Dysferlin Gene Research Articles

Limb Girdle Muscular Dystrophy Type 2B (LGMD2B): Diagnosis and Therapeutic Possibilities.

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.

A novel homozygous variant (c.5876T > C: p. Leu1959Pro) in DYSF segregates with limb-girdle muscular dystrophy: a case report

BackgroundLimb girdle muscular dystrophies (LGMDs) constitute a heterogeneous group of neuromuscular disorders with a very variable clinical presentation and overlapping traits. The clinical symptoms of LGMD typically appear in adolescence or early adulthood. Genetic variation in the dysferlin gene (DYSF) has been associated with LGMD.MethodsWe characterized a recessive LGMD in a young adult from consanguineous Irani families using whole-exome sequencing (WES) technology. Sanger sequencing was performed to verify the identified variant. Computational modeling and protein-protein docking were used to investigate the impact of the variant on the structure and function of the DYSF protein.ResultsBy WES, we identified a novel homozygous missense variant in DYSF (NM_003494.4: c.5876T > C: p. Leu1959Pro) previously been associated with LGMD phenotypes.ConclusionsThe identification and validation of new pathogenic DYSF variant in the present study further highlight the importance of this gene in LGMD.

High Prevalence of a c.5979dupA Variant in the Dysferlin Gene (DYSF) in Individuals from a Semiarid Region of Brazil.

Dysferlinopathies represent a group of limb girdle or distal muscular dystrophies with an autosomal-recessive inheritance pattern resulting from the presence of pathogenic variants in the dysferlin gene (DYSF). In this work, we describe a population from a small city in Brazil carrying the c.5979dupA pathogenic variant of DYSF responsible for limb girdle muscular dystrophy type 2R and distal muscular dystrophy. Genotyping analyses were performed by qPCR using customized probe complementary to the region with the duplication under analysis in the DYSF. A total of 104 individuals were examined. c.5979dupA was identified in 48 (46.15%) individuals. Twenty-three (22%) were homozygotes, among whom 13 (56.5%) were female. A total of 91.3% (21) of homozygous individuals had a positive family history, and seven (30.4%) reported consanguineous marriages. Twenty-five (24%) individuals were heterozygous (25.8±16 years) for the same variant, among whom 15 (60%) were female. The mean CK level was 697 IU for homozygotes, 140.5 IU for heterozygotes and 176 IU for wild-type homo-zygotes. The weakness distribution pattern showed 17.3% of individuals with a proximal pattern, 13% with a distal pattern and 69.6% with a mixed pattern. Fatigue was present in 15 homozygotes and one heterozygote. The high prevalence of this variant in individuals from this small community can be explained by a possible founder effect associated with historical, geographical and cultural aspects.

Utilization of Targeted RNA-Seq for the Resolution of Variant Pathogenicity and Enhancement of Diagnostic Yield in Dysferlinopathy

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.

A case of dysferlinopathy (Miyoshi distal myopathy limb-girdle muscular dystrophy type 2b phenotype) from a tertiary care hospital

Limb girdle muscular dystrophy type 2B (LGMD2B) and Miyoshi myopathy are caused by similar mutations in the dysferlin gene. The phenotype of these allelic disease variants can vary considerably. We report a young male with severe and rapidly progressing muscle disorder with increased creatine phosphokinase (CPK) and confirmatory muscle biopsy findings. Genetic testing was done. A homozygous nonsense variation in exon 23 of the DYSF gene, which was consistent with the patient’s clinical reports of dysferlinopathy. Clinical phenomenology and preferential muscle involvement lead one to the gold standard genetic testing in heritable myopathies, which was well established in this report.

Transcriptome analysis of skeletal muscle in dermatomyositis, polymyositis, and dysferlinopathy, using a bioinformatics approach.

Polymyositis (PM) and dermatomyositis (DM) are two distinct subgroups of idiopathic inflammatory myopathies. Dysferlinopathy, caused by a dysferlin gene mutation, usually presents in late adolescence with muscle weakness, degenerative muscle changes are often accompanied by inflammatory infiltrates, often resulting in a misdiagnosis as polymyositis. To identify differential biological pathways and hub genes related to polymyositis, dermatomyositis and dysferlinopathy using bioinformatics analysis for understanding the pathomechanisms and providing guidance for therapy development. We analyzed intramuscular ribonucleic acid (RNA) sequencing data from seven dermatomyositis, eight polymyositis, eight dysferlinopathy and five control subjects. Differentially expressed genes (DEGs) were identified by using DESeq2. Enrichment analyses were performed to understand the functions and enriched pathways of DEGs. A protein-protein interaction (PPI) network was constructed, and clarified the gene cluster using the molecular complex detection tool (MCODE) analysis to identify hub genes. A total of 1,048, 179 and 3,807 DEGs were detected in DM, PM and dysferlinopathy, respectively. Enrichment analyses revealed that upregulated DEGs were involved in type 1 interferon (IFN1) signaling pathway in DM, antigen processing and presentation of peptide antigen in PM, and cellular response to stimuli in dysferlinopathy. The PPI network and MCODE cluster identified 23 genes related to type 1 interferon signaling pathway in DM, 4 genes (PDIA3, HLA-C, B2M, and TAP1) related to MHC class 1 formation and quality control in PM, and 7 genes (HSPA9, RPTOR, MTOR, LAMTOR1, LAMTOR5, ATP6V0D1, and ATP6V0B) related to cellular response to stress in dysferliniopathy. Overexpression of genes related to the IFN1 signaling pathway and major histocompatibility complex (MHC) class I formation was identified in DM and PM, respectively. In dysferlinopathy, overexpression of HSPA9 and the mTORC1 signaling pathway genes was detected.

Limb-girdle muscular dystrophy type 2B causes HDL-C abnormalities in patients and statin-resistant muscle wasting in dysferlin-deficient mice

Limb-girdle muscular dystrophy (MD) type 2B (LGMD2B) and Duchenne MD (DMD) are caused by mutations to the Dysferlin and Dystrophin genes, respectively. We have recently demonstrated in typically mild dysferlin- and dystrophin-deficient mouse models that increased plasma cholesterol levels severely exacerbate muscle wasting, and that DMD patients display primary dyslipidemia characterized by elevated plasma cholesterol and triglycerides. Herein, we investigate lipoprotein abnormalities in LGMD2B and if statin therapy protects dysferlin-deficient mice (Dysf) from muscle damage. Herein, lipoproteins and liver enzymes from LGMD2B patients and dysferlin-null (Dysf) mice were analyzed. Simvastatin, which exhibits anti-muscle wasting effects in mouse models of DMD and corrects aberrant expression of key markers of lipid metabolism and endogenous cholesterol synthesis, was tested in Dysf mice. Muscle damage and fibrosis were assessed by immunohistochemistry and cholesterol signalling pathways via Western blot. LGMD2B patients show reduced serum high-density lipoprotein cholesterol (HDL-C) levels compared to healthy controls and exhibit a greater prevalence of abnormal total cholesterol (CHOL)/HDL-C ratios despite an absence of liver dysfunction. While Dysf mice presented with reduced CHOL and associated HDL-C and LDL-C-associated fractions, simvastatin treatment did not prevent muscle wasting in quadriceps and triceps muscle groups or correct aberrant low-density lipoprotein receptor (LDLR) and 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) protein expression. LGMD2B patients present with reduced serum concentrations of HDL-C, a major metabolic comorbidity, and as a result, statin therapy is unlikely to prevent muscle wasting in this population. We propose that like DMD, LGMD2B should be considered as a new type of genetic dyslipidemia.

The clinical, myopathological, and molecular characteristics of 26 Chinese patients with dysferlinopathy: a high proportion of misdiagnosis and novel variants

BackgroundDysferlinopathy is an autosomal recessive muscular dystrophy caused by pathogenic variants in the dysferlin (DYSF) gene. This disease shows heterogeneous clinical phenotypes and genetic characteristics.MethodsWe reviewed the clinical and pathological data as well as the molecular characteristics of 26 Chinese patients with dysferlinopathy screened by immunohistochemistry staining and pathogenic variants in DYSF genes.ResultsAmong 26 patients with dysferlinopathy, 18 patients (69.2%) presented as Limb-girdle Muscular Dystrophy Type R2 (LGMD R2), 4 (15.4%) had a phenotype of Miyoshi myopathy (MM), and 4 (15.4%) presented as asymptomatic hyperCKemia. Fifteen patients (57.7%) were originally misdiagnosed as inflammatory myopathy or other diseases. Fifteen novel variants were identified among the 40 variant sites identified in this cohort.ConclusionDysferlinopathy is a clinically and genetically heterogeneous group of disorders with various phenotypes, a high proportion of novel variants, and a high rate of misdiagnosis before immunohistochemistry staining and genetic analysis.

Identification of a novel heterozygous DYSF variant in a large family with a dominantly-inherited dysferlinopathy.

Dysferlinopathy is an autosomal recessive muscular dystrophy, caused by bi-allelic variants in the gene encoding dysferlin (DYSF). Onset typically occurs in the second to third decade and is characterised by slowly progressive skeletal muscle weakness and atrophy of the proximal and/or distal muscles of the four limbs. There are rare cases of symptomatic DYSF variant carriers. Here, we report a large family with a dominantly inherited hyperCKaemia and late-onset muscular dystrophy. Genetic analysis identified a co-segregating novel DYSF variant [NM_003494.4:c.6207del p.(Tyr2070Metfs*4)]. No secondary variants in DYSF or other dystrophy-related genes were identified on whole genome sequencing and analysis of the proband's DNA. Skeletal muscle involvement was milder and later onset than typical dysferlinopathy presentations; these clinical signs manifested in four individuals, all between the fourth and sixth decades of life. All individuals heterozygous for the c.6207del variant had hyperCKaemia. Histological analysis of skeletal muscle biopsies across three generations showed clear dystrophic signs, including inflammatory infiltrates, regenerating myofibres, increased variability in myofibre size and internal nuclei. Muscle magnetic resonance imaging revealed fatty replacement of muscle in two individuals. Western blot and immunohistochemical analysis of muscle biopsy demonstrated consistent reduction of dysferlin staining. Allele-specific quantitative PCR analysis of DYSF mRNA from patient muscle found that the variant, localised to the extreme C-terminus of dysferlin, does not activate post-transcriptional mRNA decay. We propose that this inheritance pattern may be underappreciated and that other late-onset muscular dystrophy cases with mono-allelic DYSF variants, particularly C-terminal premature truncation variants, may represent dominant forms of disease.

Cells corresponding to telocites have been detected in pathologically altered skeletal muscle

A relatively recently described population of cells, apparently belonging to the tissue system of the internal environment, are the telocytes. Their peculiarities are not only the co-expression of CD117- and CD34-molecules, but also thin, indistinguishable processes at the light-optical level, whose length can be many times greater than the diameter of the cell body. In this regard, transmission electron microscopy remains the method of choice for their detection in tissues. Telocytes were found in the myocardium, connective tissue of the gallbladder, in gastrointestinal tract, in the stroma of the exocrine glands, the placenta, some vessels. However, data on the detection of telocytes in striated skeletal muscle tissue are either absent or still rare. This brief report demonstrates cells that, by their ultrastructural characteristics, can be identified as telocytes in the endomysium of gastrocnemius muscle in Bla/J mice (mutation in the dysferlin gene, DYSF).

Clinical, Neurophysiological, Radiological, Pathological, and Genetic Features of Dysferlinopathy in Saudi Arabia.

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.

Secreted acid sphingomyelinase as a potential gene therapy for limb girdle muscular dystrophy 2B.

Efficient sarcolemmal repair is required for muscle cell survival, with deficits in this process leading to muscle degeneration. Lack of the sarcolemmal protein dysferlin impairs sarcolemmal repair by reducing secretion of the enzyme acid sphingomyelinase (ASM), and causes limb girdle muscular dystrophy 2B (LGMD2B). The large size of the dysferlin gene poses a challenge for LGMD2B gene therapy efforts aimed at restoring dysferlin expression in skeletal muscle fibers. Here, we present an alternative gene therapy approach targeting reduced ASM secretion, the consequence of dysferlin deficit. We showed that the bulk endocytic ability is compromised in LGMD2B patient cells, which was addressed by extracellularly treating cells with ASM. Expression of secreted human ASM (hASM) using a liver-specific adeno-associated virus (AAV) vector restored membrane repair capacity of patient cells to healthy levels. A single in vivo dose of hASM-AAV in the LGMD2B mouse model restored myofiber repair capacity, enabling efficient recovery of myofibers from focal or lengthening contraction–induced injury. hASM-AAV treatment was safe, attenuated fibro-fatty muscle degeneration, increased myofiber size, and restored muscle strength, similar to dysferlin gene therapy. These findings elucidate the role of ASM in dysferlin-mediated plasma membrane repair and to our knowledge offer the first non–muscle-targeted gene therapy for LGMD2B.

Integrated Genome and Transcriptome Sequencing to Solve a Neuromuscular Puzzle: Miyoshi Muscular Dystrophy and Early Onset Primary Dystonia in Siblings of the Same Family.

BackgroundNeuromuscular disorders (NMD), many of which are hereditary, affect muscular function. Due to advances in high-throughput sequencing technologies, the diagnosis of hereditary NMDs has dramatically improved in recent years.Methods and ResultsIn this study, we report an family with two siblings exhibiting two different NMD, Miyoshi muscular dystrophy (MMD) and early onset primary dystonia (EOPD). Whole exome sequencing (WES) identified a novel monoallelic frameshift deletion mutation (dysferlin: c.4404delC/p.I1469Sfs∗17) in the Dysferlin gene in the index patient who suffered from MMD. This deletion was inherited from his unaffected father and was carried by his younger sister with EOPD. However, immunostaining staining revealed an absence of dysferlin expression in the proband’s muscle tissue and thus suggested the presence of the second underlying mutant allele in dysferlin. Using integrated RNA sequencing (RNA-seq) and whole genome sequencing (WGS) of muscle tissue, a novel deep intronic mutation in dysferlin (dysferlin: c.5341-415A > G) was discovered in the index patient. This mutation caused aberrant mRNA splicing and inclusion of an additional pseudoexon (PE) which we termed PE48.1. This PE was inherited from his unaffected mother. PE48.1 inclusion altered the Dysferlin sequence, causing premature termination of translation.ConclusionUsing integrated genome and transcriptome sequencing, we discovered hereditary MMD and EOPD affecting two siblings of same family. Our results added further weight to the combined use of RNA-seq and WGS as an important method for detection of deep intronic gene mutations, and suggest that integrated sequencing assays are an effective strategy for the diagnosis of hereditary NMDs.

Эндотелиальная дисфункция при мышечных дистрофиях

Endotheliocytes are the key elements of the vascular wall and are involved in regulation of vascular tone and permeability, inflammation, hemostasis, angiogenesis etc. Impaired function of endothelial cells universally recognized as endothelial dysfunction is associated with a number of common diseases such as ischemic heart disease, arterial hypertension, atherosclerosis, peripheral arterial disease, septic shock, chronic kidney disease, obesity, oncological and autoimmune diseases. Less is known about the role of endothelial cells in pathogenesis of development and progression of rare diseases, such as muscular dystrophies. Muscular dystrophies involve over 30 genetically determined diseases, which are characterized by the development of a progressive muscular weakness and skeletal muscle degeneration. Presence of a nucleotide variant associated with a certain muscular dystrophy is primarily marked by a limited potential of skeletal muscle regeneration due to the impaired structure and function of myogenic cells. Inherited myopathies include a group of severe neuromuscular diseases caused by a mutation in the dysferlin gene DYSF, which leads to the synthesis of a dysfunctional dysferlin. Complex molecular and cellular interactions involved in skeletal muscle damage and endothelial dysfunction play an important role in the pathogenesis of dysferlinopathies. The possibility to produce an effect on different pathological aspects of dysferlinassociated myopathies such as complement system activation, inflammation, impaired function of endothelial cells, damage and necrosis of myofibrils are extensively studied in vitro and in vivo. This article is dedicated to the current understanding of relationship between the endothelium and its dysfunction in myogenesis and skeletal muscle regeneration in normal and pathological conditions caused by a group of inherited progressive myodystrophies, dysferlinopathies in particular, as well as possible clinical application of endothelial cells in treatment of muscular dystrophies.

Generation and Analysis of Genetically Modified CD14+ Monocytes with Dysferlin Overexpression

IntroductionDysferlinopathies is a group of orphan neuromuscular diseases associated with impairment of mRNA and defect in dysferlin protein expression in skeletal muscle tissue due to mutations in DYSF (Dystrophy-associated fer-1-like) gene. This group of diseases is having autosomal recessive type of inheritance and the frequency of occurrence is 1/1,300-1/200,000 in different populations. There are three main clinical phenotypes associated with dysferlin gene mutations: Limb girdle muscular dystrophy type 2B (LGMD2B), Miyoshi myopathy (MM) and distal myopathy with anterior tibial onset (DMAT) (Yakovlev et. al. Human Gene Therapy 2016). Currently there is no treatment for these diseases. Hence, the development of new methods of treating this group of diseases remains an urgent task, in particular, with the use of gene therapy. According to the literature, CD14+ cells play a key role in the pathogenesis of human dysferlinopathies and are involved in inflammatory reactions and pathological angiogenesis.MethodsIn the present work, by using previously created recombinant adenovirus Ad5-Dysf (Starostina et. al. Medicine 2016) that encodes human cDNA dysferlin gene and enables the expression of the recombinant dysferlin protein, genetic modification of CD14 + monocytes was carried out. Therefore, the following aims were set:1. Isolation of CD14+ cells from a patient with dysferlinopathy and a healthy donor.2. Genetic modification of CD14+ monocytes.3. Conduct western blot analysis of mRNA expression of recombinant dysferlin protein in modified cells after viral transduction. Evaluate the effectiveness of viral transduction by analyzing the expression of EGFP protein (adenovirus Ad5-EGFP) which was taken as a positive control by fluorescence microscopy.ResultsCD14+ cells were isolated from the freshly prepared peripheral blood leukocyte buffy coat layer of the patient with dysferlinopathy and a healthy donor by use of EasySep ™ Human Buffy Coat CD14 Positive Selection Kit. Since the correct assembly and effective DYSF gene delivery of the adenovirus Ad5-Dysf was confirmed in previous work, the viral transduction of human monocytes was carried out successfully. Ad5-EGFP adenovirus was used as a positive control to evaluate the effectiveness of viral transduction. EGFP protein expression analysis was conducted by fluorescence microscopy on an inverted fluorescent microscope of the research class AxioObserver.Z1 (Carl Zeiss) and demonstrated 100% of EGFP protein expression (Fig. 1). After genetic modification of the cells with resulting Ad5-Dysf adenovirus the evaluation of the recombinant dysferlin protein expression in cells was carried out by western blot analysis. Positive reaction was revealed with rabbit polyclonal antibodies to dysferlin (Fig. 2).ConclusionsIn this work we demonstrated that genetically modified CD14+ cells with DYSF overexpression can be successfully geberated and considered as one of the approaches for the therapy of human dysferlinopathies. Future studies are needed to analyze the functionality of the obtained genetic construct in animal models. SVV was supported by RFBR grant 16-34-00657.Fig. 1. CD14+ monocytes genetically modified with Ad5-EGFP. A - phase-contrast microscopy. B - fluorescence microscopy. 48 hours after genetic modification. Magnification 200X.Fig. 2. Western blot analysis of DYSF protein expression in CD14+ monocytes isolated from the peripheral blood of a patient with dysferlinopathy. Electrophoresis in 12% SDS-PAGE gel according to the Laemmli system. Antibodies to dysferlin (Abcam, ab15108) were used in a dilution of 1:250. The expected size of DYSF protein is 231 kDa. Well 1 - transduced CD14+ monocytes with Ad5-Dysf. Well 2 - native CD14+ monocytes. M - molecular weight marker. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Frequent DYSF rare variants/mutations in 152 Han Chinese samples with ovarian endometriosis.

Endometriosis is a common chronic gynecological disease greatly affecting women health. Prior studies have implicated that dysferlin (DYSF) aberration might be involved in the pathogenesis of ovarian endometriosis. In the present study, we explore the potential presence of DYSF mutations in a total of 152 Han Chinese samples with ovarian endometriosis. We analyze the potential presence of DYSF mutations by direct DNA sequencing. A total of seven rare variants/mutations in the DYSF gene in 10 out of 152 samples (6.6%) were identified, including 5 rare variants and 2 novel mutations. For the 5 rare variants, p.R334W and p.G941S existed in 2 samples, p.R865W, p.R1173H and p.G1531S existed in single sample, respectively; for the two novel mutations, p.W352* and p.I1642F, they were identified in three patients. These rare variants/mutations were absent or existed at extremely low frequency either in our 1006 local control women without endometriosis, or in the China Metabolic Analytics Project (ChinaMAP) and Genome Aggregation Database (gnomAD) databases. Evolutionary conservation analysis results suggested that all of these rare variants/mutations were evolutionarily conserved among 11 vertebrate species from Human to Fox. Furthermore, in silico analysis results suggested these rare variants/mutations were disease-causing. Nevertheless, we find no significant association between DYSF rare variants/mutations and the clinical features in our patients. To our knowledge, this is the first report revealing frequent DYSF mutations in ovarian endometriosis. We identified a high frequency of DYSF rare variants/mutations in ovarian endometriosis for the first time. This study suggests a new correlation between DYSF rare variants/mutations and ovarian endometriosis, implicating DYSF rare variants/mutations might be positively involved in the pathogenesis of ovarian endometriosis.

Functional recovery of a novel knockin mouse model of dysferlinopathy by readthrough of nonsense mutation

Biallelic mutations in the dysferlin gene cause limb-girdle muscular dystrophy 2B or Miyoshi distal myopathy. We found that nonsense mutations are the most common mutation type among Korean patients with dysferlinopathy; more than half of the patients have at least one nonsense allele, which may be amenable to readthrough therapy. We generated a knockin mouse, dqx, harboring DYSF p.Q832∗ mutation. Homozygous dqx mice lacked dysferlin in skeletal muscle, while 2 weeks of oral ataluren restored dysferlin expression and ameliorated skeletal muscle pathology. Their physical performance improved, and protection against eccentric contractions was noted. The improvement was most evident in mice treated with oral ataluren of 0.9 mg/mL. These improvements were sustained for 8 weeks in ataluren-treated dqx mice, while the parameters of A/J mice treated with ataluren over the same period did not improve. These results support that readthrough therapy by oral ataluren may also be applicable to dysferlinopathy patients with nonsense mutation.

БИОХИМИЧЕСКИЕ И ПАТОФИЗИОЛОГИЧЕСКИЕ АСПЕКТЫ ДИСФЕРЛИН-АССОЦИИРОВАННЫХ МЫШЕЧНЫХ ДИСТРОФИЙ

Dysferlin is a transmembrane calcium-binding protein of the ferlin-1-like protein family, which includes myoferlin Fer1L3, otoferlin Fer1L2 and other ferlins Fer1L4, Fer1L5, and Fer1L6 along with dysferlin itself. Dysferlin is synthesized in all cells of the body, but most actively – in the symplasts of striated muscle tissue. The full range of functions performed by this protein is not fully clarified, but the following have been clearly shown: participation in the post-injury repair of sarcolemma and intracellular vesicular systems, generation and maintenance of the correct functioning of the sarcolemmal T-tubule system, regulation of endo- and exocytosis and inflammation, participation in phagocytosis. Mutations in the dysferlin gene lead to the development of a number of neuromuscular diseases of the autosomal recessive type of inheritance called dysferlinopathies: Miyoshi myopathy, limb-girdle muscular dystrophy type 2B, and distal anterior compartment myopathy. These diseases are characterized by the impaired expression of mRNA and/or the function of the protein dysferlin in skeletal muscles, which is caused by mutations in the DYSF (dystrophy-associated fer-1-like) gene. Although rare, dysferlinopathy is characterized by continuous progression causing severe disability, which explains the importance of research and development of appropriate treatments, including gene therapy. The article presents information on the structure of dysferlin, the mechanisms of its normal functions, as well as the biochemical basis of the pathogenesis of dysferlinopathies.

Calcium binds and rigidifies the dysferlin C2A domain in a tightly coupled manner.

The membrane protein dysferlin (DYSF) is important for calcium-activated plasma membrane repair, especially in muscle fibre cells. Nearly 600 mutations in the DYSF gene have been identified that are causative for rare genetic forms of muscular dystrophy. The dysferlin protein consists of seven C2 domains (C2A-C2G, 13%-33% identity) used to recruit calcium ions and traffic accessory proteins and vesicles to injured membrane sites needed to reseal a wound. Amongst these, the C2A is the most prominent facilitating the calcium-sensitive interaction with membrane surfaces. In this work, we determined the calcium-free and calcium-bound structures of the dysferlin C2A domain using NMR spectroscopy and X-ray crystallography. We show that binding two calcium ions to this domain reduces the flexibility of the Ca2+-binding loops in the structure. Furthermore, calcium titration and mutagenesis experiments reveal the tight coupling of these calcium-binding sites whereby the elimination of one site abolishes calcium binding to its partner site. We propose that the electrostatic potential distributed by the flexible, negatively charged calcium-binding loops in the dysferlin C2A domain control first contact with calcium that promotes subsequent binding. Based on these results, we hypothesize that dysferlin uses a 'calcium-catching' mechanism to respond to calcium influx during membrane repair.

Fer1L5, a Dysferlin Homologue Present in Vesicles and Involved in C2C12 Myoblast Fusion and Membrane Repair.

Simple SummaryFer1L5 is a dysferlin and myoferlin homologue and has been implicated in muscle membrane fusion events; myoblast fusion and membrane repair respectively during C2C12 skeletal muscle development. The role of Fer1L5 was analyzed by immunoblot analysis, biochemical fractionation, confocal microscopy and electroporation method. We demonstrated that Fer1L5 is present in low density vesicles and resistant to non-ionic detergent and shows overlapping properties with dysferlin and myoferlin. The expression of Fer1L5 was highly observed at the fusing myoblasts membranes and its expression level is gradually increase at the early stages multinucleated myotube formation. Fusion defects were observed in the Fer1L5 deficient C2C12 cells. Fer1L5 shows impaired membrane repair. Our data provide evidence that Fer1L5 is involved in aligning the adjacent myotubes close to each other for membrane—membrane fusion to increase the muscle mass for contraction during muscle development. Our data for Fer1L5 will be of great importance in the dysferlinopathy research in near future.Fer1L5 is a dysferlin and myoferlin related protein, which has been predicted to have a role in vesicle trafficking and muscle membrane fusion events. Mutations in dysferlin and otoferlin genes cause heredity diseases: muscular dystrophy and deafness in humans, respectively. Dysferlin is implicated in membrane repair. Myoferlin has a role in myogenesis. In this study, we investigated the role of the Fer1L5 protein during myoblast fusion and membrane repair. To study the functions of Fer1L5 we used confocal microscopy, biochemical fractionation, Western blot analysis and multiphoton laser wounding assay. By immunolabelling, Fer1L5 was detected in vesicular structures. By biochemical fractionation Fer1L5 was observed in low density vesicles. Our studies show that the membranes of Fer1L5 vesicles are non-resistant to non-ionic detergent. Partial co-staining of Fer1L5 with other two ferlin vesicles, respectively, was observed. Fer1L5 expression was highly detected at the fusion sites of two apposed C2C12 myoblast membranes and its expression level gradually increased at D2 and reached a maximum at day 4 before decreasing during further differentiation. Our studies showed that Fer1L5 has fusion defects during myoblast fusion and impaired membrane repair when the C2C12 cultures were incubated with inhibitory Fer1L5 antibodies. In C2C12 cells Fer1L5 vesicles are involved in two stages, the fusion of myoblasts and the formation of large myotubes. Fer1L5 also plays a role in membrane repair.