Performance of upper limb entry item to predict forced vital capacity in dysferlin-deficient limb girdle muscular dystrophy.

Dysferlin-deficient limb girdle muscular dystrophy (LGMD R2), also referred to as dysferlinopathy, can be associated with respiratory muscle weakness as the disease progresses. Clinical practice guidelines recommend biennial lung function assessments in patients with dysferlinopathy to screen for respiratory impairment. However, lack of universal access to spirometry equipment and trained specialists makes regular monitoring challenging. This study investigated the use of the Performance of Upper Limb (PUL) clinical scale entry item as a low-cost screening tool to identify patients with dysferlinopathy at risk of respiratory impairment. Using data from 193 patients from the Jain Foundation's International Clinical Outcomes Study, modelling identified a significant positive relationship between the PUL entry item and forced vital capacity (FVC). Eighty-eight percent of patients with the lowest PUL entry item score of 1 presented with FVC % predicted values of <60 %, suggestive of respiratory impairment. By contrast, only 10 % of the remainder of the cohort (PUL entry item of 2 or more) had an FVC of <60 %. This relationship also held true when accounting for ambulatory status, age, and sex as possible confounding factors. In summary, our results suggest that the PUL entry item could be implemented in clinical practice to screen for respiratory impairment where spirometry is not readily available.

Brain of miyoshi myopathy/dysferlinopathy patients presents with structural and metabolic anomalies.

Miyoshi myopathy/dysferlinopathy (MMD) is a rare muscle disease caused by DYSF gene mutations. Apart from skeletal muscles, DYSF is also expressed in the brain. However, the impact of MMD-causing DYSF variants on brain structure and function remains unexplored. To investigate this, we utilized magnetic resonance (MR) modalities (MR volumetry and 31P MR spectroscopy) in a family with seven children, four of whom have the illness. The MMD siblings showed distinct differences from healthy controls: (1) a significant (p < 0.001) right-sided volume asymmetry (+ 232 mm3) of the inferior lateral ventricles; and (2) a significant (p < 0.001) decrease in [Mg2+], along with a modified energy metabolism profile and altered membrane turnover in the hippocampus and motor and premotor cortices. The patients' [Mg2+], energy metabolism, and membrane turnover measures returned to those of healthy relatives after a month of 400 mg/day magnesium supplementation. This work is the first to describe anatomical and functional abnormalities characteristic of neurodegeneration in the MMD brain. Therefore, we call for further examination of brain functions in larger cohorts of MMD patients and testing of magnesium supplementation, which has proven to be an effective corrective approach in our study.

Apolipoprotein E knockout, but not cholesteryl ester transfer protein (CETP)-associated high-density lipoprotein cholesterol (HDL-C) lowering, exacerbates muscle wasting in dysferlin-null mice.

BACKGROUND: Dysferlin-deficient limb-girdle muscular dystrophy type 2B (Dysf) mice are notorious for their mild phenotype. Raising plasma total cholesterol (CHOL) via apolipoprotein E (ApoE) knockout (KO) drastically exacerbates muscle wasting in Dysf mice. However, dysferlinopathic patients have abnormally reduced plasma high-density lipoprotein cholesterol (HDL-C) levels. The current study aimed to determine whether HDL-C lowering can exacerbate the mild phenotype of dysferlin-null mice. METHODS: Human cholesteryl ester transfer protein (CETP), a plasma lipid transfer protein not found in mice that reduces HDL-C, and/or its optimal adapter protein human apolipoprotein B (ApoB), were overexpressed in Dysf mice. Mice received a 2% cholesterol diet from 2 months of age and characterized through ambulatory and hanging functional tests, plasma analyses, and muscle histology. RESULTS: CETP/ApoB expression in Dysf mice caused reduced HDL-C (54.5%) and elevated ratio of CHOL/HDL-C (181.3%) compared to control Dysf mice in plasma, but without raising CHOL. Compared to the severe muscle pathology found in high CHOL Dysf/ApoE double knockout mice, Dysf/CETP/ApoB mice did not show significant changes in ambulation, hanging capacity, increases in damaged area, collagen deposition, or decreases in cross-sectional area and healthy myofibre coverage. CONCLUSIONS: CETP/ApoB over-expression in Dysf mice decreases HDL-C without increasing CHOL or exacerbating muscle pathology. High CHOL or nonHDL-C caused by ApoE KO, rather than low HDL-C, likely lead to rodent muscular dystrophy phenotype humanization.

Diagnosis of dysferlinopathy masked by a superimposed hypothyroid myopathy.

We report a woman in her 30s with dysferlinopathy whose diagnosis was masked by superimposed hypothyroidism. Laboratory studies revealed Hashimoto's thyroiditis and markedly raised serum creatine kinase (CK of 6255 U/L; reference range 0-170 U/L). Electromyography, nerve conduction studies and MRI of the hip and thigh were consistent with a diagnosis of hypothyroid myopathy, but thyroxine failed to resolve her clinical presentation or normalise the CK level. Immunohistochemical (IHC) staining of right vastus lateralis muscle biopsy revealed the selective absence of dysferlin leading to a diagnosis of limb-girdle muscular dystrophy type IIB. Dysferlinopathy is a challenging diagnosis due to a varied clinical picture and low incidence. Misdiagnosis is common even in uncomplicated presentations, and this case outlines the need for routine inclusion of IHC and a low threshold for genetic testing, in the workup of complex myopathy.

Pilot investigations into the mechanistic basis for adverse effects of glucocorticoids in dysferlinopathy.

BACKGROUND: Dysferlinopathies are a clinically heterogeneous group of muscular dystrophies caused by gene mutations resulting in deficiency of the membrane-associated protein dysferlin. They manifest post-growth and are characterised by muscle wasting (primarily in the limb and limb-gridle muscles), inflammation, and replacement of myofibres with adipose tissue. The precise pathomechanism for dysferlinopathy is currently unclear; as such there are no treatments currently available. Glucocorticoids (GCs) are widely used to reduce inflammation and treat muscular dystrophies, but when administered to patients with dysferlinopathy, they have unexpected adverse effects, with accelerated loss of muscle strength. METHODS: To investigate the mechanistic basis for the adverse effects of GCs in dysferlinopathy, the potent GC dexamethasone (Dex) was administered for 4-5 weeks (0.5-0.75 µg/mL in drinking water) to dysferlin-deficient BLA/J and normal wild-type (WT) male mice, sampled at 5 (Study 1) or 10 months (Study 2) of age. A wide range of analyses were conducted. Metabolism- and immune-related gene expression was assessed in psoas muscles at both ages and in quadriceps at 10 months of age. For the 10-month-old mice, quadriceps and psoas muscle histology was assessed. Additionally, we investigated the impact of Dex on the predominantly slow and fast-twitch soleus and extensor digitorum longus (EDL) muscles (respectively) in terms of contractile function, myofibre-type composition, and levels of proteins related to contractile function and metabolism, plus glycogen. RESULTS: At both ages, many complement-related genes were highly expressed in BLA/J muscles, and WT mice were generally more responsive to Dex than BLA/J. The effects of Dex on BLA/J mice included (i) increased expression of inflammasome-related genes in muscles (at 5 months) and (ii) exacerbated histopathology of quadriceps and psoas muscles at 10 months. A novel observation was pronounced staining for glycogen in many myofibres of the damaged quadriceps muscles, with large pale vacuolated myofibres, suggesting possible myofibre death by oncosis. CONCLUSION: These pilot studies provide a new focus for further investigation into the adverse effects of GCs on dysferlinopathic muscles.

Dysferlin Enables Tubular Membrane Proliferation in Cardiac Hypertrophy.

BACKGROUND: Cardiac hypertrophy compensates for increased biomechanical stress of the heart induced by prevalent cardiovascular pathologies but can result in heart failure if left untreated. Here, we hypothesized that the membrane fusion and repair protein dysferlin is critical for the integrity of the transverse-axial tubule (TAT) network inside cardiomyocytes and contributes to the proliferation of TAT endomembranes during pressure overload-induced cardiac hypertrophy. METHODS: Stimulated emission depletion and electron microscopy were used to localize dysferlin in mouse and human cardiomyocytes. Data-independent acquisition mass spectrometry revealed the cardiac dysferlin interactome and proteomic changes of the heart in dysferlin-knockout mice. After transverse aortic constriction, we compared the hypertrophic response of wild-type versus dysferlin-knockout hearts and studied TAT network remodeling mechanisms inside cardiomyocytes by live-cell membrane imaging. RESULTS: We localized dysferlin in a vesicular compartment in nanometric proximity to contact sites of the TAT network with the sarcoplasmic reticulum, a.k.a. junctional complexes for Ca2+-induced Ca2+ release. Interactome analyses demonstrated a novel protein interaction of dysferlin with the membrane-tethering sarcoplasmic reticulum protein juncophilin-2, a putative interactor of L-type Ca2+ channels and ryanodine receptor Ca2+ release channels in junctional complexes. Although the dysferlin-knockout caused a mild progressive phenotype of dilated cardiomyopathy, global proteome analysis revealed changes preceding systolic failure. Following transverse aortic constriction, dysferlin protein expression was significantly increased in hypertrophied wild-type myocardium, while dysferlin-knockout animals presented markedly reduced left-ventricular hypertrophy. Live-cell membrane imaging showed a profound reorganization of the TAT network in wild-type left-ventricular myocytes after transverse aortic constriction with robust proliferation of axial tubules, which critically depended on the increased expression of dysferlin within newly emerging tubule components. CONCLUSIONS: Dysferlin represents a new molecular target in cardiac disease that protects the integrity of tubule-sarcoplasmic reticulum junctional complexes for regulated excitation-contraction coupling and controls TAT network reorganization and tubular membrane proliferation in cardiomyocyte hypertrophy induced by pressure overload.

Bioengineered Model of Human LGMD2B Skeletal Muscle Reveals Roles of Intracellular Calcium Overload in Contractile and Metabolic Dysfunction in Dysferlinopathy.

Dysferlin is a multi-functional protein that regulates membrane resealing, calcium homeostasis, and lipid metabolism in skeletal muscle. Genetic loss of dysferlin results in limb girdle muscular dystrophy 2B/2R (LGMD2B/2R) and other dysferlinopathies - rare untreatable muscle diseases that lead to permanent loss of ambulation in humans. The mild disease severity in dysferlin-deficient mice and diverse genotype-phenotype relationships in LGMD2B patients have prompted the development of new in vitro models for personalized studies of dysferlinopathy. Here the first 3-D tissue-engineered hiPSC-derived skeletal muscle ("myobundle") model of LGMD2B is described that exhibits compromised contractile function, calcium-handling, and membrane repair, and transcriptomic changes indicative of impaired oxidative metabolism and mitochondrial dysfunction. In response to the fatty acid (FA) challenge, LGMD2B myobundles display mitochondrial deficits and intracellular lipid droplet (LD) accumulation. Treatment with the ryanodine receptor (RyR) inhibitor dantrolene or the dissociative glucocorticoid vamorolone restores LGMD2B contractility, improves membrane repair, and reduces LD accumulation. Lastly, it is demonstrated that chemically induced chronic RyR leak in healthy myobundles phenocopies LGMD2B contractile and metabolic deficit, but not the loss of membrane repair capacity. Together, these results implicate intramyocellular Ca2+ leak as a critical driver of dysferlinopathic phenotype and validate the myobundle system as a platform to study LGMD2B pathogenesis.

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.

The extracellular matrix differentially directs myoblast motility and differentiation in distinct forms of muscular dystrophy: Dystrophic matrices alter myoblast motility.

Extracellular matrix (ECM) pathologic remodeling underlies many disorders, including muscular dystrophy. Tissue decellularization removes cellular components while leaving behind ECM components. We generated "on-slide" decellularized tissue slices from genetically distinct dystrophic mouse models. The ECM of dystrophin- and sarcoglycan-deficient muscles had marked thrombospondin 4 deposition, while dysferlin-deficient muscle had excess decorin. Annexins A2 and A6 were present on all dystrophic decellularized ECMs, but annexin matrix deposition was excessive in dysferlin-deficient muscular dystrophy. Muscle-directed viral expression of annexin A6 resulted in annexin A6 in the ECM. C2C12 myoblasts seeded onto decellularized matrices displayed differential myoblast mobility and fusion. Dystrophin-deficient decellularized matrices inhibited myoblast mobility, while dysferlin-deficient decellularized matrices enhanced myoblast movement and differentiation. Myoblasts treated with recombinant annexin A6 increased mobility and fusion like that seen on dysferlin-deficient decellularized matrix and demonstrated upregulation of ECM and muscle cell differentiation genes. These findings demonstrate specific fibrotic signatures elicit effects on myoblast activity.

On the role of dysferlin in striated muscle: membrane repair, t-tubules and Ca2+ handling.

Dysferlin is a 237 kDa membrane-associated protein characterised by multiple C2 domains with a diverse role in skeletal and cardiac muscle physiology. Mutations in DYSF are known to cause various types of human muscular dystrophies, known collectively as dysferlinopathies, with some patients developing cardiomyopathy. A myriad of in vitro membrane repair studies suggest that dysferlin plays an integral role in the membrane repair complex in skeletal muscle. In comparison, less is known about dysferlin in the heart, but mounting evidence suggests that dysferlin's role is similar in both muscle types. Recent findings have shown that dysferlin regulates Ca2+ handling in striated muscle via multiple mechanisms and that this becomes more important in conditions of stress. Maintenance of the transverse (t)-tubule network and the tight coordination of excitation-contraction coupling are essential for muscle contractility. Dysferlin regulates the maintenance and repair of t-tubules, and it is suspected that dysferlin regulates t-tubules and sarcolemmal repair through a similar mechanism. This review focuses on the emerging complexity of dysferlin's activity in striated muscle. Such insights will progress our understanding of the proteins and pathways that regulate basic heart and skeletal muscle function and help guide research into striated muscle pathology, especially that which arises due to dysferlin dysfunction.

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

BACKGROUND: Limb 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. METHODS: We 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. RESULTS: By WES, we identified a novel homozygous missense variant in DYSF (NM_003494.4: c.5876T > C: p. Leu1959Pro) previously been associated with LGMD phenotypes. CONCLUSIONS: The identification and validation of new pathogenic DYSF variant in the present study further highlight the importance of this gene in LGMD.

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

Dysferlin protein deficiency can cause neuromuscular dysfunction, resulting in autosomal recessive dysferlinopathy, which is caused by DYSF gene mutation. Dysferlin proteins belongs to the Ferlin1-like protein family and are associated with muscle membrane repair and regeneration. In China, pathogenic mutations of the protein often result in two clinical phenotypes of Miyoshi muscular or limb band muscular dystrophy type 2B. It is clinically characterized by progressive muscle weakness and elevated serum creatine kinase. The data of the child were collected, blood samples of the child and his family members were collected, and whole exome sequencing (WES) was performed. The recombinant expression vector was constructed, the function of the mutation was verified by minigene, and the pathogenicity of the mutation was further analyzed by combining with biological information analysis. The patient initially presented with asymptomatic elevation of serum creatine kinase（CK）. Then progressive lower limb weakness, mainly distal limb weakness. Large amounts of scattered necrosis, myogenic lesions, and complete deletion of dysferlin protein were observed under muscle biopsy, which further improved genetic detection. Whole exome sequencing showed compound mutations (c.1397 + 1_1397 + 3del and c.1375dup p.M459Nfs*15) in DYSF gene. c.1375dup p.M459Nfs*15 have been reported. The other mutation is the deletion of c.1397 + 1_1397 + 3 in Intron15, which is an intron mutation that may affect splicing and the pathogenesis is still unknown. Minigene splicing assay verified that c.1397 + 1_1397 + 3del resulted in exon15 skipping and produced a premature termination codon. We report a novel pathogenic mutation in DYSF gene with Miyoshi myopathy and demonstrate this variant causes skipping of exon15 by minigene splicing assay. We point out the need of conducting functional analysis to verify the pathogenicity of intronic mutation. The finding enriches the mutation spectrum of DYSF gene and laid a foundation for future studies on the correlation between genotype and phenotype.

Magnetic resonance imaging-based criteria to differentiate dysferlinopathy from other genetic muscle diseases.

The identification of disease-characteristic patterns of muscle fatty replacement in magnetic resonance imaging (MRI) is helpful for diagnosing neuromuscular diseases. In the Clinical Outcome Study of Dysferlinopathy, eight diagnostic rules were described based on MRI findings. Our aim is to confirm that they are useful to differentiate dysferlinopathy (DYSF) from other genetic muscle diseases (GMD). The rules were applied to 182 MRIs of dysferlinopathy patients and 1000 MRIs of patients with 10 other GMD. We calculated sensitivity (S), specificity (Sp), positive and negative predictive values (PPV/NPV) and accuracy (Ac) for each rule. Five of the rules were more frequently met by the DYSF group. Patterns observed in patients with FKRP, ANO5 and CAPN3 myopathies were similar to the DYSF pattern, whereas patterns observed in patients with OPMD, laminopathy and dystrophinopathy were clearly different. We built a model using the five criteria more frequently met by DYSF patients that obtained a S 95.9%, Sp 46.1%, Ac 66.8%, PPV 56% and NPV 94% to distinguish dysferlinopathies from other diseases. Our findings support the use of MRI in the diagnosis of dysferlinopathy, but also identify the need to externally validate "disease-specific" MRI-based diagnostic criteria using MRIs of other GMD patients.

Unexpected extra exon skipping in the DYSF gene during restoring the reading frame by CRISPR/Cas9.

The DYSF gene encoding dysferlin protein is one of the largest and has many transcripts. Pathogenic variants in the gene can lead to various types of myopathies, which makes it a good object for studying the events occurring in it during genome editing by the CRISPR/Cas method. In this study, we evaluated the possibility of permanent skipping of exons 3-4, and 26-27 which deletion does not violate the reading frame and allows to eliminate truncated variants within exons. Editing was performed with simultaneous transfection of two sgRNA- and sa/spCas9-containing plasmids on HEK293T cell cultures and healthy donor myoblasts. Skipping of exons 3-4 was performed by destroying the splicing acceptor sites, and exons 26-27 by cuts in the flanking exons with the corresponding deletion in the DNA. Some unexpected results were obtained, when exons 26-27 were skipped, exon 30 was also absent in the transcript, although it is not alternatively spliced and is normally present in all transcripts. This event indicates that DNA changes near splicing sites can affect adjacent exons and the whole gene. However, this fact requires further study.

Novel five nucleotide deletion in dysferlin leads to autosomal recessive limb-girdle muscular dystrophy.

Muscular dystrophy (MD) is a genetic disorder that causes progressive muscle weakness and degeneration. Limb-girdle muscular dystrophy (LGMD) is a type of MD that mainly causes muscle atrophy within the shoulder and pelvic girdles. LGMD is classified into autosomal dominant (LGMD-D) and autosomal recessive (LGMD-R) inheritance patterns. Mutations in the Dysferlin gene (DYSF) are common causes of LGMD-R. However, genetic screening of DYSF mutations is rare in Taiwan. Herein, we identified a novel c.2867_2871del ACCAG deletion and a previously reported c.937+1G>A mutation in DYSF from a Taiwanese family with LGMD. The primary symptoms of both siblings were difficulty climbing stairs, walking on the toes, and gradually worsening weakness in the proximal muscles and increased creatine kinase level. Through pedigree analysis and sequencing, two siblings from this family were found to have compound heterozygous DYSF mutations (c. 937+1G>A and c. 2867_2871del ACCAG) within the separated alleles. These mutations induced early stop codons; if translated, truncated DYSF proteins will be expressed. Or, the mRNA products of these two mutations will merit the nonsense-mediated decay, might result in no dysferlin protein expressed. To our knowledge, this is the first report of a novel c.2867_2871del ACCAG deletion in DYSF. Further research is required to examine the effects of the novel DYSF mutation in Taiwanese patients with LGMD.

Dual Adeno-Associated Virus 9 with Codon-Optimized DYSF Gene Promotes In Vivo Muscle Regeneration and May Decrease Inflammatory Response in Limb Girdle Muscular Dystrophy Type R2.

Dysferlinopathy treatment is an active area of investigation. Gene therapy is one potential approach. We studied muscle regeneration and inflammatory response after injection of an AAV-9 with a codon-optimized DYSF gene. A dual-vector system AAV.DYSF.OVERLAP with overlapping DYSF cDNA sequences was generated. Two AAV vectors were separately assembled by a standard triple-transfection protocol from plasmids carrying parts of the DYSF gene. Artificial myoblasts from dysferlin-deficient fibroblasts were obtained by MyoD overexpression. RT-PCR and Western blot were used for RNA and protein detection in vitro. A dysferlinopathy murine model (Bla/J) was used for in vivo studies. Histological assay, morphometry, and IHC were used for the muscle tissue analysis. Dysferlin was detected in vitro and in vivo at subphysiological levels. RT-PCR and Western Blot detected dysferlin mRNA and protein in AAV.DYSF.OVERLAP-transduced cells, and mRNA reached a 7-fold elevated level compared to the reference gene (GAPDH). In vivo, the experimental group showed intermediate median values for the proportion of necrotic muscle fibers, muscle fibers with internalized nuclei, and cross-sectional area of muscle fibers compared to the same parameters in the control groups of WT and Bla/J mice, although the differences were not statistically significant. The inverse relationship between the dosage and the severity of inflammatory changes in the muscles may be attributed to the decrease in the number of necrotic fibers. The share of transduced myofibers reached almost 35% in the group with the highest dose. The use of two-vector systems based on AAV is justified in terms of therapeutic efficacy. The expression of dysferlin at a subphysiological level, within a short observation period, is capable of inducing the restoration of muscle tissue structure, reducing inflammatory activity, and mitigating necrotic processes. Further research is needed to provide a more detailed assessment of the impact of the transgene and viral vector on the inflammatory component, including longer observation periods.

Dysferlinopathy in Tunisia: clinical spectrum, genetic background and prognostic profile.

Dysferlinopathy is a rare group of hereditary muscular dystrophy with an autosomal recessive mode of inheritance caused by a mutation in the DYSF gene. It encodes for the dysferlin protein, which has a crucial role in multiple cellular processes, including muscle fiber membrane repair. This deficit has heterogeneous clinical presentations. In this study, we collected 20 Tunisian patients with a sex ratio of 1 and a median age of 50.5 years old (Interquartile range (IQR) = [36,5-54,75]). They were followed for periods ranging from 5 to 48 years. The median age at onset was 17 years old (IQR = [16,8-28,4]). Five major phenotypes were identified: Limb-girdle muscular dystrophy (LGMDR2) (35%), a proximodistal phenotype (35%), Miyoshi myopathy (10%),  Distal myopathy with anterior tibial onset (DMAT) (10%), and asymptomatic HyperCKemia (10%). At the last evaluation, more than half of patients (55%) were on wheelchair. Loss of ambulation occurred generally during the fourth decade. After 20 years of disease progression, two patients with a proximodistal phenotype (10%) developed dilated cardiomyopathy and mitral valve regurgitation. Restrictive respiratory syndrome was observed in three patients (DMAT: 1 patient, proximodistal phenotype: 1 patient, LGMDR2: 1 patient). Genetic study disclosed five mutations. We observed clinical heterogeneity between families and even within the same family. Disease progression was mainly slow to intermediate regardless of the phenotype.

The Dysferlin C2A Domain Binds PI(4,5)P2 and Penetrates Membranes.

Dysferlin is a large membrane protein found most prominently in striated muscle. Loss of dysferlin activity is associated with reduced exocytosis, abnormal intracellular Ca2+ and the muscle diseases limb-girdle muscular dystrophy and Miyoshi myopathy. The cytosolic region of dysferlin consists of seven C2 domains with mutations in the C2A domain at the N-terminus resulting in pathology. Despite the importance of Ca2+ and membrane binding activities of the C2A domain for dysferlin function, the mechanism of the domain remains poorly characterized. In this study we find that the C2A domain preferentially binds membranes containing PI(4,5)P2 through an interaction mediated by residues Y23, K32, K33, and R77 on the concave face of the domain. We also found that subsequent to membrane binding, the C2A domain inserts residues on the Ca2+ binding loops into the membrane. Analysis of solution NMR measurements indicate that the domain inhabits two distinct structural states, with Ca2+ shifting the population between states towards a more rigid structure with greater affinity for PI(4,5)P2. Based on our results, we propose a mechanism where Ca2+ converts C2A from a structurally dynamic, low PI(4,5)P2 affinity state to a high affinity state that targets dysferlin to PI(4,5)P2 enriched membranes through interaction with Tyr23, K32, K33, and R77. Binding also involves changes in lipid packing and insertion by the third Ca2+ binding loop of the C2 domain into the membrane, which would contribute to dysferlin function in exocytosis and Ca2+ regulation.

Tetraspanin CD82 Associates with Trafficking Vesicle in Muscle Cells and Binds to Dysferlin and Myoferlin.

Tetraspanins organize protein complexes at the cell membrane and are responsible for assembling diverse binding partners in changing cellular states. Tetraspanin CD82 is a useful cell surface marker for prospective isolation of human myogenic progenitors and its expression is decreased in Duchenne muscular dystrophy (DMD) cell lines. The function of CD82 in skeletal muscle remains elusive, partly because the binding partners of this tetraspanin in muscle cells have not been identified. CD82-associated proteins are sought to be identified in human myotubes via mass spectrometry proteomics, which identifies dysferlin and myoferlin as CD82-binding partners. In human dysferlinopathy (Limb girdle muscular dystrophy R2, LGMDR2) myogenic cell lines, expression of CD82 protein is near absent in two of four patient samples. In the cell lines where CD82 protein levels are unaffected, increased expression of the ≈72 kDa mini-dysferlin product is identified using an antibody recognizing the dysferlin C-terminus. These data demonstrate that CD82 binds dysferlin/myoferlin in differentiating muscle cells and its expression can be affected by loss of dysferlin in human myogenic cells.