Mutations In Dystrophin Gene Research Articles

Mutation spectrum analysis of Duchenne/Becker muscular dystrophy in 68 families in Kuwait: The era of personalized medicine.

Duchenne and Becker muscular dystrophies (DMD/BMD) are X-linked recessive neuromuscular disorders characterized by progressive irreversible muscle weakness and atrophy that affect both skeletal and cardiac muscles. DMD/BMD is caused by mutations in the Dystrophin gene on the X chromosome, leading to the absence of the essential muscle protein Dystrophin in DMD. In BMD, Dystrophin is partially functioning with a shorter protein product. Recent advances in molecular therapies for DMD require precise genetic diagnoses because most therapeutic strategies are mutation-specific. Hence, early diagnosis is crucial to allow appropriate planning for patient care and treatment. In this study, data from DMD/BMD patients who attended the Kuwait Medical Genetic Center during the last 20 years was retrieved from a Kuwait neuromuscular registry and analyzed. We combined multiplex PCR and multiplex ligation-dependent probe amplification (MLPA) with Sanger sequencing to detect Dystrophin gene mutations. A total of 35 different large rearrangements, 2 deletion-insertions (Indels) and 4 substitution mutations were identified in the 68 unrelated families. The deletion and duplication rates were 66.2% and 4.4%, respectively. The analyzed data from our registry revealed that 11 (16%) of the DMD families will benefit from newly introduced therapies (Ataluren and exon 51 skipping). At the time of submitting this paper, two cases have already enrolled in Ataluren (Tranlsarna™) therapy, and one case has been enrolled in exon 51 skipping therapy.

Molecular Analysis-Based Genetic Characterization of a Cohort of Patients with Duchenne and Becker Muscular Dystrophy in Eastern China.

Background:Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are common X-linked recessive neuromuscular disorders caused by mutations in dystrophin gene. Multiplex polymerase chain reaction (multiplex PCR) and multiplex ligation-dependent probe amplification (MLPA) are the most common methods for detecting dystrophin gene mutations. This study aimed to contrast the two methods and discern the genetic characterization of patients with DMD/BMD in Eastern China.Methods:We collected 121 probands, 64 mothers of probands, and 15 fetuses in our study. The dystrophin gene was detected by multiplex PCR primarily in 28 probands, and MLPA was used in multiplex PCR-negative cases subsequently. The dystrophin gene of the remaining 93 probands and 62 female potential carriers was tested by MLPA directly. In fetuses, multiplex PCR and MLPA were performed on 4 fetuses and 10 fetuses, respectively. In addition, sequencing was also performed in 4 probands with negative MLPA.Results:We found that 61.98% of the subjects had genetic mutations including deletions (50.41%) and duplications (11.57%). There were 43.75% of mothers as carriers of the mutation. In 15 fetuses, 2 out of 7 male fetuses were found to be unhealthy and 2 out of 8 female fetuses were found to be carriers. Exons 3–26 and 45–52 have the maximum frequency in mutation regions. In the frequency of exons individually, exon 47 and exon 50 were the most common in deleted regions and exons 5, 6, and 7 were found most frequently in duplicated regions.Conclusions:MLPA has better productivity and sensitivity than multiplex PCR. Prenatal diagnosis should be applied in DMD high-risk fetuses to reduce the disease incidence. Furthermore, it is the responsibility of physicians to inform female carriers the importance of prenatal diagnosis.

Creation of Dystrophin Expressing Chimeric Cells of Myoblast Origin as a Novel Stem Cell Based Therapy for Duchenne Muscular Dystrophy

Over the past decade different stem cell (SC) based approaches were tested to treat Duchenne Muscular Dystrophy (DMD), a lethal X-linked disorder caused by mutations in dystrophin gene. Despite research efforts, there is no curative therapy for DMD. Allogeneic SC therapies aim to restore dystrophin in the affected muscles; however, they are challenged by rejection and limited engraftment. Thus, there is a need to develop new more efficacious SC therapies. Chimeric Cells (CC), created via ex vivo fusion of donor and recipient cells, represent a promising therapeutic option for tissue regeneration and Vascularized Composite Allotransplantation (VCA) due to tolerogenic properties that eliminate the need for lifelong immunosuppression. This proof of concept study tested feasibility of myoblast fusion for Dystrophin Expressing. Chimeric Cell (DEC) therapy through in vitro characterization and in vivo assessment of engraftment, survival, and efficacy in the mdx mouse model of DMD. Murine DEC were created via ex vivo fusion of normal (snj) and dystrophin–deficient (mdx) myoblasts using polyethylene glycol. Efficacy of myoblast fusion was confirmed by flow cytometry and dystrophin immunostaining, while proliferative and myogenic differentiation capacity of DEC were assessed in vitro. Therapeutic effect after DEC transplant (0.5 × 106) into the gastrocnemius muscle (GM) of mdx mice was assessed by muscle functional tests. At 30 days post-transplant dystrophin expression in GM of injected mdx mice increased to 37.27 ± 12.1% and correlated with improvement of muscle strength and function. Our study confirmed feasibility and efficacy of DEC therapy and represents a novel SC based approach for treatment of muscular dystrophies.

Role of miR-200c in Myogenic Differentiation Impairment via p66Shc: Implication in Skeletal Muscle Regeneration of Dystrophic mdx Mice.

Duchenne muscular dystrophy (DMD) is a genetic disease associated with mutations of Dystrophin gene that regulate myofiber integrity and muscle degeneration, characterized by oxidative stress increase. We previously published that reactive oxygen species (ROS) induce miR-200c that is responsible for apoptosis and senescence. Moreover, we demonstrated that miR-200c increases ROS production and phosphorylates p66Shc in Ser-36. p66Shc plays an important role in muscle differentiation; we previously showed that p66Shc−/− muscle satellite cells display lower oxidative stress levels and higher proliferation rate and differentiated faster than wild-type (wt) cells. Moreover, myogenic conversion, induced by MyoD overexpression, is more efficient in p66Shc−/− fibroblasts compared to wt cells. Herein, we report that miR-200c overexpression in cultured myoblasts impairs skeletal muscle differentiation. Further, its overexpression in differentiated myotubes decreases differentiation indexes. Moreover, anti-miR-200c treatment ameliorates myogenic differentiation. In keeping, we found that miR-200c and p66Shc Ser-36 phosphorylation increase in mdx muscles. In conclusion, miR-200c inhibits muscle differentiation, whereas its inhibition ameliorates differentiation and its expression levels are increased in mdx mice and in differentiated human myoblasts of DMD. Therefore, miR-200c might be responsible for muscle wasting and myotube loss, most probably via a p66Shc-dependent mechanism in a pathological disease such as DMD.

Early Diagnosis and Treatment – The Use of Ataluren in the Effective Management of Duchenne Muscular Dystrophy

The understanding of the natural history of Duchenne muscular dystrophy (DMD) is increasing rapidly and new treatments are emerging that have the potential to substantially improve the prognosis for patients with this disabling and life-shortening disease. For many, however, there is a long delay between the appearance of symptoms and DMD diagnosis, which reduces the possibility of successful treatment. DMD results from mutations in the large dystrophin gene of which one-third are de novo mutations and two-thirds are inherited from a female carrier. Roughly 75% of mutations are large rearrangements and 25% are point mutations. Certain deletions and nonsense mutations can be treated whereas many other mutations cannot currently be treated. This emphasises the need for early genetic testing to identify the mutation, guide treatment and inform genetic counselling. Treatments for DMD include corticosteroids and more recently, ataluren has been approved in Europe, the first disease-modifying therapy for treating DMD caused by nonsense mutations. The use of ataluren in DMD is supported by positive results from phase IIb and phase III studies in which the treatment produced marked improvements in the 6-minute walk test, timed function tests such as the 10 m walk/run test and the 4-stair ascent/descent test compared with placebo. In these trials, ataluren was well tolerated and adverse event profiles were similar to placebo. As such disease-modifying treatments become more widely available, the outlook for children with DMD will improve but physicians must be aware of the disease, rapidly initiate testing where it is suspected and promptly begin appropriate treatment.

Analysis of 12 patients with novel mutations of Dystrophin gene

To study the characteristics, location, and amino acid changes of novel mutations of the Dystrophin gene. Twelve patients in whom no deletion or duplication of the Dystrophin gene was detected were analyzed with next-generation sequencing. Fifty healthy adult males were recruited as the controls. All patients were detected with mutations of the Dystrophin gene, which included c.33C>G, c.583C>T, c.1333C>T, c.2593C>T, c.5731A>T, c.7288G>T, c.2803+1G>T, c.10034G>A, c.4289A>G, c.1905_906delAG, c.5017delC, c.5768_5771delAAGA, and c.6261_6262insA. No similar mutations were found among the controls. Our data has enriched the mutation spectrum of the Dystrophin gene and may provide an important basis for genetic diagnosis.

Single-cut genome editing restores dystrophin expression in a new mouse model of muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a severe, progressive muscle disease caused by mutations in the dystrophin gene. The majority of DMD mutations are deletions that prematurely terminate the dystrophin protein. Deletions of exon 50 of the dystrophin gene are among the most common single exon deletions causing DMD. Such mutations can be corrected by skipping exon 51, thereby restoring the dystrophin reading frame. Using clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9), we generated a DMD mouse model by deleting exon 50. These ΔEx50 mice displayed severe muscle dysfunction, which was corrected by systemic delivery of adeno-associated virus encoding CRISPR/Cas9 genome editing components. We optimized the method for dystrophin reading frame correction using a single guide RNA that created reframing mutations and allowed skipping of exon 51. In conjunction with muscle-specific expression of Cas9, this approach restored up to 90% of dystrophin protein expression throughout skeletal muscles and the heart of ΔEx50 mice. This method of permanently bypassing DMD mutations using a single cut in genomic DNA represents a step toward clinical correction of DMD mutations and potentially those of other neuromuscular disorders.

Duchenne muscular dystrophy: A immunohistochemical profile and deletion pattern in dystrophin gene in North Indian population

Background: Duchenne muscular dystrophy (DMD), one of the most common X linked muscular disorder, affecting 1 in 3500 male births and is caused by mutation in dystrophin gene. 65% of DMD cases are caused by large deletion of dystrophin gene, followed by duplication (5-10%) or point mutation (25-30%). There is wide mutation spectrum of the mutations in dystrophin gene. Hence, population specific information is needed on mutation spectrum and frequency of common mutations occurring in that particular population for appropriate counseling, prenatal diagnosis and for developing genetic therapy in future. Aims and Objectives: To find out the frequency and distribution of deletion in dystrophin gene in DMD patients along with contribution of pathology and genetic testing in diagnosis of DMD and Becker muscular dystrophy (BMD) in North Indian population.Materials and Methods: Dystrophin gene was screened for deletion by multiplex polymerase chain reaction (PCR). Out of 41 patients, 09 patients underwent muscle biopsy, on which immunohistochemistry was performed for dystrophin, sarcoglycan, dysferlin and merosin.Results: Majority of the deletions were located in distal hotspot region (26/39 ~66.66%) which includes the exons 45-55 and 15.38% of deletions were located at the proximal hotspot region (2- 19 exons).Conclusion: In the present study, 34% patients only showed deletion. Hence complete work up of any muscular dystrophy requires immnohistochemical analysis to see the expression of muscle proteins along with multipleplex PCR test to detect any exon deletion, multiplex ligation-dependent probe amplification (MLPA) to detect point mutation and duplication and western blotting to quantify the dystrophin protein.Asian Journal of Medical Sciences Vol.8(6) 2017 13-18

A De novo Mutation in Dystrophin Causing Muscular Dystrophy in a Female Patient.

Background:Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are X-linked recessive neuromuscular diseases resulting from dystrophin (DMD) gene mutations. It has been known that the carrier of DMD mutations may also have symptoms of the disease. While de novo mutation is quite common in BMD/DMD patients, it is rarely reported in the female carriers.Methods:Two sporadic Chinese patients with progressive muscular dystrophy and their familial members were recruited. The targeted next-generation sequencing (NGS) and the multiplex ligation-dependent probe analysis (MLPA) were performed in the proband. Blood tests, electrocardiography, echocardiography, and electromyography were also evaluated.Results:Two novel mutations of DMD gene were identified, c.7318C>T (p.Q2440*) in the male proband and c.4983dupA (p.A1662Sfs*24) in the female carrier. The MLPA analysis did not detect any large rearrangements. The haplotype analysis indicated that the two mutations were derived from de novo mutagenesis.Conclusions:We identified two novel de novo mutations of DMD gene in two Chinese pedigrees, one of which caused a female patient with muscular dystrophy. The mutational analysis is important for DMD patients and carriers in the absence of a family history. The NGS can help detect the mutations in MLPA-negative patients.

P.246 - Characterization of molecular pathophysiology in muscular dystrophy by next generation RNA sequencing using DMD and CMD mouse models

Duchenne muscular dystrophy (DMD) caused by mutations in Dystrophin gene and congenital muscular dystrophy type 1A (MDC1A) caused by mutations in LAMA2, are incurable childhood forms of muscular dystrophy. Although both disorders result from mutations in proteins strengthening the muscle sarcolemma, they are implicated in clinically distinct phenotypes. We used RNA deep sequencing (RNA-Seq) of dy2J/dy2J, MDC1A mouse model, skeletal muscle to elucidate disease pathophysiology. This study is the first report of comparing the transcriptome profile of dy2J/dy2J mouse model to WT. RNA-Seq of the mdx mouse model of DMD and its control was carried as well in order to enable a comparison of the two mouse models. 3844 genes were significantly differentially expressed in mdx versus WT mice (2695 upregulated and 1149 downregulated, FDR (false detection rate) < 0.05) and 2675 genes in dy2J/dy2J versus WT (1727 upregulated and 948 downregulated, FDR < 0.05). 1834 common differentially expressed genes between mdx and dy2J/dy2J were found (1285 upregulated and 549 downregulated, FDR < 0.05). Enrichment pathway analysis using Ingenuity Pathway Analysis (IPA) software of the common differentially expressed genes showed enrichment of inflammation, fibrosis, adhesion, apoptosis, muscle cell structure and metabolism (p-values 3E-10 – 9E-37). Canonical pathways, such as ILK, NF-kB, Wnt, calcium signaling and others were found to be enriched in both models (FDR < 0.05). Our data deepen our understanding of the molecular pathophysiology of each disease in order to select new pathways or targets for additional therapies in CMD and DMD.

P.337 - Multilevel molecular analysis identifies all dystrophin gene mutations pointing out that DMD is a genetically homogenous disease: repercussions on diagnosis, prevention and therapy

Dystrophinopathies are a group of allelic diseases caused by mutation in the dystrophin (DMD) gene: Deletions (~65%), duplications (~10%), small mutations (25%) and deep intronic mutations and complex rearrangements (~1%). Many exceptions do exist to the reading frame rule and a fine genetic characterization is mandatory for providing genetic diagnosis and making patients eligible for novel personalized trials. We analyzed the dystrophin gene in a large heterogeneous cohort of 891 individuals; reasons for testing were: clinical diagnosis of DMD (485), clinical diagnosis of BMD (226), symptomatic female carriers (43), asymptomatic patient with high CK (38), limb girdle muscle dystrophy (24) and at risk females tested for known familial mutation (78). We identified the dystrophin causative mutation in all the probands except for two boys with a clinical diagnosis of DMD and absence of dystrophin in the biopsy; in both the transcript analysis pinpointed the presence of a dystrophin gene mutation. Our data demonstrate that dystrophinopathies are genetically homogenous and a multilevel diagnostic approach is able to identify all DMD mutations; therefore a monogenic NGS approach should be endorsed in this disease. These findings carry some consequences in the address and interpretation of dystrophin genetic tests in clinical genetic counseling, in the definition of the role of modifiers, in the design of new therapies and in the plan of clinical care flowcharts.

Ayurvedic management of Ducchen’s Muscular Dystrophy - A Case report

Ducchen’s muscular dystrophy is most common X-linked recessive disorder affecting 30 in 100,000 live male births. The primary cause of this disease is mutations in Dystrophin gene which is essential for the structural and functional integrity of muscle. It is a progressive muscle wasting disease in which patients frequently develop contractures and lose the ability to walk between 6 and 12 years of age. With progressive disease most patients succumb to death from respiratory failure and cardiac dysfunction in their twenties. As this is a genetic disorder we can consider it as Adibala Pravritta Vyadhi. As Mamsa Kshaya is seen at some muscles and Mamsa Vriddhi at other this is an Avarana Vata Vyadhi. In both Upsthambha and Nirupasthmbha Vatavyadhi, Basthi is considered as prime choice of treatment. A Variety of Ksheerabasti in the form of Kalabasti is studied in this condition by taking subjective and objective parameters. As this has given better improvement with no adverse effects in the patient, it can be tried in large number of patients.

Female Duchenne

Objective: Duchenne muscular dystrophy (DMD) is a X-linked recessive disease caused by mutations in dystrophin gene and is characterized by progressive muscle degeneration and weakness resulting in poor disease outcome. The disease primarily affects boys. Females are usually only asymptomatic carriers of mutations. Manifesting carriers are those with muscle weakness and/or dilated cardiomyopathy and asymptomatic are considered those with elevated levels of creatine kinase, and/or minor myopathic signs such as myalgia or muscle cramps. Methods: Our patient is a girl in which disease manifested at the age of 13, with muscle weakness and exercise tolerance. During the last two years her problems became more pronounced and we started diagnostic workup under the suspicion of neuromuscular disease. Besides the clear signs of muscle weakness, particularly expressed in the proximal muscles of the lower extremities with positive Gowers sign, she had hypertrophy of the calves. Repeated cardiac examination was normal. Results: We performed initial laboratory testing and observed elevated creatine kinase (CK) 10 times more than the reference value. EMG analysis was of regular neurographic parameters but needle EMG showed pronounced spontaneous outbreak with myopathic pattern. We performed muscle biopsy and histological examination revealed the destructive myopathic process and immunohistochemistry complete absence of dystrophin expression. MLPA revealed a heterozygous deletion of exon 3 and 4 of dystrophin gene. Conclusion: Our female patient most likely has de novo mutation of dystrophin gene that led to expression of symptoms at an earlier age than the average mentioned in literature, symptoms are progressive, and the girl is already difficult to stay ambulatory and we think that she has atypical form of dystrophinopathy with complete absence of dystrophin in muscle biopsy. Nowadays, for our patient besides genetic counseling it is very important to consider medicamentous treatment.

Nanotherapy for Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is a lethal X-linked childhood muscle wasting disease caused by mutations in the dystrophin gene. Nanobiotechnology-based therapies (such as synthetic nanoparticles and naturally existing viral and nonviral nanoparticles) hold great promise to replace and repair the mutated dystrophin gene and significantly change the disease course. While a majority of DMD nanotherapies are still in early preclinical development, several [such as adeno-associated virus (AAV)-mediated systemic micro-dystrophin gene therapy] are advancing for phase I clinical trials. Recent regulatory approval of Ataluren (a nonsense mutation read-through chemical) in Europe and Exondys51 (an exon-skipping antisense oligonucleotide drug) in the United States shall offer critical insight in how to move DMD nanotherapy to human patients. Progress in novel, optimized nano-delivery systems may further improve emerging molecular therapeutic modalities for DMD. Despite these progresses, DMD nanotherapy faces a number of unique challenges. Specifically, the dystrophin gene is one of the largest genes in the genome while nanoparticles have an inherent size limitation per definition. Furthermore, muscle is the largest tissue in the body and accounts for 40% of the body mass. How to achieve efficient bodywide muscle targeting in human patients with nanomedication remains a significant translational hurdle. New creative approaches in the design of the miniature micro-dystrophin gene, engineering of muscle-specific synthetic AAV capsids, and novel nanoparticle-mediated exon-skipping are likely to result in major breakthroughs in DMD therapy. WIREs Nanomed Nanobiotechnol 2018, 10:e1472. doi: 10.1002/wnan.1472 This article is categorized under: Biology-Inspired Nanomaterials > Protein and Virus-Based Structures Therapeutic Approaches and Drug Discovery > Emerging Technologies.

CRISPR-Cpf1 correction of muscular dystrophy mutations in human cardiomyocytes and mice.

Duchenne muscular dystrophy (DMD), caused by mutations in the X-linked dystrophin gene (DMD), is characterized by fatal degeneration of striated muscles. Dilated cardiomyopathy is one of the most common lethal features of the disease. We deployed Cpf1, a unique class 2 CRISPR (clustered regularly interspaced short palindromic repeats) effector, to correct DMD mutations in patient-derived induced pluripotent stem cells (iPSCs) and mdx mice, an animal model of DMD. Cpf1-mediated genomic editing of human iPSCs, either by skipping of an out-of-frame DMD exon or by correcting a nonsense mutation, restored dystrophin expression after differentiation to cardiomyocytes and enhanced contractile function. Similarly, pathophysiological hallmarks of muscular dystrophy were corrected in mdx mice following Cpf1-mediated germline editing. These findings are the first to show the efficiency of Cpf1-mediated correction of genetic mutations in human cells and an animal disease model and represent a significant step toward therapeutic translation of gene editing for correction of DMD.

CRISPR/Cas9-Mediated Genome Editing Corrects Dystrophin Mutation in Skeletal Muscle Stem Cells in a Mouse Model of Muscle Dystrophy.

Muscle stem cells (MuSCs) hold great therapeutic potential for muscle genetic disorders, such as Duchenne muscular dystrophy (DMD). The CRISP/Cas9-based genome editing is a promising technology for correcting genetic alterations in mutant genes. In this study, we used fibrin-gel culture system to selectively expand MuSCs from crude skeletal muscle cells of mdx mice, a mouse model of DMD. By CRISP/Cas9-based genome editing, we corrected the dystrophin mutation in expanded MuSCs and restored the skeletal muscle dystrophin expression upon transplantation in mdx mice. Our studies established a reliable and feasible platform for gene correction in MuSCs by genome editing, thus greatly advancing tissue stem cell-based therapies for DMD and other muscle disorders.

Cardiac involvement in female carriers of duchenne or becker muscular dystrophy.

The significance of abnormal cardiac measures in asymptomatic females who harbor dystrophin gene mutations is controversial. Echo-measures of ventricular function were compared with published norms in a cross-sectional study of 130 (age, 39 ± 15.7 years) "carriers" of Duchenne or Becker muscular dystrophy (DMD/BMD). Correlations between cardiomyopathy (CM) and mutation, creatine kinase (CK) levels, age, and muscle symptoms were investigated. Depending on definition, CM prevalence was 3-33%. Ejection fraction (Simpson method) was < 55% in 9 (13%) and < 40% in 2 (2.9%). Eleven (8.5%) had wall motion abnormalities. Left ventricular end-systolic dimensions were increased in 7 (5.7%) and end-diastolic in 17 (13.9%). CM did not correlate with mutation type, DMD or BMD phenotype, CK level, muscle symptoms, or age. Occult CM can be found by screening in DMD/BMD carriers. Its lack of age-correlation suggests that not all abnormalities progress. Optimum screening schedules require a better understanding of progressive CM. Muscle Nerve 55: 810-818, 2017.

Advances in genome editing technologies for treating muscular dystrophy.

Recent advances in genome editing technologies have opened the possibility for treating genetic diseases, such as Duchenne muscular dystrophy(DMD), by correcting the causing gene mutations in dystrophin gene. In fact, there are several reports that demonstrated the restoration of the mutated dystrophin gene in DMD patient-derived iPS cell or functional recovery of forelimb grip strength in DMD model mice. For future clinical applications, there are several aspects that need to be taken into consideration:efficient delivery of the genome editing components, risk of off-target mutagenesis and immunogenicity against genome editing enzyme. In this review, we summarize the current status and future prospective of the research in applying genome editing technologies to DMD.

Pharmacological Inhibition of PKCθ Counteracts Muscle Disease in a Mouse Model of Duchenne Muscular Dystrophy

Inflammation plays a considerable role in the progression of Duchenne Muscular Dystrophy (DMD), a severe muscle disease caused by a mutation in the dystrophin gene. We previously showed that genetic ablation of Protein Kinase C θ (PKCθ) in mdx, the mouse model of DMD, improves muscle healing and regeneration, preventing massive inflammation. To establish whether pharmacological targeting of PKCθ in DMD can be proposed as a therapeutic option, in this study we treated young mdx mice with the PKCθ inhibitor Compound 20 (C20). We show that C20 treatment led to a significant reduction in muscle damage associated with reduced immune cells infiltration, reduced inflammatory pathways activation, and maintained muscle regeneration. Importantly, C20 treatment is efficient in recovering muscle performance in mdx mice, by preserving muscle integrity. Together, these results provide proof of principle that pharmacological inhibition of PKCθ in DMD can be considered an attractive strategy to modulate immune response and prevent the progression of the disease. Research in contextDuchenne muscular dystrophy (DMD) is a severe muscle disease affecting 1:3500 male births. DMD is caused by a mutation in dystrophin gene, coding for a protein required for skeletal and cardiac muscle integrity. Lack of a functional dystrophin is primarily responsible for the muscle eccentric contraction-induced muscle damage, observed in dystrophic muscle. However, inflammation plays a considerable role in the progression of DMD. Glucocorticoids, which have anti-inflammatory properties, are being used to treat DMD with some success; however, long term treatment with these drugs induces muscle atrophy and wasting, outweighing their benefit. The identification of specific targets for anti-inflammatory therapies is one of the ongoing therapeutic options. Although blunting inflammation would not be a “cure” for the disease, the emerging clue is that multiple strategies, addressing different aspects of the pathology, which may eventually converge, may be successful. In this context, we previously showed that genetic ablation of Protein Kinase C θ (PKCθ), an enzyme known to be involved in immune response, in mdx, the mouse model of DMD, improves muscle healing and regeneration, preventing massive inflammation. To establish whether pharmacological targeting of PKCθ in DMD can be proposed as a therapeutic option, in this study we treated young mdx mice with the PKCθ inhibitor Compound 20 (C20). We show that C20 treatment led to a significant reduction in muscle damage associated with reduced immune cells infiltration, reduced inflammatory pathways activation, and maintained muscle regeneration. Importantly, C20 treatment is efficient in recovering muscle performance in mdx mice, by preserving muscle integrity. Together, these results provide proof of principle that pharmacological inhibition of PKCθ in DMD can be considered an attractive strategy to modulate immune response and prevent the progression of the disease.

Multiplex Ligation Dependent Probe Amplification Based Mutation Analysis of Dystrophin Gene in Nepalese Patients with Duchenne Muscular Dystrophy

Duchenne muscular dystrophy (DMD) is X-linked recessive neuromuscular disorders caused due to mutation in dystrophin gene, leading to progressive muscle weakness. This study was done to identify mutation in dystrophin gene in Nepalese patients with DMD using Multiplex Ligation Dependent Probe Amplification (MLPA) assay in Nepal. Twenty one patients from different regions of Nepal, who were clinically diagnosed as DMD were enrolled in the study. Peripheral blood samples were collected in EDTA vials, gDNA was extracted, and deletion mutation in the dystrophin gene was analysed using Multiplex Ligation Dependent Probe Amplification (MLPA) assay.Exon deletion mutation in the dystrophin gene was observed in 14 (66.6%) out of 21 DMD cases. The most common exon deletion was observed and confined in exon 7-14 and 45-53 of dystrophin gene. The location of deletion in dystrophin gene is apparently non-random with a preponderance found in the hot spot regions. Use of MLPA is useful in detecting copy number changes in DMD proband and suspected carriers in Nepal.