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1.
Circ Res ; 135(5): 554-574, 2024 Aug 16.
Article in English | MEDLINE | ID: mdl-39011635

ABSTRACT

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.


Subject(s)
Cardiomegaly , Dysferlin , Mice, Knockout , Myocytes, Cardiac , Sarcoplasmic Reticulum , Animals , Dysferlin/metabolism , Dysferlin/genetics , Myocytes, Cardiac/metabolism , Myocytes, Cardiac/pathology , Cardiomegaly/metabolism , Cardiomegaly/pathology , Cardiomegaly/genetics , Cardiomegaly/physiopathology , Humans , Mice , Sarcoplasmic Reticulum/metabolism , Sarcoplasmic Reticulum/pathology , Mice, Inbred C57BL , Male , Membrane Proteins/metabolism , Membrane Proteins/genetics , Cell Proliferation , Cells, Cultured , Muscle Proteins/metabolism , Muscle Proteins/genetics , Myosin-Light-Chain Kinase
2.
J Physiol ; 602(9): 1893-1910, 2024 May.
Article in English | MEDLINE | ID: mdl-38615232

ABSTRACT

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.


Subject(s)
Calcium , Dysferlin , Humans , Calcium/metabolism , Dysferlin/metabolism , Membrane Proteins/metabolism , Membrane Proteins/genetics , Membrane Proteins/physiology , Muscle Proteins/metabolism , Muscle Proteins/genetics , Muscle Proteins/physiology , Muscle, Skeletal/metabolism , Muscle, Skeletal/physiology , Muscle, Striated/metabolism , Muscle, Striated/physiology
3.
Lipids Health Dis ; 23(1): 247, 2024 Aug 13.
Article in English | MEDLINE | ID: mdl-39138561

ABSTRACT

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.


Subject(s)
Apolipoproteins E , Cholesterol Ester Transfer Proteins , Cholesterol, HDL , Dysferlin , Mice, Knockout , Muscular Dystrophies, Limb-Girdle , Animals , Humans , Male , Mice , Apolipoproteins B/blood , Apolipoproteins B/genetics , Apolipoproteins E/genetics , Apolipoproteins E/deficiency , Cholesterol Ester Transfer Proteins/genetics , Cholesterol Ester Transfer Proteins/deficiency , Cholesterol, HDL/blood , Disease Models, Animal , Dysferlin/genetics , Dysferlin/deficiency , Muscle, Skeletal/metabolism , Muscle, Skeletal/pathology , Muscular Atrophy/genetics , Muscular Atrophy/pathology , Muscular Atrophy/metabolism , Muscular Dystrophies, Limb-Girdle/genetics , Muscular Dystrophies, Limb-Girdle/pathology
4.
BMC Musculoskelet Disord ; 25(1): 241, 2024 Mar 27.
Article in English | MEDLINE | ID: mdl-38539162

ABSTRACT

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.


Subject(s)
Muscular Dystrophies, Limb-Girdle , Adult , Humans , Young Adult , Dysferlin/genetics , Muscular Dystrophies, Limb-Girdle/genetics , Mutation , Mutation, Missense , Phenotype
5.
Int J Mol Sci ; 25(11)2024 May 21.
Article in English | MEDLINE | ID: mdl-38891760

ABSTRACT

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.


Subject(s)
Dysferlin , Genetic Therapy , Muscular Dystrophies, Limb-Girdle , Mutation , Humans , Muscular Dystrophies, Limb-Girdle/therapy , Muscular Dystrophies, Limb-Girdle/genetics , Muscular Dystrophies, Limb-Girdle/diagnosis , Dysferlin/genetics , Dysferlin/metabolism , Genetic Therapy/methods , Oligonucleotides, Antisense/therapeutic use , Animals
6.
Int J Mol Sci ; 24(17)2023 Aug 31.
Article in English | MEDLINE | ID: mdl-37686363

ABSTRACT

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.


Subject(s)
Dependovirus , Muscular Dystrophies, Limb-Girdle , Animals , Mice , Dependovirus/genetics , Dysferlin/genetics , Muscular Dystrophies, Limb-Girdle/genetics , Muscular Dystrophies, Limb-Girdle/therapy , Codon , Muscle Fibers, Skeletal , RNA, Messenger
7.
Int J Mol Sci ; 24(5)2023 Feb 28.
Article in English | MEDLINE | ID: mdl-36902136

ABSTRACT

Quantitative surface plasmon resonance (SPR) was utilized to determine binding strength and calcium dependence of direct interactions between dysferlin and proteins likely to mediate skeletal muscle repair, interrupted in limb girdle muscular dystrophy type 2B/R2. Dysferlin canonical C2A (cC2A) and C2F/G domains directly interacted with annexin A1, calpain-3, caveolin-3, affixin, AHNAK1, syntaxin-4, and mitsugumin-53, with cC2A the primary target and C2F lesser involved, overall demonstrating positive calcium dependence. Dysferlin C2 pairings alone showed negative calcium dependence in almost all cases. Like otoferlin, dysferlin directly interacted via its carboxy terminus with FKBP8, an anti-apoptotic outer mitochondrial membrane protein, and via its C2DE domain with apoptosis-linked gene (ALG-2/PDCD6), linking anti-apoptosis with apoptosis. Confocal Z-stack immunofluorescence confirmed co-compartmentalization of PDCD6 and FKBP8 at the sarcolemmal membrane. Our evidence supports the hypothesis that prior to injury, dysferlin C2 domains self-interact and give rise to a folded, compact structure as indicated for otoferlin. With elevation of intracellular Ca2+ in injury, dysferlin would unfold and expose the cC2A domain for interaction with annexin A1, calpain-3, mitsugumin 53, affixin, and caveolin-3, and dysferlin would realign from its interactions with PDCD6 at basal calcium levels to interact strongly with FKBP8, an intramolecular rearrangement facilitating membrane repair.


Subject(s)
Apoptosis Regulatory Proteins , Calcium-Binding Proteins , Calcium , Dysferlin , Muscle, Skeletal , Regeneration , Tacrolimus Binding Proteins , Annexin A1/metabolism , Calcium/metabolism , Calpain/metabolism , Caveolin 3/metabolism , Dysferlin/metabolism , Muscle, Skeletal/injuries , Muscle, Skeletal/physiology , Sarcolemma/metabolism , Calcium-Binding Proteins/metabolism , Apoptosis Regulatory Proteins/metabolism , Surface Plasmon Resonance , Tacrolimus Binding Proteins/metabolism , Apoptosis , Signal Transduction , Animals , Rats
8.
Bull Exp Biol Med ; 174(6): 768-773, 2023 Apr.
Article in English | MEDLINE | ID: mdl-37160600

ABSTRACT

We studied the effects of a dual-vector DYSF gene delivery system based on adeno-associated virus serotype 9 capsids on pathological manifestations of dysferlinopathy in skeletal muscles of Bla/J mice lacking DYSF expression. The mice received intravenous injection of 3×1013 genomic copies of the virus containing the dual-vector system. M. gastrocnemius, m. psoas major, m. vastus lateralis, and m. gluteus superficialis were isolated for histological examination in 3, 6, and 12 weeks after treatment. Healthy wild-type (C57BL/6) mice served as positive control and were sacrificed 3 weeks after injection of 150 µl of 0.9% NaCl into the caudal vein. To detect dysferlin in muscle cryosections, immunohistochemical analysis with diagnostic antibodies was performed; paraffin sections were stained with hematoxylin and eosin for morphometric analysis. After administration of gene-therapeutic constructs, muscle fibers with membrane or cytoplasmic dysferlin location were detected in all examined muscles. The proportion of necrotic muscle fibers decreased, the number of muscle fibers with central location of the nucleus increased, and the mean cross-section area of the muscle fibers decreased.


Subject(s)
Muscle, Skeletal , Muscular Dystrophies, Limb-Girdle , Mice , Animals , Dysferlin/genetics , Dysferlin/metabolism , Mice, Inbred C57BL , Muscle, Skeletal/metabolism , Muscular Dystrophies, Limb-Girdle/genetics , Muscular Dystrophies, Limb-Girdle/therapy , Muscular Dystrophies, Limb-Girdle/metabolism , Muscle Fibers, Skeletal/metabolism , Gene Transfer Techniques
9.
J Physiol ; 600(8): 1953-1968, 2022 04.
Article in English | MEDLINE | ID: mdl-35156706

ABSTRACT

Dysferlin is an integral membrane protein of the transverse tubules of skeletal muscle that is mutated or absent in limb girdle muscular dystrophy 2B and Miyoshi myopathy. Here we examine the role of dysferlin's seven C2 domains, C2A through C2G, in membrane repair and Ca2+ release, as well as in targeting dysferlin to the transverse tubules of skeletal muscle. We report that deletion of either domain C2A or C2B inhibits membrane repair completely, whereas deletion of C2C, C2D, C2E, C2F or C2G causes partial loss of membrane repair that is exacerbated in the absence of extracellular Ca2+ . Deletion of C2C, C2D, C2E, C2F or C2G also causes significant changes in Ca2+ release, measured as the amplitude of the Ca2+ transient before or after hypo-osmotic shock and the appearance of Ca2+ waves. Most deletants accumulate in endoplasmic reticulum. Only the C2A domain can be deleted without affecting dysferlin trafficking to transverse tubules, but Dysf-ΔC2A fails to support normal Ca2+ signalling after hypo-osmotic shock. Our data suggest that (i) every C2 domain contributes to repair; (ii) all C2 domains except C2B regulate Ca2+ signalling; (iii) transverse tubule localization is insufficient for normal Ca2+ signalling; and (iv) Ca2+ dependence of repair is mediated by C2C through C2G. Thus, dysferlin's C2 domains have distinct functions in Ca2+ signalling and sarcolemmal membrane repair and may play distinct roles in skeletal muscle. KEY POINTS: Dysferlin, a transmembrane protein containing seven C2 domains, C2A through C2G, concentrates in transverse tubules of skeletal muscle, where it stabilizes voltage-induced Ca2+ transients and participates in sarcolemmal membrane repair. Each of dysferlin's C2 domains except C2B regulate Ca2+ signalling. Localization of dysferlin variants to the transverse tubules is not sufficient to support normal Ca2+ signalling or membrane repair. Each of dysferlin's C2 domains contributes to sarcolemmal membrane repair. The Ca2+ dependence of membrane repair is mediated by C2C through C2G. Dysferlin's C2 domains therefore have distinct functions in Ca2+ signalling and sarcolemmal membrane repair.


Subject(s)
C2 Domains , Membrane Proteins , Dysferlin/genetics , Membrane Proteins/genetics , Membrane Proteins/metabolism , Muscle, Skeletal/metabolism , Sarcolemma/metabolism
10.
Neuropathol Appl Neurobiol ; 48(7): e12846, 2022 12.
Article in English | MEDLINE | ID: mdl-35962550

ABSTRACT

AIMS: 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. METHODS AND RESULTS: 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. CONCLUSIONS: 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.


Subject(s)
Dysferlin , Muscular Dystrophies, Limb-Girdle , Muscular Dystrophies , Humans , Dysferlin/genetics , Membrane Proteins/genetics , Muscle Proteins/genetics , Muscle, Skeletal/pathology , Muscular Dystrophies, Limb-Girdle/genetics , Pedigree , Male , Female
11.
Clin Genet ; 102(6): 465-473, 2022 12.
Article in English | MEDLINE | ID: mdl-36029111

ABSTRACT

Dysferlinopathies are a clinically heterogeneous group of diseases caused by mutations in the DYSF gene encoding the dysferlin protein. Dysferlin is mostly expressed in muscle tissues and is localized in the sarcolemma, where it performs its main function of resealing and maintaining of the integrity of the cell membrane. At least four forms of dysferlinopathies have been described: Miyoshi myopathy, limb-girdle muscular dystrophy type 2B, distal myopathy with anterior tibial onset, and isolated hyperCKemia. Here we review the clinical features of different forms of dysferlinopathies and attempt to identify genotype-phenotype correlations. Because of the great clinical variability and rarety of the disease and mutations little is known, how different phenotypes develop as a result of different mutations. However, missense mutations seem to induce more severe disease than LoF, which is typical for many muscle dystrophies. The role of several specific mutations and possible gene modifiers is also discussed in the paper.


Subject(s)
Distal Myopathies , Muscular Dystrophies, Limb-Girdle , Humans , Dysferlin/genetics , Muscle Proteins/genetics , Membrane Proteins/genetics , Muscular Dystrophies, Limb-Girdle/genetics , Mutation
12.
Muscle Nerve ; 66(4): 513-522, 2022 10.
Article in English | MEDLINE | ID: mdl-35859452

ABSTRACT

INTRODUCTION/AIMS: Most mouse models of muscular dystrophy (MD) show mild phenotypes, which limits the translatability of experimental therapies to patients. A growing body of evidence suggests that MD is accompanied by metabolic abnormalities that could potentially exacerbate the primary muscle wasting process. Since thermoneutral (TN) housing of mice (~30°C) has been shown to affect many metabolic parameters, particularly when combined with a Western diet (WD), our aim was to determine whether the combination of TN and WD exacerbates muscle wasting in dysferlin-deficient BLAJ mice, a common model of limb-girdle MD type 2b (LGMD2b). METHODS: The 2-mo-old wild-type (WT) and BLAJ mice were housed at TN or room temperature (RT) and fed a WD or regular chow for 9 mo. Ambulatory function, muscle histology, and protein immunoblots of skeletal muscle were assessed. RESULTS: BLAJ mice at RT and fed a chow diet showed normal ambulation function similar to WT mice, whereas 90% of BLAJ mice under WD and TN combination showed ambulatory dysfunction (p < 0.001), and an up to 4.1-fold increase in quadriceps and gastrocnemius fat infiltration. Western blotting revealed decreased autophagy marker microtubules-associated protein 1 light chain 3-B (LC3BII/LC3BI) ratio and up-regulation of protein kinase B/AKT and ribosomal protein S6 phosphorylation, suggesting inefficient cellular debris and protein clearance in TN BLAJ mice fed a WD. Male and female BLAJ mice under TN and WD combination showed heterogenous fibro-fatty infiltrate composition. DISCUSSION: TN and WD combination exacerbates rodent LGMD2b without affecting WT mice. This improves rodent modeling of human MD and helps elucidate how metabolic abnormalities may play a causal role in muscle wasting.


Subject(s)
Muscular Dystrophies, Limb-Girdle , Muscular Dystrophies , Animals , Diet, Western/adverse effects , Dysferlin/genetics , Dysferlin/metabolism , Female , Housing , Humans , Male , Mice , Muscle, Skeletal , Muscular Atrophy/genetics , Muscular Atrophy/metabolism , Muscular Dystrophies/pathology , Muscular Dystrophies, Limb-Girdle/pathology , Proto-Oncogene Proteins c-akt/metabolism , Ribosomal Protein S6/metabolism
13.
BMC Neurol ; 22(1): 398, 2022 Nov 01.
Article in English | MEDLINE | ID: mdl-36319958

ABSTRACT

BACKGROUND: Dysferlinopathy is an autosomal recessive muscular dystrophy caused by pathogenic variants in the dysferlin (DYSF) gene. This disease shows heterogeneous clinical phenotypes and genetic characteristics. METHODS: We 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. RESULTS: Among 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. CONCLUSION: Dysferlinopathy 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.


Subject(s)
Distal Myopathies , Muscular Dystrophies, Limb-Girdle , Humans , China , Diagnostic Errors , Distal Myopathies/genetics , Distal Myopathies/pathology , Dysferlin/genetics , Membrane Proteins/genetics , Muscle Proteins/genetics , Muscular Dystrophies, Limb-Girdle/genetics , Mutation
14.
Biochem J ; 478(1): 197-215, 2021 01 15.
Article in English | MEDLINE | ID: mdl-33449082

ABSTRACT

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.


Subject(s)
Calcium-Binding Proteins/chemistry , Calcium/chemistry , Dysferlin/chemistry , Muscle Proteins/chemistry , Calcium/metabolism , Calcium-Binding Proteins/metabolism , Crystallography, X-Ray , Dysferlin/genetics , Dysferlin/metabolism , Gene Expression , Models, Molecular , Muscle Proteins/metabolism , Mutagenesis , Mutation , Nuclear Magnetic Resonance, Biomolecular , Protein Binding , Protein Domains , Protein Structure, Tertiary , Static Electricity
15.
BMC Pediatr ; 22(1): 515, 2022 08 30.
Article in English | MEDLINE | ID: mdl-36042458

ABSTRACT

BACKGROUND: Dysferlinopathy refers to a heterogenous group of autosomal recessive disorders that affect a skeletal muscle protein called dysferlin. These mutations are associated with limb-girdle muscular dystrophy type 2B, Miyoshi myopathy, asymptomatic hyperCKemia, and distal myopathy with anterior tibial onset. CASE PRESENTATION: A 16 year old female presented with myalgia, weakness and dark urine one week after her second BNT162b2 mRNA (Pfizer) vaccine. Initial serum creatine kinase (CK) was measured at 153,000 IU/L, eventually up-trending to over 200,000 IU/L. However, stable renal function precluded hemodialysis allowing discharge after 10 days of intravenous (IV) hydration and alkaline diuresis. Just two years prior to the current presentation, the patient was hospitalized following Group A Streptococcal pharyngitis infection complicated by rhabdomyolysis. She presented with fatigue, lower extremity weakness, and dark oliguria with CK measuring 984,800 IU/L. IV hydration was attempted however hemodialysis was ultimately required throughout her 24-day hospital stay. Her episode was presumed to be idiopathic and no further work-up was performed at that time. During the patient's current hospitalization, she reported similar symptomology (myalgias and weakness) following her first quadrivalent Gardasil vaccine at age 11. No hospitalization was required at that time. A comprehensive workup was now initiated while the patient was being treated for her suspected second or third non-exertional, non-traumatic rhabdomyolysis. Rheumatologic, metabolic, infectious, and endocrinologic workup were all unremarkable. Patient eventually had whole exome sequencing performed which revealed a heterozygous pathogenic variant in the DYSF gene (DYSF c.2643 + 1G > A) encoding dysferlin. No clinically significant sequelae occurred thus far. CONCLUSIONS: While there have been reports of symptomatic heterozygote carriers of dysferlinopathies, to our knowledge none have been associated with recurrent rhabdomyolysis after immunogenic stimuli. This unique case presentation highlights the importance of a multi-disciplinary care team, the utility of modern whole-exome gene sequencing, and the future challenges of balancing vaccine risk vs benefit.


Subject(s)
Muscular Dystrophies, Limb-Girdle , Rhabdomyolysis , Adolescent , BNT162 Vaccine , Child , Dysferlin/genetics , Female , Humans , Membrane Proteins/genetics , Muscular Dystrophies, Limb-Girdle/genetics , Muscular Dystrophies, Limb-Girdle/pathology , Mutation , Rhabdomyolysis/etiology
16.
Ultrastruct Pathol ; 46(4): 359-367, 2022 Jul 04.
Article in English | MEDLINE | ID: mdl-35880824

ABSTRACT

A number of sarcolemma proteins are responsible for muscle fiber repair. Dysferlin encoded by the DYSF gene is one of these proteins. Dysferlin promotes membrane repair in striated muscle fibers (MFs). Mutations in DYSF lead to loss of or decreased dysferlin expression, impaired membrane repair in MF, and its destruction, clinically manifesting as dysferlinopathy. Preclinical studies of cell and gene therapies aimed at restoring impaired muscle regeneration require well-characterized small animal models. Our investigation aimed to distinguish the histopathological features of a mouse strain lacking dysferlin expression (Bla/J strain). Ultrastructural changes in the sarcolemma, mitochondria and contractile apparatus were observed. It was shown that postnatal histogenesis of skeletal muscles in genetically determined dysferlin deficiency is characterized by a higher proportion of necrotic muscle fibers, compensatory hypertrophy of muscle fibers with their subsequent atrophy, and decreases in proliferative activity and the level of myogenic differentiation of myogenic progenitor cells compared to wild-type mice (C57Bl/6).


Subject(s)
Dysferlin , Muscle, Skeletal , Muscular Dystrophies, Limb-Girdle , Animals , Dysferlin/genetics , Dysferlin/metabolism , Mice , Mice, Inbred C57BL , Muscle Fibers, Skeletal/pathology , Muscle, Skeletal/pathology , Muscular Dystrophies, Limb-Girdle/pathology
17.
Int J Mol Sci ; 24(1)2022 Dec 21.
Article in English | MEDLINE | ID: mdl-36613515

ABSTRACT

Dysferlinopathies are a clinically heterogeneous group of muscular dystrophies caused by a genetic deficiency of the membrane-associated protein dysferlin, which usually manifest post-growth in young adults. The disease is characterized by progressive skeletal muscle wasting in the limb-girdle and limbs, inflammation, accumulation of lipid droplets in slow-twitch myofibers and, in later stages, replacement of muscles by adipose tissue. Previously we reported myofiber-type specific differences in muscle contractile function of 10-month-old dysferlin-deficient BLAJ mice that could not be fully accounted for by altered myofiber-type composition. In order to further investigate these findings, we examined the impact of dysferlin deficiency on the abundance of calcium (Ca2+) handling and glucose/glycogen metabolism-related proteins in predominantly slow-twitch, oxidative soleus and fast-twitch, glycolytic extensor digitorum longus (EDL) muscles of 10-month-old wild-type (WT) C57BL/6J and dysferlin-deficient BLAJ male mice. Additionally, we compared the Ca2+ activation properties of isolated slow- and fast-twitch myofibers from 3-month-old WT and BLAJ male mice. Differences were observed for some Ca2+ handling and glucose/glycogen metabolism-related protein levels between BLAJ soleus and EDL muscles (compared with WT) that may contribute to the previously reported differences in function in these BLAJ muscles. Dysferlin deficiency did not impact glycogen content of whole muscles nor Ca2+ activation of the myofilaments, although soleus muscle from 10-month-old BLAJ mice had more glycogen than EDL muscles. These results demonstrate a further impact of dysferlin deficiency on proteins associated with excitation-contraction coupling and glycogen metabolism in skeletal muscles, potentially contributing to altered contractile function in dysferlinopathy.


Subject(s)
Calcium , Dysferlin , Glycogen , Animals , Male , Mice , Calcium/metabolism , Dysferlin/deficiency , Glucose/metabolism , Glycogen/metabolism , Mice, Inbred C57BL , Muscle, Skeletal/metabolism
18.
Int J Mol Sci ; 23(16)2022 Aug 11.
Article in English | MEDLINE | ID: mdl-36012197

ABSTRACT

Mutations in the DYSF gene, encoding dysferlin, are responsible for Limb Girdle Muscular Dystrophy type R2/2B (LGMDR2/2B), Miyoshi myopathy (MM), and Distal Myopathy with Anterior Tibialis onset (MDAT). The size of the gene and the reported inter and intra familial phenotypic variability make early diagnosis difficult. Genetic analysis was conducted using Next Gene Sequencing (NGS), with a panel of 40 Muscular Dystrophies associated genes we designed. In the present study, we report a new missense variant c.5033G>A, p.Cys1678Tyr (NM_003494) in the exon 45 of DYSF gene related to Limb Girdle Muscular Dystrophy type R2/2B in a 57-year-old patient affected with LGMD from a consanguineous family of south Italy. Both healthy parents carried this variant in heterozygosity. Genetic analysis extended to two moderately affected sisters of the proband, showed the presence of the variant c.5033G>A in both in homozygosity. These data indicate a probable pathological role of the variant c.5033G>A never reported before in the onset of LGMDR2/2B, pointing at the NGS as powerful tool for identifying LGMD subtypes. Moreover, the collection and the networking of genetic data will increase power of genetic-molecular investigation, the management of at-risk individuals, the development of new therapeutic targets and a personalized medicine.


Subject(s)
Distal Myopathies , Muscular Dystrophies, Limb-Girdle , Dysferlin/genetics , Homozygote , Humans , Middle Aged , Muscular Atrophy , Muscular Dystrophies, Limb-Girdle/diagnosis , Muscular Dystrophies, Limb-Girdle/genetics , Muscular Dystrophies, Limb-Girdle/pathology , Mutation
19.
Hum Mutat ; 42(12): 1615-1623, 2021 12.
Article in English | MEDLINE | ID: mdl-34559919

ABSTRACT

Dysferlinopathy is one of the most common subgroup of autosomal recessive limb-girdle muscular dystrophies that is caused by mutations in DYSF gene. However, there is currently no worldwide comprehensive genetic analysis of DYSF variants. Through a national multicenter collaborative effort in China, we identified 222 DYSF variants with 40 novel variants from 245 patients. We then integrated DYSF variants from disease-related genetic databases including LOVD (n = 1020) and Clinvar (n = 1179), to depict the global landscape of disease-related DYSF variants. Normal-population-derived DSYF variants from gnomAD (n = 4318) and ChinaMAP (n = 13,330) were also analyzed in comparison. In Chinese patients, gender instead of genotype showed influence on the onset age of dysferlinopathy, with males showing an earlier age of onset. After integrative analysis, we identified two hotspot DYSF mutations, c.2997G>T in world patients and c.1375dup in Chinese patients, respectively. Both the pathogenic and likely pathogenic variants scattered on the whole gene length of DYSF. However, three specific domains (C2F-C2G-TM, DysF, and C2B-Ferl-C2C) contained variants at higher frequencies than reported in both the databases and Chinese patients. This study comprehensively collected available DYSF variant data, which may pave way for genetic counselling and future clinical trial design for gene therapies in dysferlinopathy.


Subject(s)
Muscular Dystrophies, Limb-Girdle , Asian People/genetics , Dysferlin/genetics , Humans , Male , Muscular Dystrophies, Limb-Girdle/genetics , Mutation
20.
Genet Med ; 23(8): 1574-1577, 2021 08.
Article in English | MEDLINE | ID: mdl-33927379

ABSTRACT

PURPOSE: Recent evolution of sequencing technologies and the development of international standards in variant interpretation have profoundly changed the diagnostic approaches in clinical genetics. As a consequence, many variants that were initially claimed to be disease-causing can be now reclassified as benign or uncertain in light of the new data available. Unfortunately, the misclassified variants are still present in the scientific literature and variant databases, greatly interfering with interpretation of diagnostic sequencing results. Despite the urgent need, large-scale efforts to update the classifications of these variants are still not sufficient. METHODS: We retrospectively analyzed 176 DYSF gene variants that were identified in dysferlinopathy patients referred to the Marseille Medical Genetics Department for diagnostic sequencing since 2001. RESULTS: We reclassified all variants into five-tier American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP) pathogenicity classes, revealing changed pathogenicity for 17 variants. We then updated the information for the variants that have been previously published in the variant database and submitted 46 additional DYSF variants. CONCLUSION: Besides direct benefit for dysferlinopathy diagnostics, our study contributes to the much needed effort to reanalyze variants from previously published cohorts and to work with curators of variant databases to update the entries for erroneously classified variants.


Subject(s)
Genetic Variation , Muscular Dystrophies, Limb-Girdle , Dysferlin/genetics , Genetic Testing , Genetic Variation/genetics , Humans , Retrospective Studies
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