Next-generation sequencing identified a novel DYSF variant in a patient with limb-girdle muscular dystrophy type 2B: A case report.
ABSTRACT: RATIONALE:Limb-girdle muscular dystrophy (LGMD) is a genetic disease, which is characterized by muscle atrophy and weakness mainly involving proximal muscles. Accurate diagnosis of LGMD patient is very important for the appropriate management and long-term prognosis. PATIENT CONCERNS:An 18-year-old woman presented with progressive weakness of limbs, persistent elevated serum creatine kinase, myogenic damages in electromyography, and dysferlin protein deficiency in muscle biopsy. Further next-generation sequencing (NGS) revealed a compound heterozygous variant in dysferlin gene (DYSF), including a novel frameshift variant of c.4010delT. DIAGNOSIS:The patient was diagnosed with LGMD2B clinically and genetically. INTERVENTIONS:Oral levocarnitine and coenzyme Q10 were prescribed to the patient. OUTCOMES:After symptomatic treatments for 1 week, the patient's symptoms were not improved. LESSONS:NGS might be a helpful tool for the diagnosis of LGMD. A novel variant of c.4010delT in DYSF was identified in this case, which broadens the genetic spectrum of LGMD2B.
Project description:Limb girdle muscular dystrophy 2B (LGMD2B) is without treatment and caused by mutations in the dysferlin gene (DYSF). One-third is missense mutations leading to dysferlin aggregation and amyloid formation, in addition to defects in sarcolemmal repair and progressive muscle wasting. Dysferlin-null mouse models do not allow study of the consequences of missense mutations. We generated a new mouse model (MMex38) carrying a missense mutation in exon 38 in analogy to a clinically relevant human DYSF variant (DYSF p.Leu1341Pro). The targeted mutation induces all characteristics of missense mutant dysferlinopathy, including a progressive dystrophic pattern, amyloid formation, and defects in membrane repair. We chose U7 small nuclear RNA (snRNA)-based splice switching to demonstrate a possible exon-skipping strategy in this new animal model. We show that Dysf exons 37 and 38 can successfully be skipped in vivo. Overall, the MMex38 mouse model provides an ideal tool for preclinical development of treatment strategies for dysferlinopathy.
Project description:Dysferlin encoded by DYSF deficiency leads to two main phenotypes, limb girdle muscular dystrophy (LGMD) 2B and Miyoshi myopathy. To reveal in detail the mutational and clinical features of LGMD2B in Japan, we observed 40 Japanese patients in 36 families with LGMD2B in whom dysferlin mutations were confirmed.Three mutations (c.1566C>G, c.2997G>T and c.4497delT) were relatively more prevalent. The c.2997G>T mutation was associated with late onset, proximal dominant forms of dysferlinopathy, a high probability that muscle weakness started in an upper limb and lower serum creatine kinase (CK) levels. The clinical features of LGMD2B are as follows: (1) onset in the late teens or early adulthood, except patients homozygous for the c.2997G>T mutation; (2) lower limb weakness at onset; (3) distal change of lower limbs on muscle CT at an early stage; (4) impairment of lumbar erector spinal muscles on muscle CT at an early stage; (5) predominant involvement of proximal upper limbs; (6) preservation of function of the hands at late stage; (7) preservation of strength in neck muscles at late stage; (8) lack of facial weakness or dysphagia; (9) avoidance of scoliosis; (10) hyper-Ckaemia; (11) preservation of cardiac function; and (12) a tendency for respiratory function to decline with disease duration. It is important that the late onset phenotype is found with prevalent mutations.
Project description:Mutations of the DYSF gene leading to reduced dysferlin protein level causes limb girdle muscular dystrophy type 2B (LGMD2B). Dysferlin facilitates sarcolemmal membrane repair in healthy myofibers, thus its deficit compromises myofiber repair and leads to chronic muscle inflammation. An experimental therapeutic approach for LGMD2B is to protect damage or improve repair of myofiber sarcolemma. Here, we compared the effects of prednisolone and vamorolone (a dissociative steroid; VBP15) on dysferlin-deficient myofiber repair. Vamorolone, but not prednisolone, stabilized dysferlin-deficient muscle cell membrane and improved repair of dysferlin-deficient mouse (B6A/J) myofibers injured by focal sarcolemmal damage, eccentric contraction-induced injury or injury due to spontaneous in vivo activity. Vamorolone decreased sarcolemmal lipid mobility, increased muscle strength, and decreased late-stage myofiber loss due to adipogenic infiltration. In contrast, the conventional glucocorticoid prednisolone failed to stabilize dysferlin deficient muscle cell membrane or improve repair of dysferlinopathic patient myoblasts and mouse myofibers. Instead, prednisolone treatment increased muscle weakness and myofiber atrophy in B6A/J mice-findings that correlate with reports of prednisolone worsening symptoms of LGMD2B patients. Our findings showing improved cellular and pre-clinical efficacy of vamorolone compared to prednisolone and better safety profile of vamorolone indicates the suitability of vamorolone for clinical trials in LGMD2B.
Project description: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.
Project description:BACKGROUND:Limb-girdle muscular dystrophy (LGMD) is the most common adult-onset class of muscular dystrophies in India, but a majority of suspected LGMDs in India remain unclassified to the genetic subtype level. The next-generation sequencing (NGS)-based approaches have allowed molecular characterization and subtype diagnosis in a majority of these patients in India. MATERIALS AND METHODS:(I) To select probable dysferlinopathy (LGMD2B) cases from other LGMD subtypes using two screening methods (i) to determine the status of dysferlin protein expression in blood (peripheral blood mononuclear cell) by monocyte assay (ii) using a predictive algorithm called automated LGMD diagnostic assistant (ALDA) to obtain possible LGMD subtypes based on clinical symptoms. (II) Identification of gene pathogenic variants by NGS for 34 genes associated with LGMD or LGMD like muscular dystrophies, in cases showing: absence of dysferlin protein by the monocyte assay and/or a typical dysferlinopathy phenotype, with medium to high predictive scores using the ALDA tool. RESULTS:Out of the 125 patients screened by NGS, 96 were confirmed with two dysferlin variants, of which 84 were homozygous. Single dysferlin pathogenic variants were seen in 4 patients, whereas 25 showed no variants in the dysferlin gene. CONCLUSION:In this study, 98.2% of patients with absence of the dysferlin protein showed one or more variants in the dysferlin gene and hence has a high predictive significance in diagnosing dysferlinopathies. However, collection of blood samples from all over India for protein analysis is expensive. Our analysis shows that the use of the "ALDA tool" could be a cost-effective alternative method. Identification of dysferlin pathogenic variants by NGS is the ultimate method for diagnosing dysferlinopathies though follow-up with the monocyte assay can be useful to understand the phenotype in relation to the dysferlin protein expression and also be a useful biomarker for future clinical trials.
Project description:Limb-girdle muscular dystrophy type R2 dysferin-related (LGMD R2 dysferin-related), a phenotype of dysferlinopathy, usually begins with pelvic girdle weakness. A 35-year-old male presented with right leg pain for 2 weeks without a previous history of limb weakness. Magnetic resonance imaging of the lumbar spine showed disc extrusion at L5-S1 and incidental severe fatty degeneration of the lumbar erector spinae. Physical examination demonstrated no definite limb weakness. Serum creatine kinase levels were elevated. Genetic testing using a targeted gene-sequencing panel identified compound heterozygous variants NM_003494.3(DYSF) c.[1284+2T>C]; [5303G>A]. Computed tomography revealed fatty degeneration of lower-limb muscles, which was mild in the adductor muscles and severe in the gluteus minimus. Immunohistochemistry staining of the vastus lateralis showed under-expression of dysferlin. This patient was diagnosed with LGMD R2 dysferin-related. Thus, unusual fatty degeneration of the lumbar paraspinalis can be a manifestation of dysferlinopathy.
Project description:Mutations in dysferlin, the first protein linked with the cell membrane repair mechanism, causes a group of muscular dystrophies called dysferlinopathies. Dysferlin is a type two-anchored membrane protein, with a single C terminal trans-membrane helix, and most of the protein lying in cytoplasm. Dysferlin contains several C2 domains and two DysF domains which are nested one inside the other. Many pathogenic point mutations fall in the DysF domain region.We describe the crystal structure of the human dysferlin inner DysF domain with a resolution of 1.9 Ångstroms. Most of the pathogenic mutations are part of aromatic/arginine stacks that hold the domain in a folded conformation. The high resolution of the structure show that these interactions are a mixture of parallel ring/guanadinium stacking, perpendicular H bond stacking and aliphatic chain packing.The high resolution structure of the Dysferlin DysF domain gives a template on which to interpret in detail the pathogenic mutations that lead to disease.
Project description:Dysferlinopathy is a progressive myopathy caused by mutations in the dysferlin (DYSF) gene. Dysferlin protein plays a major role in plasma-membrane resealing. Some patients with DYSF deletion mutations exhibit mild symptoms, suggesting some regions of DYSF can be removed without significantly impacting protein function. Antisense-mediated exon-skipping therapy uses synthetic molecules called antisense oligonucleotides to modulate splicing, allowing exons harboring or near genetic mutations to be removed and the open reading frame corrected. Previous studies have focused on DYSF exon 32 skipping as a potential therapeutic approach, based on the association of a mild phenotype with the in-frame deletion of exon 32. To date, no other DYSF exon-skipping targets have been identified, and the relationship between DYSF exon deletion pattern and protein function remains largely uncharacterized. In this study, we utilized a membrane-wounding assay to evaluate the ability of plasmid constructs carrying mutant DYSF, as well as antisense oligonucleotides, to rescue membrane resealing in patient cells. We report that multi-exon skipping of DYSF exons 26-27 and 28-29 rescues plasma-membrane resealing. Successful translation of these findings into the development of clinical antisense drugs would establish new therapeutic approaches that would be applicable to ?5%-7% (exons 26-27 skipping) and ?8% (exons 28-29 skipping) of dysferlinopathy patients worldwide.
Project description:<h4>Background</h4>Mouse models of dysferlinopathies are valuable tools with which to investigate the pathomechanisms underlying these diseases and to test novel therapeutic strategies. One such mouse model is the Dysf (tm1Kcam) strain, which was generated using a targeting vector to replace a 12-kb region of the dysferlin gene and which features a progressive muscular dystrophy. A prerequisite for successful animal studies using genetic mouse models is an accurate genotyping protocol. Unfortunately, the lack of robustness of currently available genotyping protocols for the Dysf (tm1Kcam) mouse has prevented efficient colony management. Initial attempts to improve the genotyping protocol based on the published genomic structure failed. These difficulties led us to analyze the targeted locus of the dysferlin gene of the Dysf (tm1Kcam) mouse in greater detail.<h4>Methods</h4>In this study we resequenced and analyzed the targeted locus of the Dysf (tm1Kcam) mouse and developed a novel PCR protocol for genotyping.<h4>Results</h4>We found that instead of a deletion, the dysferlin locus in the Dysf (tm1Kcam) mouse carries a targeted insertion. This genetic characterization enabled us to establish a reliable method for genotyping of the Dysf (tm1Kcam) mouse, and thus has made efficient colony management possible.<h4>Conclusion</h4>Our work will make the Dysf (tm1Kcam) mouse model more attractive for animal studies of dysferlinopathies.
Project description:Dysferlin (DYSF) is a type II transmembrane protein implicated in surface membrane repair of muscle. Mutations in dysferlin lead to Limb Girdle Muscular Dystrophy 2B (LGMD2B), Miyoshi Myopathy (MM), and Distal Myopathy with Anterior Tibialis onset (DMAT). The DYSF protein complex is not well understood, and only a few protein-binding partners have been identified thus far. To increase the set of interacting protein partners for DYSF we recovered a list of predicted interacting protein through a systems biology approach. The predictions are part of a "reverse-engineered" genome-wide human gene regulatory network obtained from experimental data by computational analysis. The reverse-engineering algorithm behind the analysis relates genes to each other based on changes in their expression patterns. DYSF and AHNAK were used to query the system and extract lists of potential interacting proteins. Among the 32 predictions the two genes share, we validated the physical interaction between DYSF protein with moesin (MSN) and polymerase I and transcript release factor (PTRF) in mouse heart lysate, thus identifying two novel Dysferlin-interacting proteins. Our strategy could be useful to clarify Dysferlin function in intracellular vesicles and its implication in muscle membrane resealing.