

Case presentation: A 24-year-old male presented with a four-year history of progressive proximal lower limb weakness and gait difficulty. He had previously been active and engaged in high-impact exercise, but he had discontinued exercise and gained weight over the past four years. His gait became increasingly impaired, and he reported worsening limb weakness Laboratory tests revealed elevated muscle enzymes, including CPK (16,907), aldolase (141), and DHL (878). EMG showed a myopathic pattern in all four limbs, with evidence of membrane instability in the paraspinal and proximal lower limb muscles. Magnetic resonance imaging of the thigh revealed mild fatty replacement of the posterior and medial compartments. Based on the clinical presentation, laboratory findings, and EMG results, a diagnosis of inflammatory myopathy was suspected. The patient was treated with a pulse of corticosteroids and immunoglobulin, but his symptoms did not improve significantly. Other immunosuppressive therapies were initiated, without any improvement in the condition. Due to the progressive worsening of his symptoms, the patient underwent a muscle biopsy which revealed a marked reduction in dysferlin staining, suggesting dysferlinopathy. There was no CD8 staining, moderate CD68 staining, and mild MHC-I staining in some fibers. The histologic pattern was myopathic with rare necrosis and dysferlin deficiency. The features of the biopsy suggest the diagnosis of dysferlinopathy. Patient maintains clinical stability and is being weaned off immunosuppressive drugs. Discussion: Dysferlinopathies are autosomal recessive neuromuscular disorders caused by mutations in the DYSF gene, which encodes the dysferlin protein. The dysferlin protein is involved in muscle membrane repair, and mutations in the DYSF gene lead to a spectrum of phenotypes, including Miyoshi myopathy and limb-girdle muscular dystrophy type 2B (LGMD2B). Inflammatory myopathies are a group of autoimmune disorders that also cause muscle weakness and inflammation. Both diseases can share many clinical features, including muscle weakness, elevated muscle enzymes, and a myopathic EMG pattern. However, there are also some key differences. In inflammatory myopathies, there is typically an inflammatory infiltrate present in the muscle biopsy. In dysferlinopathies, there is no inflammatory infiltrate, but there is a deficiency of the dysferlin protein. The initial diagnostic hypothesis in the reported case was inflammatory myopathy, however the lack of response to immunosuppression raised suspicion of an alternative diagnosis, with muscle biopsy being of extreme importance for diagnostic guidance. Final comments: This case highlights the importance of careful differential diagnosis in patients with myopathy. The combination of clinical history, laboratory findings, EMG results, muscle biopsy findings, and response to therapy can help to distinguish between inflammatory myopathies and other disorders, such as dysferlinopathies.

Phenotypic and genotypic analysis of a patient with Miyoshi myopathy caused by truncated protein

Objective To diagnose and differentially diagnose limb-girdle muscular dystrophy type 2B(LGMD2B)and polymyositis (PM) based on clinical and pathological characteristics. Methods Muscle biopsics were obtained from 8 patients suspected with LGMD2B who were initially diagnosed with PM.The clinical and pathological data from 8 cases of LGMD2B and 4 cases of PM by using histo-and immunohistochemistry with anti-dysferlin,dystrophins,sarcoglycans,MHC-Ⅰ,CD8 monoclonal antibodies were compared.Results (1) LGMD2B and PM shared similar pathological presentations including muscle fibet degeneration and necrosis in various degree,proliferation of connective tissue,and inflammatory cell infiltration.Normal stains of dystrophins and sarcoglycans were observed.whereas absent or very faint staining of dysfedin observed in muscle biopsies of 8 patients confirmed the diagnosis of LGMD2B.while normal stains of dysferlin on sarcolemma were observed in the 4 cases of PM.MHC-Ⅰ was weakly expressed or absent in LGMD2B.while strongly expressed on sarcolemma in PM and the infiltration area of inflammation cells.The expression of CD8 on a few inflammatory cells were positive in LGMD2B.while some inflammatory cells were positive in PM.(2)Both LGMD2B and PM shared similar presentation,including proximal muscle weakness,remarkable elevation of CK,myopathic changes in electromyography.Patients with LGMD2B did not complain of apparent muscle pain.and their erythrocyte sedimentation rate and Creactive protein were in normal range.which could be used as marker to differentiate from patients with PM.Conclusions Clinically and pathologically LGMD2B and PM are presented similarly and likely to be misdiagnosed.The absence of dysferlin in LGMD2B and high expression of MHC-Ⅰ and CD8 in PM are the key index of the diagnosis and differential diagnosis between LGMD2B and PM. Key words: Muscular dystrophies,limb-girdle; Polymyositis; Diagnosis,differential; Membrane proteins; Muscle proteins; Major histocompatibility complex; Antigens,CD8

Dysferlinopathies are rare disorders of muscle that present two main phenotypes: Miyoshi myopathy with primarily distal weakness and limb-girdle muscular dystrophy type 2B (LGMD2B) with primarily proximal weakness. They are caused by mutations in the gene encoding the skeletal muscle protein dysferlin, which is involved in muscle repair. The clinical presentation of the disease is rather uncharacteristic, and molecular genetic testing is long-lasting; thus muscle biopsy may be essential in the diagnostic process. Histology itself reveals non-specific changes, but a variety of currently available muscle protein immunostains may be very helpful. We present a 19-year-old girl with epilepsy and elevated creatine phosphokinase (CPK) concentration. Due to increased CPK, myopathy was suspected and muscle biopsy was performed. Light microscopy showed no distinctive myopathic changes, and electron microscopy showed no abnormalities. Extended immunohistochemistry, performed much later, showed complete absence of dysferlin immunostaining. Based on that result, the diagnosis of LGMD2B was made, with subsequent genetic testing to be done. Two known pathogenic variants were found in the DYSF gene, confirming the diagnosis of LGMD2B and allowing proper genetic counseling.

Limb girdle muscular dystrophy type 2B (LGMD2B) and Miyoshi myopathy (MM), a distal muscular dystrophy, are both caused by mutations in the recently cloned gene dysferlin, gene symbol DYSF. Two large pedigrees have been described which have both types of patient in the same families. Moreover, in both pedigrees LGMD2B and MM patients are homozygous for haplotypes of the critical region. This suggested that the same mutation in the same gene would lead to both LGMD2B or MM in these families and that additional factors were needed to explain the development of the different clinical phenotypes. In the present paper we show that in one of these families Pro791 of dysferlin is changed to an Arg residue. Both the LGMD2B and MM patients in this kindred are homozygous for this mutation, as are four additional patients from two previously unpublished families. Haplotype analyses suggest a common origin of the mutation in all the patients. On western blots of muscle, LGMD2B and MM patients show a similar abundance in dysferlin staining of 15 and 11%, respectively. Normal tissue sections show that dysferlin localizes to the sarcolemma while tissue sections from MM and LGMD patients show minimal staining which is indistinguishable between the two types. These findings emphasize the role for the dysferlin gene as being responsible for both LGMD2B and MM, but that the distinction between these two clinical phenotypes requires the identification of additional factor(s), such as modifier gene(s).

Dysferlinopathies are autosomal recessive disorders caused by mutations in the dysferlin (DYSF) gene, encoding the dysferlin protein. DYSF mutations lead to a wide range of muscular phenotypes, with the most prominent being Miyoshi myopathy (MM) and limb girdle muscular dystrophy type 2B (LGMD2B) and the second most common being LGMD. Symptoms generally appear at the end of childhood and, although disease progression is typically slow, walking impairments eventually result. Dysferlin is a modular type II transmembrane protein for which numerous binding partners have been identified. Although dysferlin function is only partially elucidated, this large protein contains seven calcium sensor C2 domains, shown to play a key role in muscle membrane repair. On the basis of this major function, along with detailed clinical observations, it has been possible to design various therapeutic approaches for dysferlin-deficient patients. Among them, exon-skipping and minigene transfer strategies have been evaluated at the preclinical level and, to date, represent promising approaches for clinical trials. This review aims to summarize the pathophysiology of dysferlinopathies and to evaluate the therapeutic potential for treatments currently under development.

Rationale:Nemaline myopathy (NM) is a rare inherited muscle disorder characterized by nemaline rods in muscle fibers and clinically presenting with muscle weakness and hypotonia. This report describes a case of NM caused by a mutation in the ACTA1 (actin alpha 1) gene to enhance the understanding of its diagnosis and clinical course, particularly in the context of pregnancy.Patient concerns:A 33-year-old female presented with progressive lower limb weakness and muscle atrophy. Symptom onset occurred during pregnancy. Laboratory tests revealed an elevated serum creatine kinase level (1011 U/L).Diagnoses:Genetic testing identified a heterozygous missense mutation (Q139H, c.417G>C) in exon 3 of the ACTA1 gene, confirming the diagnosis of NM. A muscle biopsy to definitively identify nemaline rods was indicated but was declined by the patient.Interventions:The diagnostic evaluation included clinical examination, serum creatine kinase testing, electromyography, and genetic analysis. Following diagnosis, the patient was managed with a regimen of supportive care.Outcomes:Electromyography confirmed myopathic changes. With supportive care, the patient remained clinically stable at follow-up.Lessons:This case underscores the diagnostic utility of genetic testing for NM when a muscle biopsy is not feasible. It also demonstrates the potential for symptom exacerbation or onset during pregnancy and highlights the importance of multidisciplinary supportive care and genetic counseling in the management of ACTA1-related NM.

Objective To clarify the differential diagnosis between limb-girdle muscular dystrophy type 2B (LGMD2B) and polymyositis (PM). Methods Analyzed the character of clinical, histochemical and immunohistochemical pathologic with biopsied skeletal muscle in 5 patients with LGMD2B and the relationship with PM. Results Both LGMD2B and PM showed the muscle weakness in proximal limbs and elevated creatinkinase level and electromyogram showed myogenic damage. Histochemical stains: it was similar to PM, that was the muscle fibers degenerating, regenerating and necrotic and the infiltrating inflammatory cells in different extent in 5 patients. Immunohistoehemical stains: anfi-Dysferlin monoelonal antibody showed that Dysferlin was deficient on sarcolemma in LGMD2B, but it was normal in PM. Conclusions It is resemble on the clinical and histoehemieal pathologic characters in both LGMD2B and PM. It is key to antidiastole LGMD2B and PM by the immunohistochemieal stain of anti-Dysferlin monoelonal antibody. Key words: Muscular dystrophies; Polymyositis; Immunohistochemistry

BackgroundDysferlinopathies are autosomal recessive disorders caused by mutations in the dysferlin (DYSF) gene encoding the dysferlin protein. DYSF mutations lead to a wide range of muscular phenotypes, with the most prominent being Miyoshi myopathy (MM) and limb girdle muscular dystrophy type 2B (LGMD2B).MethodsWe assessed the one-year-natural course of dysferlinopathy, and the safety and efficacy of deflazacort treatment in a double-blind, placebo-controlled cross-over trial. After one year of natural course without intervention, 25 patients with genetically defined dysferlinopathy were randomized to receive deflazacort and placebo for six months each (1 mg/kg/day in month one, 1 mg/kg every 2nd day during months two to six) in one of two treatment sequences.ResultsDuring one year of natural course, muscle strength declined about 2% as measured by CIDD (Clinical Investigation of Duchenne Dystrophy) score, and 76 Newton as measured by hand-held dynamometry. Deflazacort did not improve muscle strength. In contrast, there is a trend of worsening muscle strength under deflazacort treatment, which recovers after discontinuation of the study drug. During deflazacort treatment, patients showed a broad spectrum of steroid side effects.ConclusionDeflazacort is not an effective therapy for dysferlinopathies, and off-label use is not warranted. This is an important finding, since steroid treatment should not be administered in patients with dysferlinopathy, who may be often misdiagnosed as polymyositis.Trial registrationThis clinical trial was registered at http://www.ClincalTrials.gov, identifier: NCT00527228, and was always freely accessible to the public.

Rationale:Limb-girdle muscular dystrophy type 2B (LGMD2B) is a degenerative muscle disorder induced by mutations in the dysferlin gene. Dysferlin is involved in membrane repair and vesicle fusion through its 7 C2 calcium-binding domains, which mediate these calcium-dependent processes. It is currently considered an untreatable neuromuscular condition with a poor prognosis. The estimated incidence of this disease is 1 to 6.5 per 100,000 individuals. The primary clinical features of LGMD2B include proximal muscle weakness and elevated serum creatine kinase (CK) levels; occasionally, patients may present with elevated transaminase and hematuria. It is often misdiagnosed as polymyositis or liver disease. Herein, we report a case of LGMD2B initially presenting with elevated transaminase levels and hematuria.Patient concerns:A 30-year-old woman was found to have elevated transaminases and subsequent hematuria.Diagnoses:The patient was ultimately diagnosed with LGMD2B after muscle biopsy and genetic testing were performed.Interventions:In the patient’s first hospitalization, she was found to have elevated transaminase levels and untested CK levels, and no pathogenic findings were identified after a liver biopsy. Four years later, the patient was admitted to the Nephrology Department with gross hematuria. After hospitalization, serum CK levels were elevated. She was misdiagnosed with polymyositis and treated with oral prednisone; however, her condition did not improve, muscle strength declined, and hematuria persisted. Muscle biopsy and genetic testing were performed, and the patient was ultimately diagnosed with LGMD2B. Supportive therapy with coenzyme Q10, idebenone, and creatine monohydrate was initiated, and she was advised to avoid strenuous physical activity.Outcomes:Currently, the patient exhibits generalized muscle weakness, unstable walking gait, and urine positive (+++) for occult blood. Her muscle strength has gradually declined over the past 13 years.Lessons:LGMD2B initially presents with atypical clinical manifestations. In some cases, elevated transaminase levels can be the first manifestation of the disease; therefore, any unexplained elevated transaminase levels should prompt evaluation of underlying muscle diseases. A 13-year follow-up of a female patient demonstrated progressive muscle atrophy, emphasizing the importance of considering muscle diseases in patients with unexplained elevated serum transaminase levels. Subsequent dark brown urine and hematuria were likely caused by increased myoglobin levels and dysferlin deficiency in podocytes, which may be associated with minimal change nephropathy.

P.P.6 08 Clinical features of the limb-girdle muscular dystrophy type 2B

The dysferlin gene or the DYSF gene encodes the Ca2+ -dependent phospholipid-binding protein dysferlin, which belongs to the ferlin family and is associated with muscle membrane regeneration and repair. Variants in the DYSF gene are responsible for limb-girdle muscular dystrophy type 2B (LGMD2B), also called limb-girdle muscular dystrophy recessive 2 (LGMDR2), a rare subtype of muscular dystrophy involving progressive muscle weakness and atrophy. The present study aimed to identify the variants responsible for the clinical symptoms of a Chinese patient with limb girdle muscular dystrophies (LGMDs) and to explore the genotype-phenotype associations of LGMD2B. A series of clinical examinations, including blood tests, magnetic resonance imaging scans for the lower legs, electromyography and muscle biopsy, was performed on the proband diagnosed with muscular dystrophies. Whole exome sequencing was conducted to detect the causative variants, followed by Sanger sequencing to validate these variants. We identified two compound heterozygous variants in the DYSF gene, c.1058 T>C, p.(Leu353Pro) in exon 12 and c.1461C>A/p.Cys487* in exon 16 in this proband, which were inherited from the father and mother, respectively. In silico analysis for these variants revealed deleterious results by PolyPhen-2 (Polymorphism Phenotyping v2; http://genetics.bwh.harvard.edu/pph2), SIFT (Sorting Intolerant From Tolerant; https://sift.bii.a-star.edu.sg), PROVEAN (Protein Variation Effect Analyzer; http://provean.jcvi.org/seq_submit.php) and MutationTaster (http://www.mutationtaster.org). In addition, the two compound heterozygous variants in the proband were absent in 100 control individuals who had an identical ethnic origin and were from the same region, suggesting that these variants may be the pathogenic variants responsible for the LGMD2B phenotypes for this proband. The present study broadens our understanding of the mutational spectrum of the DYSF gene, which provides a deep insight into the pathogenesis of LGMDs and accelerates the development of a prenatal diagnosis.

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 LGMD21 (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 (approximately 14%), thus representing the second most frequent known LGMD form, after calpainopathy, in our population.

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.