Domain Of Dystrophin Research Articles

Computational analysis of missense mutations in dystrophin protein: Insights into domain-specific effects and functional implications

BackgroundDystrophin is a structural protein primarily found in skeletal muscles that connects trans-membrane component of dystrophin-glycoprotein complex to the intracellular cytoskeleton network. N-terminal domain of dystrophin binds to F-actin and its C-terminal domain attaches to dystrophin-associated complex (DAG) in the membrane. It serves as the bridge connecting the extracellular matrix to the actin based cytoskeleton of muscle cells across the plasma membrane. This complex works together to strengthen muscle fibers and shield them from damage as they contract and relax. ObjectiveOur Objective is to investigate the different pathogenic mutations in four distinct domains of dystrophin protein and to decipher how these mutations impact protein structure and function. In this study, we investigated the impact of disease causing missense mutations in isoforms of dystrophin – P.11532–1 (K18N.A165V, D3187G, F3228L); P11532–4 (Y223N, D3179G) and P-11532-11 (Y227N,D3183G).Occurrence of pathogenic mutations in dystrophin might compromise structural stability, interfere with protein-protein interactions or alter cellular signaling pathways collectively. All these can contribute to the progressive muscle degeneration observed in Duchene or Becker muscular dystrophy(DMD or BMD) and X-lined dilated cardiomyopathy(CMD3B) affected individuals. MethodsEvolutionary conservation analysis using multiple sequence alignment followed by pathogenicity prediction using web based server was performed. Homology modeling of mutants was performed bySWISS MODEL a fully automated homology-modeling server, accessible through the Expasy web server. These models were then analyzed using various servers such as Dynamut. Pymol was used for visualization and structural analysis. ResultsAnalyzed mutations cause structural instability by either gaining or losing specific interactions involved in the folding of protein. Loss of pi-pi stacking interactions has the potential to significant impact the overall stability of the protein. It is important to note that the majority of the mutations lead to stability defects rather than functional defects, ultimately contributing to different forms of muscular dystrophy. ConclusiónThe mutations in different domains of Dystrophin protein significantly alters the protein structure. This may in turn impact its ability to interact with other proteins. Understanding the specific impacts of these mutations can pave the way for development of targeted therapies that aims at restoring functionality of dystrophin and ameliorating the debilitating effects of muscular dystrophy.

SGCD Missense Variant in a Lagotto Romagnolo Dog with Autosomal Recessively Inherited Limb-Girdle Muscular Dystrophy.

An 8-month-old female Lagotto Romagnolo dog was presented for a 1-month history of an initial severe reluctance to move, rapidly progressing to a marked stiff gait and progressive muscular weakness and evolving to tetraparesis, which persuaded the owner to request euthanasia. A primary muscle pathology was supported by necropsy and histopathological findings. Macroscopically, the muscles were moderately atrophic, except for the diaphragm and the neck muscles, which were markedly thickened. Histologically, all the skeletal muscles examined showed atrophy, hypertrophy, necrosis with calcification of the fibers, and mild fibrosis and inflammation. On immunohistochemistry, all three dystrophin domains and sarcoglycan proteins were absent. On Western blot analysis, no band was present for delta sarcoglycan. We sequenced the genome of the affected dog and compared the data to more than 900 control genomes of different dog breeds. Genetic analysis revealed a homozygous private protein-changing variant in the SGCD gene encoding delta- sarcoglycan in the affected dog. The variant was predicted to induce a SGCD:p.(Leu242Pro) change in the protein. In silico tools predicted the change to be deleterious. Other 770 Lagotto Romagnolo dogs were genotyped for the variant and all found to be homozygous wild type. Based on current knowledge of gene function in other mammalian species, including humans, hamsters, and dogs, we propose the SGCD missense variant as the causative variant of the observed form of muscular dystrophy in the index case. The absence of the variant allele in the Lagotto Romagnolo breeding population indicates a rare allele that has appeared recently.

One-year Data from ENDEAVOR, a Phase 1b Trial of Delandistrogene Moxeparvovec (SRP-9001) in Patients with Duchenne Muscular Dystrophy (DMD) (S48.003)

One-year Data from ENDEAVOR, a Phase 1b Trial of Delandistrogene Moxeparvovec (SRP-9001) in Patients with Duchenne Muscular Dystrophy (DMD) (S48.003)

PK/PD Modeling to Inform Clinical Development of an Adeno-associated Virus Gene Transfer Therapy for Duchenne Muscular Dystrophy (S48.005)

<h3>Objective:</h3> To evaluate the pharmacokinetic (PK)/pharmacodynamic (PD) relationship between tissue vector genome exposure, biological efficacy and functional outcome in an animal model of Duchenne muscular dystrophy (DMD) (DMD^mdx mice) following treatment with delandistrogene moxeparvovec (SRP-9001). <h3>Background:</h3> Delandistrogene moxeparvovec is an investigational gene transfer therapy developed to address the root cause of DMD through targeted skeletal and cardiac muscle expression of SRP-9001 dystrophin protein, which contains key functional domains of dystrophin. <h3>Design/Methods:</h3> We evaluated vector biodistribution, expression, and clinical dose selection of delandistrogene moxeparvovec using a novel application of a PK and PD modeling approach applied to data collected from DMD^mdx mice. We analyzed PK/PD relationships between dose, tissue vector genome exposure, SRP-9001 dystrophin protein expression (percent dystrophin positive fibers [PDPF] and western blot), and functional improvement (relative specific force from tibialis anterior and diaphragm). <h3>Results:</h3> Linear kinetics with a dose-proportional increase in tissue drug exposure were demonstrated across the nearly 10-fold dose range (4.43×10¹³–4.01×10¹⁴ vg/kg), and in all tissues. The relationship between tissue vector exposure and PD endpoints (PDPF, motor function outcome) showed a saturable response across a wide range of vector exposures. The vector exposure at 1.33×10¹⁴ vg/kg (the clinical dose) approached the maximum treatment response. Relative specific force and PDPF were significantly correlated (<i>P</i>=4.43×10⁻⁶). However, the relationship appeared to be nonlinear, with increased PDPF expression approaching the maximal functional improvement. Relative specific force and western blot were not significantly correlated. <h3>Conclusions:</h3> For the first time, biodistribution, biomarker and functional efficacy data were used to quantify and demonstrate PK/PD relationships for an adeno-associated virus (AAV)-based gene transfer therapy in a DMD animal model. The results continue to support the expected therapeutic benefit and clinical dose of delandistrogene moxeparvovec, an AAV-based gene transfer therapy. <b>Disclosure:</b> Mrs. East has received personal compensation for serving as an employee of Sarepta Therapeutics. Rachael Potter has received stock or an ownership interest from Sarepta Therapeutics. Dr. Snedeker has received personal compensation for serving as an employee of Sarepta Therapeutics. Dr. Snedeker has stock in Sarepta Therapeutics. Dr. Haile has nothing to disclose. Christopher Wier has received personal compensation for serving as an employee of Sarepta Therapeutics. Christopher Wier has stock in Sarepta Therapeutics. Ms. Rodino-Klapac has received personal compensation for serving as an employee of Sarepta Therapeutics, Inc.. Ms. Rodino-Klapac has received stock or an ownership interest from Sarepta Therapeutics. Ms. Rodino-Klapac has received intellectual property interests from a discovery or technology relating to health care.

Long-term Safety and Efficacy in Patients with DMD 4 Years Post-Treatment with Delandistrogene Moxeparvovec (SRP-9001) in a Phase 1/2a Study (P3-8.006)

Long-term Safety and Efficacy in Patients with DMD 4 Years Post-Treatment with Delandistrogene Moxeparvovec (SRP-9001) in a Phase 1/2a Study (P3-8.006)

EMBARK, a Phase 3 Trial Evaluating Safety and Efficacy of Delandistrogene Moxeparvovec (SRP- 9001) in Duchenne Muscular Dystrophy (DMD): Study Design and Baseline Characteristics (P5-8.012)

EMBARK, a Phase 3 Trial Evaluating Safety and Efficacy of Delandistrogene Moxeparvovec (SRP- 9001) in Duchenne Muscular Dystrophy (DMD): Study Design and Baseline Characteristics (P5-8.012)

Analysis of Vector Shedding Following Treatment with Delandistrogene Moxeparvovec (SRP-9001), an Investigational rAAVrh74-Based Gene Therapy for Duchenne Muscular Dystrophy (DMD) (P5-8.013)

<h3>Objective:</h3> To evaluate extent and magnitude of vector shedding following delandistrogene moxeparvovec (SRP-9001) administration. <h3>Background:</h3> Delandistrogene moxeparvovec is an investigational gene transfer therapy developed to address the root cause of DMD through targeted skeletal and cardiac muscle expression of SRP-9001 dystrophin protein, which contains key functional domains of dystrophin. The hypothetical risk of seroconversion in naive individuals through exposure to vector shed from treated patients is a point to consider for healthcare providers and the Duchenne community. <h3>Design/Methods:</h3> We evaluated extent of vector shedding and clearance following a single administration of delandistrogene moxeparvovec (1.33×10¹⁴ vg/kg body weight) in ENDEAVOR (NCT04626674; Study 103; N=20), a Phase 1 study in patients with DMD. Delandistrogene moxeparvovec vector exposure in saliva, urine, and feces was quantified by droplet digital polymerase chain reaction. In a nonclinical study, we tested naive mice to determine risk of AAVrh74 seroconversion following mucosal vector exposure, with doses based on exposure levels (vector genome copies) demonstrated in nonclinical and clinical studies. Mice were exposed to AAVrh74.CMV.eGFP via optic exposure and intramuscularly. Antibody levels were measured by AAVrh74 ELISA at baseline and 4 weeks post-delivery. <h3>Results:</h3> Following treatment in ENDEAVOR, the percentage decrease in amount of vector shed was &gt;99% in saliva (n=12), urine (n=18), and feces (n=11) by Week 4. In mice, topical optical delivery of the AAVrh74 vector at relevant concentrations did not produce seropositivity or detectable vector genomes throughout tissues at doses based on clinical vector shedding levels. <h3>Conclusions:</h3> Clinical findings show that peak vector shedding occurs in the first few days after delandistrogene moxeparvovec administration and exponentially declines to insignificant levels by Week 4. In mice, seroconversion was not observed by relevant exposure route at relevant concentrations. Results suggest that the risk of seroconversion following exposure to vector shed by individuals treated with AAVrh74-based gene therapy may be very low. <b>Disclosure:</b> Jyoti Malhotra has nothing to disclose. Dr. Smith has received personal compensation for serving as an employee of Sarepta Therapeutics, Inc. Dr. Smith has stock in Sarepta Therapeutics. Sarah Lewis has received stock or an ownership interest from Sarepta. Mrs. Zhang has nothing to disclose. Mr. Asher has received personal compensation for serving as an employee of Sarepta Therapeutics. Mr. Asher has stock in Sarepta Therapeutics. Shufang Wang has received personal compensation for serving as an employee of Sarepta Therapeutics INC. Shufang Wang has received stock or an ownership interest from Sarepta Therapeutics INC. Mrs. East has received personal compensation for serving as an employee of Sarepta Therapeutics. Rachael Potter has received stock or an ownership interest from Sarepta Therapeutics. Ms. Rodino-Klapac has received personal compensation for serving as an employee of Sarepta Therapeutics, Inc.. Ms. Rodino-Klapac has received stock or an ownership interest from Sarepta Therapeutics. Ms. Rodino-Klapac has received intellectual property interests from a discovery or technology relating to health care.

Integrated Analyses of Data from Clinical Trials of Delandistrogene Moxeparvovec (SRP-9001) in Duchenne Muscular Dystrophy (DMD) (S48.006)

Integrated Analyses of Data from Clinical Trials of Delandistrogene Moxeparvovec (SRP-9001) in Duchenne Muscular Dystrophy (DMD) (S48.006)

Evaluating Pharmacology and Efficacy of Delandistrogene Moxeparvovec in Young and Aged DMDmdx Rats (P3-8.008)

<h3>Objective:</h3> The purpose of this preclinical study is to evaluate the myocardial efficacy and safety of intended commercial process delandistrogene moxeparvovec (SRP-9001) material in Duchenne muscular dystrophy (DMD)^mdx rats. DMD^mdx rats are a valuable alternative animal model of DMD, as they demonstrate cardiac dysfunction that recapitulates cardiac dysfunction of patients with DMD. <h3>Background:</h3> Gene transfer therapy is a promising treatment for patients with DMD. Delandistrogene moxeparvovec is an investigational gene transfer therapy developed to address the root cause of DMD through targeted skeletal and cardiac muscle expression of SRP-9001 dystrophin protein, which contains key functional domains of dystrophin. <h3>Design/Methods:</h3> We performed systemic, intravenous delivery of intended commercial process delandistrogene moxeparvovec material in young (21–35 days old) and aged (3–5 months old) DMD^mdx rats. The older rats demonstrate a more severe phenotype in terms of fibrosis or cardiac disease progression. Rats received a dose of 1.33×10¹⁴ vg/kg or 7.00×10¹³ vg/kg. Ambulation activity was recorded via the Photobeam Activity System Open Field. Echocardiograms and histological analysis of fibrosis were used to evaluate cardiac disease. <h3>Results:</h3> Data from 12 weeks and 24 weeks post-systemic delivery demonstrated no evidence of cardiac toxicity. Importantly, there were no deaths attributed to treatment. Compared with the saline control, intended commercial process delandistrogene moxeparvovec material increased ambulation and vertical activity in young DMD^mdx rats and improved cardiac function. Protein expression was broadly distributed across skeletal muscle, the diaphragm and the heart. <h3>Conclusions:</h3> Taken together, these findings confirm expression in cardiac muscle of rats, as expected, and support the potential myocardial efficacy and safety of delandistrogene moxeparvovec. Further results of cardiac disease phenotypes at 12 and 24 weeks post-systemic delivery utilizing several indicators of cardiac function will also be presented. <b>Disclosure:</b> Rachael Potter has received stock or an ownership interest from Sarepta Therapeutics. Christopher Wier has received personal compensation for serving as an employee of Sarepta Therapeutics. Christopher Wier has stock in Sarepta Therapeutics. Dr. Cooper-Olson has received personal compensation for serving as an employee of Sarepta Therapeutics. Dr. Cooper-Olson has stock in Sarepta Therapeutics. Ms. Wheeler has nothing to disclose. Miss Anderbery has nothing to disclose. Ms. Kempton has received personal compensation for serving as an employee of Sarepta Therapeutics. Ms. Kempton has stock in Sarepta Therapeutics. Ms. Clements has received personal compensation for serving as an employee of Sarepta Therapeutics . Ms. Clements has stock in Sarepta Therapeutics. Mrs. Adegboye has nothing to disclose. Dr. Haile has nothing to disclose. Ms. Peterson has nothing to disclose. Ms. Rodino-Klapac has received personal compensation for serving as an employee of Sarepta Therapeutics, Inc.. Ms. Rodino-Klapac has received stock or an ownership interest from Sarepta Therapeutics. Ms. Rodino-Klapac has received intellectual property interests from a discovery or technology relating to health care.

Sub-region analysis of DMD gene in cases with idiopathic generalized epilepsy.

Gene sub-region encoded protein domain is the basic unit for protein structure and function. The DMD gene is the largest coding gene in humans, with its phenotype relevant to idiopathic generalized epilepsy. We hypothesized variants clustered in sub-regions of idiopathic generalized epilepsy genes and investigated the relationship between the DMD gene and idiopathic generalized epilepsy. Whole exome sequencing was performed in 106 idiopathic generalized epilepsy individuals. DMD variants were filtered with variant type, allele frequency, in silico prediction, hemizygous or homozygous status in the population, inheritance mode, and domain location. Variants located at the sub-regions were selected by the subRVIS software. The pathogenicity of variants was evaluated by the American College of Medical Genetics and Genomics criteria. Articles on functional studies related to epilepsy for variants clustered protein domains were reviewed. In sub-regions of the DMD gene, two variants were identified in two unrelated cases with juvenile absence epilepsy or juvenile myoclonic epilepsy. The pathogenicity of both variants was uncertain significance. Allele frequency of both variants in probands with idiopathic generalized epilepsy reached statistical significance compared with the population (Fisher's test, p = 2.02 × 10-6, adjusted α = 4.52 × 10-6). The variants clustered in the spectrin domain of dystrophin, which binds to glycoprotein complexes and indirectly affects ion channels contributing to epileptogenesis. Gene sub-region analysis suggests a weak association between the DMD gene and idiopathic generalized epilepsy. Functional analysis of gene sub-region helps infer the pathogenesis of idiopathic generalized epilepsy.

Dystrophin (DMD) Missense Variant in Cats with Becker-Type Muscular Dystrophy

Muscular dystrophy due to dystrophin deficiency in humans is phenotypically divided into a severe Duchenne and milder Becker type. Dystrophin deficiency has also been described in a few animal species, and few DMD gene variants have been identified in animals. Here, we characterize the clinical, histopathological, and molecular genetic aspects of a family of Maine Coon crossbred cats with clinically mild and slowly progressive muscular dystrophy. Two young adult male littermate cats exhibited abnormal gait and muscular hypertrophy with macroglossia. Serum creatine kinase activities were highly increased. Histopathologically, dystrophic skeletal muscle exhibited marked structural changes including atrophic, hypertrophic, and necrotic muscle fibers. Immunohistochemistry showed irregularly reduced expression of dystrophin but the staining of other muscle proteins such as β- and γ-sarcoglycans as well as desmin was also diminished. Whole genome sequencing of one affected cat and genotyping of the littermate found both to be hemizygous mutant at a single DMD missense variant (c.4186C>T). No other protein-changing variants in candidate genes for muscular dystrophy were detected. In addition, one clinically healthy male littermate was hemizygous wildtype, while the queen and one female littermate were clinically healthy, but heterozygous. The predicted amino acid exchange (p.His1396Tyr) resides in a conserved central rod spectrin domain of dystrophin. Various protein modeling programs did not predict major disruption of the dystrophin protein by this substitution, but the altered charge of the region may still affect protein function. This study represents the first genotype-to-phenotype correlation of Becker-type dystrophin deficiency in companion animals.

Dystrophin myonuclear domain restoration governs treatment efficacy in dystrophic muscle

Dystrophin is essential for muscle health: its sarcolemmal absence causes the fatal, X-linked condition, Duchenne muscular dystrophy (DMD). However, its normal, spatial organization remains poorly understood, which hinders the interpretation of efficacy of its therapeutic restoration. Using female reporter mice heterozygous for fluorescently tagged dystrophin (DmdEGFP), we here reveal that dystrophin distribution is unexpectedly compartmentalized, being restricted to myonuclear-defined sarcolemmal territories extending ~80 µm, which we called "basal sarcolemmal dystrophin units (BSDUs)." These territories were further specialized at myotendinous junctions, where both Dmd transcripts and dystrophin protein were enriched. Genome-level correction in X-linked muscular dystrophy mice via CRISPR/Cas9 gene editing restored a mosaic of separated dystrophin domains, whereas transcript-level Dmd correction, following treatment with tricyclo-DNAantisense oligonucleotides, restored dystrophin initially at junctions before extending along the entire fiber-with levels ~2% sufficient to moderate the dystrophic process. We conclude that widespread restoration of fiber dystrophin is likely critical for therapeutic success in DMD, perhaps most importantly, at muscle-tendon junctions.

A rare missense Duchenne muscular dystrophy gene variant in a family with muscular dystrophy from Turkey

Objectives: Duchenne and Becker muscular dystrophies (DMD/BMD) are muscle diseases that show X-linked recessive inheritance. The disease occurs depending on large mutations, deletions/duplications, small mutations, point mutations and mid-intronic mutations of the gene encoding the protein called dystrophin. Therefore, in this study, we aimed to investigate the pathogenic variants of DMD in the affected family. Methods: A 23-year-old male who had weakness in the muscles, difficulty climbing the stairs, frequent falls at the age of seven was referred to the Medical Genetics department for an initial diagnosis of DMD/BMD. His siblings also suffered from similar symptoms. Therefore, eight individuals from the same family were included in the study. MLPA analysis was performed to evaluate deletion/duplication and variants of the DMD gene were evaluated by targeted NGS. Sophia DDM algorithms were used for the bioinformatics analysis of data, and the pathogenicity of the mutations was evaluated based on in silico prediction tools. Results: Allelic variants were identified in 8 individuals of the family including two suspected patients and six suspected obligatory carriers. NGS analysis revealed that proband and his nephew were hemizygous for pathogenic c.10018T&amp;gt; C (p.Cys3340Arg, C3340R) mutation and mother, two sisters and niece were carriers. Conclusions: C3340R mutation was first reported in a Taiwanese BMD patient among the 23 different pathologic changes. This variant identified as pathogenic because of being highly conserved cysteine substitution in the dystroglycan-binding domain of dystrophin. This study has the importance of reporting an infrequent pathogenic mutation, C3340R, in two patients and four suspected obligatory carriers of a Turkish family.

Molecular Fingerprint of BMD Patients Lacking a Portion in the Rod Domain of Dystrophin.

BMD is characterized by a marked heterogeneity of gene mutations resulting in many abnormal dystrophin proteins with different expression and residual functions. The smaller dystrophin molecules lacking a portion around exon 48 of the rod domain, named the D8 region, are related to milder phenotypes. The study aimed to determine which proteins might contribute to preserving muscle function in these patients. Patients were subdivided, based on the absence or presence of deletions in the D8 region, into two groups, BMD1 and BMD2. Muscle extracts were analyzed by 2-D DIGE, label-free LC-ESI-MS/MS, and Ingenuity pathway analysis (IPA). Increased levels of proteins typical of fast fibers and of proteins involved in the sarcomere reorganization characterize BMD2. IPA of proteomics datasets indicated in BMD2 prevalence of glycolysis and gluconeogenesis and a correct flux through the TCA cycle enabling them to maintain both metabolism and epithelial adherens junction. A 2-D DIGE analysis revealed an increase of acetylated proteoforms of moonlighting proteins aldolase, enolase, and glyceraldehyde-3-phosphate dehydrogenase that can target the nucleus promoting stem cell recruitment and muscle regeneration. In BMD2, immunoblotting indicated higher levels of myogenin and lower levels of PAX7 and SIRT1/2 associated with a set of proteins identified by proteomics as involved in muscle homeostasis maintenance.

Protein Expression of Canine and Feline Muscular Dystrophies

Muscular dystrophies in dogs and cats represent a heterogeneous group of inherited, sometimes congenital, but infrequently diagnosed, progressive neuromuscular disorders. A correct identification and characterization of canine and feline muscular dystrophies could increase diagnostic and treatment strategies for veterinary neurologists and could identify useful animal models for the study of human dystrophies. However, in dogs and cats, diagnosis of muscular dystrophies is challenging due to a nonspecific clinical phenotype and pathological lesions, thus is most likely underestimated. We performed immunofluorescence and Western blot techniques using a wide panel of antibodies against proteins involved in human dystrophies (dystrophin mid-rod and carboxyterminal domain, α, β, γ, and δ-sarcoglycan, α-dystroglycan, caveolin-3, emerin, merosin, dysferlin, calpain-3, spectrin epitopes), on 9 canine and 3 feline muscle biopsies characterized by myopathic changes. Dystrophin deficiency was detected in 3 dogs and 2 novel canine muscular dystrophies have been identified, characterized by deficiency of caveolin-3 and calpain-3, respectively. In 2 cats, deficiency of β-SG and carboxyterminal domain of dystrophin in all muscle fibers has been detected. Performing immunofluorescence and Western blot analyses with a wider panel of antibodies allowed a correct identification of muscular dystrophies in dogs and cats and provides a direction for subsequent targeted genetic testing.

Rapid, redox-mediated mechanical susceptibility of the cortical microtubule lattice in skeletal muscle

The highly ordered cortical microtubule lattice of skeletal muscle is disorganized in dystrophin-deficient mdx mice. Implicated mechanisms include loss of dystrophin binding, altered α-tubulin posttranslational modification, expression of a β-tubulin involved in regeneration, and reactive oxygen species (ROS). Here we show that the transverse microtubules in mdx muscle expressing miniaturized dystrophins are rapidly lost after eccentric contraction. Analysis of mdx lines expressing different dystrophin constructs demonstrate that spectrin-like repeats R4-15 and R20-23 were required for mechanically stable microtubules. Microtubule loss was prevented by the non-specific antioxidant N-acetylcysteine while inhibition of NADPH oxidase 2 had only a partial effect, suggesting that ROS from multiple sources mediate the rapid loss of transverse microtubules after eccentric contraction. Finally, ablation of α-dystrobrevin, β- or γ-cytoplasmic actin phenocopied the transverse microtubule instability of miniaturized dystrophins. Our data demonstrate that multiple dystrophin domains, α-dystrobrevin and cytoplasmic actins are necessary for mechanically stable microtubules.

Tyrosine phosphorylation as a regulator of dystrophin and beta-dystroglycan interaction: A molecular insight

Dystrophin-β-dystroglycan interaction has gained a special attention during current years due to its association with the pathogenesis of muscular dystrophies. Dystrophin is an important component of dystrophin associated protein complex that functions in the normal physiology and cell signaling in addition to membrane stabilization and provides integrity to skeletal muscle fibers. WW, EF-hand and ZZ domains of dystrophin are known to bind with extreme C-terminal region of beta-dystroglycan (β-DG) containing PPxY motif and this interaction is experimentally proven to be coordinated and regulated by two tyrosine (Tyr886 and Tyr892) residues in the C-terminus of beta-dystroglycan. These tyrosine residues are phosphorylated in adhesion dependent manner that disrupts dystrophin-β-DG interaction. The failure of dystrophin to interact with β-DG causes muscular dystrophies. In this study, we have performed molecular docking analysis of dystrophin with phosphorylated and mutated variants of β-DG to pinpoint the actual nature of this interaction at molecular level. We have discovered significant structural and conformational changes in β-DG molecule caused by mutations and tyrosine phosphorylation that alter the nature and site of its interaction with dystrophin. Our results not only support the previous findings but also bring to attention previously unreported discoveries about the nature of this interaction and behavior of different β-DG variants with dystrophin WW, EF-hand and ZZ domains.

How the central domain of dystrophin acts to bridge F-actin to sarcolemmal lipids

Dystrophin is a large intracellular protein that prevents sarcolemmal ruptures by providing a mechanical link between the intracellular actin cytoskeleton and the transmembrane dystroglycan complex. Dystrophin deficiency leads to the severe muscle wasting disease Duchenne Muscular Dystrophy and the milder allelic variant, Becker Muscular Dystrophy (DMD and BMD). Previous work has shown that concomitant interaction of the actin binding domain 2 (ABD2) comprising spectrin like repeats 11 to 15 (R11-15) of the central domain of dystrophin, with both actin and membrane lipids, can greatly increase membrane stiffness. Based on a combination of SAXS and SANS measurements, mass spectrometry analysis of cross-linked complexes and interactive low-resolution simulations, we explored in vitro the molecular properties of dystrophin that allow the formation of ABD2-F-actin and ABD2-membrane model complexes. In dystrophin we identified two subdomains interacting with F-actin, one located in R11 and a neighbouring region in R12 and another one in R15, while a single lipid binding domain was identified at the C-terminal end of R12. Relative orientations of the dystrophin central domain with F-actin and a membrane model were obtained from docking simulation under experimental constraints. SAXS-based models were then built for an extended central subdomain from R4 to R19, including ABD2. Overall results are compatible with a potential F-actin/dystrophin/membrane lipids ternary complex. Our description of this selected part of the dystrophin associated complex bridging muscle cell membrane and cytoskeleton opens the way to a better understanding of how cell muscle scaffolding is maintained through this essential protein.

EP.66A case with dystrophinopathies mimicking metabolic myopathies

Rhabdomyolysis associated with inherited conditions refers to recurrent episodes characterized by minimal exercise, history of malignant hyperthermia, familial history, or hyperCKemia between attacks. We describe a 17-year-old male presented recurrent myalgia following exercise. He had suffered exertional rhabdomyolysis with myoglobinuria and hyperCKemia between attacks, mimicking metabolic myopathies. The neurological examination at his 17 years of age, the muscle weakness was not present. The ischemic forearm exercise test revealed normal response, showing elevated lactate and ammonia. The needle electromyography showed myopathic changes. The immunoreactivity to C-terminal of dystrophin was absent in pathology biopsied from left biceps muscle. Genetic analysis of whole exome sequencing revealed a novel hemizygous c.119T>A (p.Leu40His) nonsense variant in DMD, located in the N-terminal actin-binding domain of dystrophin. In-silico test for the structure of dystrophin was predicted to destabilize interaction in the vicinity at residue 40 by losing hydrophobicity. This report will raise awareness that rhabdomyolysis and exertional myalgia can be seen in patients with a missense mutation of the exons encoding the N-terminal actin-binding domain of dystrophin.

Unfolding Transitions and Interdomain Coupling in Human Dystrophin Spectrin Repeats

Dystrophin is a 427 kDa rod-shaped protein encoded by one of the largest mammalian genes. The protein is formed by 27 domains, with 24 of them being single spectrin repeats interposed by two hinge regions. Single point and missing domain mutations within Dystrophin have been linked to Becker's Muscular Dystrophy (BMD), which is a degenerative muscle disease. This suggests that one role for Dystrophin is to reduce mechanical force between the sarcolemma membrane and the cytoskeleton. The mechanism for this function is unknown. The observation that single point mutations within Dystrophin are correlated with BMD suggests there is communication between each amino acid within each Dystrophin domain and that this communication extends to its neighboring domains. This led to the hypothesis that as force is transduced through the spectrin repeats, this results in the partial unfolding of the repeats which then dissipates force. If this partial unfolding of a given domain is accentuated when another repeat domain is in tandem, then this is consistent with negative coupling. Conversely, stabilization of one domain by another is positive coupling. Molecular dynamics (MD) forced unfolding experiments were consistent with negative coupling through non-additive force trajectories for tandem domains compared to individual domains. Therefore, we designed an experimental approach to test the computational results.Using monomeric spectrin 17, monomeric spectrin 18, and dimeric spectrin 17-18, we experimentally defined the thermodynamic and structural basis of energetic coupling between the spectrin domains through differential scanning calorimetry (DSC), fluorescence lifetime (FLT), and Overhauser dynamic nuclear polarization (ODNP). A combined approach utilizing these methods will provide insight into how coupling between spectrin repeats affects signal propagation and mechanical force dissipation.