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2011 NEWS

DECEMBER

30 - N-Acetylcysteine treatment of dystrophic mdx mice results in protein thiol modifications and inhibition of exercise induced myofibre necrosis

30 - Families of Ill Children Try Drug Development

25 - Ascorbic acid protects the diaphragm muscle against myonecrosis in mdx mice

13 - Nutrition Strategies to Improve Physical Capabilities in Duchenne Muscular Dystrophy

11 - Audit finds that many emergency admissions to hospital could be avoided

3 - (Molecular Therapy, 2011) Phase 1 Gene Therapy for Duchenne Muscular Dystrophy Using a Translational Optimized AAV Vector

Dawn E Bowles, Scott WJ McPhee, Chengwen Li, Steven J Gray, Jade J Samulski, Angelique S Camp, Juan Li, Bing Wang, Paul E Monahan, Joseph E Rabinowitz, Joshua C Grieger, Lakshmanan Govindasamy, Mavis Agbandje-McKenna, Xiao Xiao and R Jude Samulski - USA

Efficient and widespread gene transfer is required for successful treatment of Duchenne muscular dystrophy (DMD). Here, we performed the first clinical trial using a chimeric adeno-associated virus (AAV) capsid variant (designated AAV2.5) derived from a rational design strategy. AAV2.5 was generated from the AAV2 capsid with five mutations from AAV1. The novel chimeric vector combines the improved muscle transduction capacity of AAV1 with reduced antigenic crossreactivity against both parental serotypes, while keeping the AAV2 receptor binding. In a randomized double-blind placebo-controlled phase I clinical study in DMD boys, AAV2.5 vector was injected into the bicep muscle in one arm, with saline control in the contralateral arm. A subset of patients received AAV empty capsid instead of saline in an effort to distinguish an immune response to vector versus minidystrophin transgene. Recombinant AAV genomes were detected in all patients with up to 2.56 vector copies per diploid genome. There was no cellular immune response to AAV2.5 capsid. This trial established that rationally designed AAV2.5 vector was safe and well tolerated, lays the foundation of customizing AAV vectors that best suit the clinical objective (e.g., limb infusion gene delivery) and should usher in the next generation of viral delivery systems for human gene transfer.

NOVEMBER

20 - Brasil will make a clinical trial with stem cells in DMD boys in 2012 (Portuguese text)

14 - (American Heart Association Meeting, Orlando 2011) NOX2 Inhibition Restores Contractility, Intracellular Calcium Handling and Reduces Arrhythmogenicity in Dystrophic Cardiomyopathy

Daniel R Gonzalez1; Adriana V Treuer1; Raul A Dulce2; Guillaume Lamirault2; Joshua M Hare2

1 Facultad de Ciencias de la Salud, Universidad de Talca, Talca, Chile
2 Interdisciplinary Stem Cell Institute, Univ of Miami, Miami, FL

Background. Dystrophic cardiomyopathy is the cardiac manifestation of three closely related diseases that include Duchenne and Becker muscular dystrophies, and X-linked dilated cardiomyopathy. Oxidative stress is characteristic of cardiomyopathies. Intracellular calcium ([Ca2+]i) handling is abnormal in the heart of the mdx mouse, a model of Duchenne muscular dystrophy. We tested the hypothesis that oxidative stress may be responsible of disturbances in Ca2+ handling and contractility in this model.

Methods and results. We used mdx mice with established cardiomyopathy: ejection fraction of 56.7 ± 0.5 % in mdx vs. 70.8 ± 2.4% in wild type. We found increased expression (fivefold) of the NADPH oxidase NOX2 in the mdx hearts compared to wild type, along with increased superoxide production, measured as intensity of dihydroethidium staining (p<0.05 vs. wild type). Treatment with apocyinin (30 min, 100 µmol/L), a NOX2 inhibitor, decreased superoxide in mdx (p<0.05 vs. untreated). Next, we studied the impact of NOX2 inhibition on contractility and calcium handling in isolated cardiomyocytes. Contractility, assessed as sarcomere shortening, was decreased in mdx myocytes compared to wild type (p<0.05). Pre-treatment with apocynin restored this response to normal levels. In addition, the amplitude of evoked [Ca2+]i transients (measured as fura-2 fluorescence) that was diminished in mdx myocytes (p<0.05), was also restored upon NOX2 inhibition. Total sarcoplasmic reticulum (SR) Ca2+ content (evaluated as caffeine-induced Ca2+ release) was reduced in mdx hearts (p<0.05). This content was normalized by apocynin treatment. At the same time, NOX2 inhibition decreased dramatically the production of spontaneous diastolic calcium release events in mdx myocytes (p<0.05, ANOVA).

Conclusions. These results indicate that in mdx hearts, NOX2 inhibition reduces oxidative stress, improving the SR [Ca2+] handling and contractility. Additionally, NOX2 inhibition reduced the sensitivity of the ryanodine receptor, reducing the incidence of diastolic Ca2+ waves. Targeting of NOX2 in may be therapeutically helpful to increase cardiac performance and avoid the incidence of ventricular arrhythmias in Duchenne and other dystrophic cardiomyopathies.

14 - (American Heart Association Meeting, Orlando 2011) Long-Term Treatment with the SIRT1 Activator Resveratrol Ameliorates Cardiomyopathy in Dystrophin-Deficient Mice

Atsushi Kuno1; Yusuke Hori2; Masaya Tanno1; Hidemichi Kouzu1; Takahito Itoh1; Daisuke Sunaga1; Yoshiyuki Horio2; Tetsuji Miura1

1 Second Dept of Internal Medicine, Sapporo Med Univ Sch of Medicine, Sapporo, Japan
2 Dept of Pharmacology, Sapporo Med Univ Sch of Medicine, Sapporo, Japan

Background: Loss of cardiac dystrophin eventually leads to cardiac dysfunction and heart failure, which is a main cause of death in patients with Duchenne muscular dystrophy (DMD). However, there is still no effective pharmacological therapy. Here, we examined the effect of resveratrol, an activator of the longevity factor SIRT1, on cardiomyopathy in dystrophin-deficient mice (mdx), a model of DMD.

Methods and Results:Mdx were left untreated or received resveratrol (400 mg/kg/day orally) for 32 weeks starting at 9 weeks of age. Age-matched C57BL10 served as controls. Mice were examined by echocardiography at 40 weeks of age and sacrificed. Compared with C57BL10, mdx showed significant cardiac hypertrophy as evidenced by higher heart-to-body weight (HW/BW) ratio (4.1±0.6 vs. 5.4±0.8 mg/g), increased diastolic ventricular thickness (0.72±0.02 vs. 0.82±0.03 mm), larger cardiomyocyte cross-sectional area (1.7 fold), and induction of atrial natriuretic peptide mRNA expression (4.3 fold). Resveratrol treatment significantly attenuated cardiac hypertrophy (HW/BW: 4.2±0.1) in mdx. Left ventricular (LV) dimension and LV ejection fraction were similar among the three study groups, but diastolic posterior wall movement, an index of LV diastolic function, was improved by resveratrol in mdx (20.8±0.7 vs. 26.2±1.3 mm/sec, p<0.05). Tissue area stained for fibronectin and tissue level of collagen mRNA, two indices of fibrosis, in mdx were reduced to 48% and 57% of untreated controls, respectively, by resveratrol. Myocardial mRNA level of the fibrogenic cytokine TGFβ1, extent of phosphorylation of extracellular signal-regulated kinase 1/2, mediators of cardiac hypertrophy and fibrosis, in mdx hearts were not reduced by resveratrol. However, resveratrol suppressed the upregulation of protein level of the transcription co-activator p300, a pro-hypertrophic and -fibrotic protein acetylase, in the mdx myocardium. In C2C12 myoblasts, resveratrol decreased p300 protein, and this effect was blocked by SIRT1 knockdown.

Conclusion: Resveratrol attenuates ventricular fibrosis and improves diastolic LV function in mdx presumably by p300 downregulation via SIRT1 activation. SIRT1 activation may be a novel strategy in treatment of cardiomyopathy in DMD.

5 - (ASCB 2011, Denver - Colorado) Arbekacin as a therapeutic readthrough inducer for treatment of nonsense mutation-mediated Duchenne muscular dystrophy

M. Shiozuka, A. Nishida, M. Matsuo, M. Yoshida, Y. Nonomura, R. Matsuda - Japan

 Translational readthrough of a premature termination codon is a promising therapeutic method in more than 2,400 distinctly inherited human diseases.  We previously reported that negamycin, a dipeptide antibiotic, that binds to the ribosomal decoding site and alters translational accuracy, successfully restored dystrophin expression with less toxicity than gentamicin in mdx mouse, which carries a premature termination codon in the dystrophin gene.  In order to measure translational readthrough activity with  quantitative accuracy, we established a  novel transgenic mouse strain, named READ (Readthrough Evaluation and Assessment by Dual reporter).  We found that arbekacin induced the in vivo nonsense suppression in READ mice dose-dependently, and promotes the accumulation of  ystrophin, reduction of serum creatine kinase activity and improvement of contractile function in  mdx mice.  Moreover, arbekacin exhibits restoration of dystrophin expression on muscle cell obtained by biopsies from Duchenne muscular dystrophy patients caused by nonsense mutations.  We have validated the efficacy of arbekacin on dystrophin-deficient muscle that we ultimately wish to treat.  Arbekacin is a brekthrough readthrough-inducing drug for muscular dystrophy patients harboring nonsense mutations.  This work was supported in part by The Ichiro Kanehara Foundation (to MS), Fugaku Foundation (to MS), Health and Labour Sciences Research Grant for Research on Psychiatric and Neurological Diseases and Mental Health (19A-020; to RM), Comprehensive Research on Disability Health and Welfare (H22-ShinkeiKin-Ippan-016; to RM), Nervous and Mental Disorders (20B-13; to RM) from the Ministry of Health, Labour and Welfare, Japan.  

5 - (ASCB 2011, Denver - Colorado) Therapeutic Multiple Exon-skipping Using Cell-penetrating Morpholinos for Dystrophic Dogs

T. Yokota, T. Nagata, A. Nakamura, N. Urasawa, T. Saito, R. Kole, P. Sazani, T. Partridge, E. Hoffman, S. Takeda – Canada, Japan, USA

 Duchenne muscular dystrophy (DMD),  the most common and fatal X-linked myopathy, and its milder form, Becker muscular dystrophy (BMD), are caused by mutations in  the dystrophin (DMD) gene. Antisense-mediated exon skipping therapy is currently one of the most promising molecular therapies for DMD. The exon skipping leads to the production of internally deleted in-frame mRNA transcripts but the truncated quasi-dystrophin retains some functions like BMD. Previously we demonstrated the first successful exon-skipping treatment in body-wide skeletal muscles in Canine X-linked muscular dystrophy (CXMD) using a cocktail of phosphorodiamidate morpholino oligomers (PMOs, morpholinos) targeting exon 6 and exon 8 of dystrophin mRNA. However, unmodified (bare) morpholino injections led to inefficient delivery to the heart, and dystrophin induction was barely detectable in the cardiac muscle. Here, we sought to recover the dystrophin expression in cardiac muscles in dystrophic dogs using morpholinos conjugated with negatively charged arginine-rich cell-penetrating peptides (PPMOs). We demonstrated that the delivery moieties significantly improved dystrophin production in both skeletal and cardiac muscles. Intravenous injections with PPMOs restored dystrophin expression in cardiac muscles accompanied by ameliorated histology. No obvious toxicity was detected by blood tests and histology. Our results show the potential of PMO conjugates as therapeutic agents for DMD.

5-  (Annals of Neurology Vol 70 (suppl 15) 2011) Improvements in muscle function and accidental falling in Ataluren-treated patients with nonsense mutation dystrophinopathy (Duchenne/Becker Muscular Dystrophy - nmDBMD)

Russman BS (Portland, OR), Mathews KD (Iowa City, IA),Sampson JB (Salt Lake City, UT), Renfroe JB (Gulf Breeze,FL), Morsy MA, Elfring GL, Barth JA, Peltz SW(South Plainfield, NJ)

Objective: Dystrophinopathy patients progressively lose muscle function and become susceptible to accidental falling, the most common cause of limb fractures in this population. Ataluren is an investigational drug designed to overcome the effects of nonsense mutations, which are responsible for 13% of dystrophinopathy cases. In a pivotal trial in nmDBMD, low-dose ataluren demonstrated a clinically meaningful difference (29.7m) in change in 6-minute walk distance (6MWD) versus placebo (p=0.0584; post-hoc analysis). Secondary endpoints included timed function tests and patient/parentreported accidental falling. Methods: Males 5 yr of age with nmDBMD were stratified by age, corticosteroid use, and baseline 6MWD;randomized 1:1:1 to placebo; low-dose ataluren (10,10,20 mg/kg); or high-dose ataluren (20,20,40 mg/kg) orally TID; and evaluated every 6 wks for 48 wks. Results: The study enrolled 174 subjects (median [range] age=8 [5–20] yr, corticosteroid use=123/174 [71%], median [range] baseline 6MWD=360 [75–554] m). Differences in mean changes from baseline to Week 48 for low dose versus placebo were -2.40s stair ascend, -1.62s stair descend, -1.35s 10-m walk/run, and -0.01s stand from supine. Over 48 weeks, accidental falling declined in the low-dose arm versus placebo (relative ratio=0.37; post-hoc analysis). High dose-treated subjects with approximate peak plasma concentrations similar to low dose-treated subjects generally had outcomes similar to low dose-treated subjects. Conclusions: Subjects treated with ataluren 10,10,20 mg/kg experienced trends toward improvements in timed function tests and had fewer accidental falls versus placebo. These findings support the primary endpoint (6MWD) results showing a positive treatment effect for low-dose ataluren in this population.

5 - (Annals of Neurology Vol 70 (suppl 15) 2011) Glucocorticoids may act through natural killer cells in Mdx Mice

Golumbek PTR (St. Louis, MO), Gutting K (St Louis, MO),Muglia LJ (Nashville, TN), Unanue ER (St Louis, MO),Calderon B (St Louis, MO), Connolly AM (St Louis, MO)

Objective: To determine whether Natural Killer (NK) cells play a role in the therapeutic effect of prednisolone on mdx mice. Twice weekly oral prednisolone improves muscle strength, running speed, and lifespan in mdx mice, through an unknown mechanism of action. Given its anti-inflammatory effects, and the abundance of inflammatory cells in dystrophic muscle, we previously showed prednisolone’s positive effect is independent of T-cells, B-cells (Ig) and the complement cascade (C3 / ).These previous studies could not eliminate the possibility that the beneficial effect of prednisolone may be through the remaining innate immune components or a direct effect on muscle itself. Methods: Male mdx mice where depleted of NK cells with biweekly IP injection of PK136 antibody starting at 3 weeks of age. Mice were given oral prednisolone (at 5 mg/kg, 2x/week) starting at 4 weeks of age. Forelimb grip strength (FGS), hanging wire time and voluntary wheel running speed were used as physiologic outcome measures.
Muscle histology and serum CK are supportive outcome measures. We assessed prednisolone’s therapeutic effect on strength in the presence and absence of NK cells. Results: Oral prednisolone increases strength in mdx mice. Removal of NK cells produces an identical effect on FGS (in magnitude and time course) as prednisolone treatment alone. After NK cell removal there is no additional effect of strength from prednisolone.

 

 

 

 

OCTOBER

19 - Human Adipose-Derived Mesenchymal Stromal cells injected systemically into GRMD dogs without immunosuppression are able to reach the host muscle and express human dystrophin

SEPTEMBER

24 - Long-term Engraftment of Multipotent Mesenchymal Stromal Cells That Differentiate to Form Myogenic Cells in Dogs With Duchenne Muscular Dystrophy

14 - (The American Journal of Pathology, Available online 13 September 2011) Systemic Delivery of Allogenic Muscle Stem (MuStem) Cells Induces Long-Term Muscle Repair and Clinical Efficacy in Duchenne Muscular Dystrophy Dogs  

Karl Rouger, Thibaut Larcher, Laurence Dubreil, Jack-Yves Deschamps, Caroline Le Guiner, Gregory Jouvion, Bruno Delorme, Blandine Lieubeau, Marine Carlus, Benoît Fornasari, Marine Theret, Priscilla Orlando, Mireille Ledevin, Céline Zuber, Isabelle Leroux, Stéphane Deleau, Lydie Guigand, Isabelle Testault, Elisabeth Le Rumeur, Marc Fiszman, et al.   - France

Here, we characterized canine delayed adherent stem cells and investigated the efficacy of their systemic delivery in the clinically relevant DMD animal model to assess potential therapeutic application in humans. Delayed adherent stem cells, named MuStem cells (muscle stem cells), were isolated from healthy dog muscle using a preplating technique. In vitro, MuStem cells displayed a large expansion capacity, an ability to proliferate in suspension, and a multilineage differentiation potential. Phenotypically, they corresponded to early myogenic progenitors and uncommitted cells. When injected in immunosuppressed dystrophic dogs, they contributed to myofiber regeneration, satellite cell replenishment, and dystrophin expression. Importantly, their systemic delivery resulted  in long-term dystrophin expression, muscle damage course limitation with an increased regeneration activity and an interstitial expansion restriction, and persisting stabilization of the dog’s clinical status. These results demonstrate that MuStem cells provide an attractive therapeutic avenue for DMD patients.

12 - Abstracts that will be presented in 16th World Muscle Society Meeting in Portugal - October 2011

10 - (Current Opinion in Neurology 2011, 24:415–422) Impending therapies for Duchenne muscular dystrophy

Terence A. Partridge - USA

Purpose of review:  As the first genetic disease for which the culpable gene was identified by positional cloning, Duchenne muscular dystrophy has served as a paradigm for therapeutic approaches to neuromuscular disease, in which role it has proved especially testing. The large mass and broad distribution of the target tissue, skeletal muscle, have stretched the patience and ingenuity of those seeking therapeutic delivery of the largest known gene. The most promising recent advances are summarized in this article. Recent findings: The main obstacle to genetic therapies has been the development of vectors able to efficiently deliver large, potentially therapeutic, genetic constructs to the large and widely dispersed mass of body musculature. Recombinant viral vectors that efficiently transduce muscle are unable to carry the full-length construct. Myogenic cells that are able both to carry full-length genes and to repair muscles are technically challenging to produce in sufficient quantity. A recent promising approach is the use of agents that obviate the mutation. Summary: Although genetic and cell-mediated approaches are currently showing genuine promise in preclinical and clinical trials, there remains considerable interest in the development of agents that ameliorate the downstream pathology. One general challenge is the three- way tension between the interests of patients, regulators, and the biotechnology industry.

Impending_therapies_DMD 

9 -  Forty-Eight Years with Duchenne Muscular Dystrophy

AUGUST

27 - The soy isoflavone genistein blunts nuclear factor kappa-B, MAPKs and TNF-a activation and ameliorates muscle function and morphology in mdx mice

17 - Researchers Combine Stem Cell with Artificial Chromosome to Treat Duchenne Muscular Dystrophy in Mice

Full article: Stem-cell- Mediated Transfer of a Human Artificial Chromosome Ameliorates Muscular Dystrophy

10 - Melatonin improves muscle function of the dystrophic mdx5Cv mouse, a model for Duchenne muscular dystrophy

JULY

25 - Exon skipping and dystrophin restoration in patients with Duchenne muscular dystrophy after systemic
phosphorodiamidate morpholino oligomer treatment: an open-label, phase 2, dose-escalation study

Exon-skipping therapy for Duchenne muscular dystrophy

25 - (Neuromuscular Disorders, 2011) Combination of steroids and ischial weight-bearing knee ankle foot orthoses in Duchenne’s muscular dystrophy prolongs ambulation past 20 years of age – A case report

Andrea C. Pardo, Twee Do, Ted Ryder, Amy Meyer, Lili Miles, Brenda L. Wong - USA

Patients with Duchenne muscular dystrophy (DMD) lose ambulation by age 12. Long-term steroids have lengthened ambulation by
2–5 years. Ischial weight-bearing knee ankle foot orthoses prolong ambulation for 2–3 years. We report the outcome of the ambulatory status of a patient with DMD treated with daily steroid therapy and orthoses. This male patient was diagnosed with DMD at age of 2. He has been treated with daily steroids since age 7 years. He lost the ability to arise from the floor and walk up steps at age 14 and lost ambulation by age 16. He was fitted with orthoses at age 16 following surgical correction of his lower extremity contractures and regained independent ambulation. At age 20, he was able to stand independently in his orthoses and take steps with moderate support. We conclude that a combination of daily steroids and orthoses prolongs ambulation beyond that of the natural history DMD

18 - (Circulation, 2011) Early Treatment With Lisinopril and Spironolactone Preserves Cardiac and Skeletal Muscle in Duchenne Muscular Dystrophy Mice

Jill A. Rafael-Fortney, Neeraj S. Chimanji, Kevin E. Schill, Christopher D. Martin, Jason D. Murray, Ranjit Ganguly, Jenna E. Stangland, Tam Tran, Ying Xu, Benjamin D. Canan, Tessily A. Mays, Dawn A. Delfín, Paul M.L. Janssen, and Subha V. Raman - USA

Background—Nearly universal cardiomyopathy in Duchenne muscular dystrophy (DMD) contributes to heart failure and death. Because DMD patients show myocardial fibrosis well before functional impairment, we postulated that earlier treatment using drugs with antifibrotic effect may be beneficial.

Methods and Results—Three groups of 10 utrn+/−;mdx, or “het” mice, deficient for dystrophin and haploinsufficient for utrophin with skeletal myopathy and cardiomyopathy that closely mimics clinical DMD were studied. One het group received spironolactone and lisinopril starting at 8 weeks of life (het-treated-8); a second received the same starting at 4 weeks of life (het-treated-4), and the third het group was untreated. At 20 weeks, all mice had normal ejection fractions though circumferential strain rate was abnormal (−0.21±0.08) in untreated hets. This improved to −0.40±0.07 in het-treated-8 mice (P=0.003) and further improved to −0.56±0.10 in het-treated-4 mice (P=0.014 for het-treated-4 versus het-treated-8). Treated mice showed less cardiomyocyte damage, with a 44% reduction in intracardiomyocyte serum immunoglobulin G localization in het-treated-8 mice (P<0.0001) and a further 53% reduction in het-treated-4 mice (P=0.0003 versus het-treated-8); matrix metalloproteinases were similarly reduced. Cardiac, limb, and diaphragm function by ex vivo muscle testing remained at 80% of normal with early treatment compared to a decline to 40% of normal skeletal muscle function without treatment.

Conclusions—These findings offer clinically available medications with proven antifibrotic effect as a new therapeutic strategy in DMD. Early initiation greatly attenuated myocardial disease and, for the first time with these drugs, improved skeletal myopathy. Thus, early initiation of such agents warrants further clinical evaluation to maintain ambulatory, respiratory, and cardiac function for patients with DMD and related myopathies

16 - Report on the Second Endocrine Aspects of Duchenne Muscular Dystrophy Conference December 1–2, 2010,
Baltimore, Maryland, USA

15 -  (Neurology, 2011) Randomized, blinded trial of weekend vs daily prednisone in Duchenne muscular dystrophy

D.M. Escolar, L.P. Hache, P.R. Clemens, A. Cnaan, C.M. McDonald, V. Viswanathan, A.J. Kornberg, T.E. Bertorini, Y. Nevo, T. Lotze, A. Pestronk, M.M. Ryan, E. Monasterio, J.W. Day, A. Zimmerman, A. Arrieta, E. Henricson, J. Mayhew, J. Florence, F. Hu, and A.M. Connolly - USA

Objective: To perform a double-blind, randomized study comparing efficacy and safety of daily and weekend prednisone in boys with Duchenne muscular dystrophy (DMD)

Methods: A total of 64 boys with DMD who were between 4 and 10 years of age were randomized at 1 of 12 centers of the Cooperative International Neuromuscular Research Group. Efficacy and safety of 2 prednisone schedules (daily 0.75 mg/kg/day and weekend 10 mg/kg/wk) were evaluated over 12 months

Results: Equivalence was met for weekend and daily dosing of prednisone for the primary outcomes of quantitative muscle testing (QMT) arm score and QMT leg score. Secondary strength scores for QMT elbow flexors also showed equivalence between the 2 treatment groups. Overall side effect profiles of height and weight, bone density, cataract formation, blood pressure, and behavior, analyzed at 12 months, did not differ between weekend and daily dosing of prednisone

Conclusions: Weekend dosing of prednisone is equally beneficial to the standard daily dosing of prednisone. Analysis of side effect profiles demonstrated overall tolerability of both dosing regimens

Classification of evidence: This study provides Class I evidence that weekend prednisone dosing is as safe and effective as daily prednisone in preserving muscle strength and preventing body mass index increases in boys with DMD over a 12-month period.

9 - Chronic losartan administration reduces mortality and preserves cardiac but not skeletal muscle function in dystrophic mice

2-  (Neuromuscular Disorders, 2011) Serum matrix metalloproteinase-9 (MMP-9) as a biomarker for monitoring disease progression in Duchenne muscular dystrophy (DMD)

V.D. Nadarajah, M. van Putten, A. Chaouch, P. Garrood, V. Straub, H. Lochmuller, H.B. Ginjaar, A.M. Aartsma-Rus,
G.J.B. van Ommen, J.T. den Dunnen, P.A.C. ’t Hoen - The Netherlands, United Kingdom

To identify serum biomarkers that allow monitoring of disease progression and treatment effects in Duchenne muscular dystrophy
(DMD) patients, levels of matrix metalloproteinase-9 (MMP-9), tissue inhibitors of metalloproteinase-1 (TIMP-1) and osteopontin
(OPN) were determined in 63 DMD patients on corticosteroid therapy. These proteins were selected for their role in the pathogenesis of muscular dystrophy. Levels of MMP-9 and TIMP-1 were significantly higher in sera of DMD patients compared to healthy controls, whereas the OPN levels showed no significant difference. MMP-9 levels were also observed to be significantly higher in older, nonambulant patients, compared to ambulant patients. Longitudinal data from a smaller cohort of DMD patients followed up for over 4 years showed that MMP-9, but not TIMP-1 increased significantly with age. Hence, MMP-9 is a potential DMD biomarker for disease progression. Future studies have to confirm whether serum MMP-9 levels can be used to monitor therapeutic response.

JUNE

25 - CINRG PILOT TRIAL OF COENZYME Q10 IN STEROID-TREATED DUCHENNE MUSCULAR DYSTROPHY

23 - CARDIOMYOPATHY OF DUCHENNE MUSCULAR DYSTROPHY: CURRENT UNDERSTANDING AND FUTURE DIRECTIONS

23 - Evaluation of the Therapeutic Utility of Phosphodiesterase 5A Inhibition in the mdx Mouse Model of Duchenne Muscular Dystrophy

23 - (Neurology, 2011) Cardiac abnormalities in a follow-up study on carriers of Duchenne and Becker muscular dystrophy

S.M. Schade van Westrum, E.M. Hoogerwaard, L. Dekker, T.S. Standaar, E. Bakker, P.F. Ippel, J.C. Oosterwijk, D.F. Majoor-Krakauer, A.J. van Essen, N.J. Leschot, A.A.M. Wilde, R.J. de Haan, M. de Visser, and A.J. van der Kooi - USA

Objectives: Cardiac involvement has been reported in carriers of dystrophin mutations giving rise to Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD). The progress of these abnormalities during long-term follow-up is unknown. We describe the long-term follow-up of dilated cardiomyopathy (DCM) in DMD/BMD carriers.

Methods: A long-term follow-up study was performed among Dutch DMD/BMD carriers first analyzed in 1995. A cardiac history was taken, and all carriers were assigned a functional score to assess skeletal muscle involvement. Electrocardiography and M-mode and 2-D echocardiography were performed. DCM was defined as an enlarged left ventricle with a global left ventricle dysfunction or fractional shortening less than 28%. Slow vital capacity of the lung was measured by a hand-held spirometer.

Results: Ninety-nine carriers were monitored with a median follow-up of 9 years (range 7.0–10.6 years). Eleven carriers with DCM (10 DMD, 1 BMD) were identified. Nine of them developed DCM in the follow-up period. One of the patients with DCM reported in the 1995 study died of cardiac failure at age 57 years. DCM was more frequently found in carriers who were functionally symptomatic

Conclusion: Cardiac abnormalities in DMD/BMD carriers are progressive, as in patients with DMD/BMD

 

15 -  (Pharmacological Research, 2011)  Enalapril treatment discloses an early role of angiotensin II in inflammation - and oxidative stress-related muscle damage in dystrophic mdx mice 

Anna Cozzoli, Beatrice Nico, Valeriana Teresa Sblendorio, Roberta Francesca Capogrosso, Maria Maddalena Dinardo, Vito Longo, Sara Gagliardi, Monica Montagnani, Annamaria De Luca - Italy

Inhibitors of angiotensin converting enzymes (ACE) are clinically used to control cardiomyopathy in patients of Duchenne muscular dystrophy. Various evidences suggest potential usefulness of long-term treatment with ACE inhibitors to reduce advanced fibrosis of dystrophic muscle in the mdx mouse model. However, angiotensin II is known to exert pro-inflammatory and pro-oxidative actions that might contribute to early events of dystrophic muscle degeneration. The present study has been aimed at evaluating the effects of an early treatment with enalapril on the pathology signs of exercised mdx mouse model. The effects of 1 and 5 mg/kg enalapril i.p. for 4-8 weeks have been compared with those of 1 mg/kg α-methyl-prednisolone (PDN), as positive control. Enalapril caused a dose-dependent increase in fore limb strength, the highest dose leading to a recovery score similar to that observed with PDN. A dose-dependent reduction of superoxide anion production was observed by dihydroethidium staining in tibialis anterior muscle of enalapril-treated mice, approaching the effect observed with PND. In parallel, a significant reduction of the activated form of the pro-inflammatory Nuclear Factor-kB has been observed in gastrocnemious muscle. Histologically, 5 mg/kg enalapril reduced the area of muscle necrosis in both gastrocnemious muscle and diaphragm, without significant effect on non-muscle area. In parallel no significant changes have been observed in both muscle TGF-β1 and myonuclei positive to phosphorylated Smad2/3. Myofiber functional indices were also monitored by microelectrodes recordings. A dose-dependent recovery of macroscopic chloride conductance has been observed upon enalapril treatment in EDL muscle, with minor effects being exerted in diaphragm. However a modest effect, if any, was found on mechanical threshold, a functional index of calcium homeostasis. No recovery was observed in creatine kinase and lactate dehydrogenase. Finally the results suggest the ability of enalapril to blunt angiotensin-II dependent activation of pro-inflammatory and pro-oxidant pathways which may be earlier events with respect to the pro-fibrotic ones, and may in part account for both functional impairment and muscle necrosis. The PDN-like profile may corroborate the combined use of the two classes of drugs in DMD patients so to potentiate the beneficial effects at skeletal muscle level, while reducing both spontaneous and PDN-aggravated cardiomyopathy.

8 - (J. Pharmacol. Exp. Ther., Jun 2011) Resveratrol ameliorates muscular pathology in the dystrophic mdx mouse, a model for Duchenne muscular dystrophy

Yusuke S Hori, Atsushi Kuno, Ryusuke Hosoda, Masaya Tanno, Tetsuji Miura, Kazuaki Shimamoto, and Yoshiyuki Horio - Japan

Muscular dystrophies are inherited myogenic disorders accompanied by progressive skeletal muscle weakness and degeneration. We previously showed that resveratrol (3,5,4'-trihydroxy-trans-stilbene), an anti-oxidant and activator of the NAD+-dependent protein deacetylase SIRT1, delays the progression of heart failure and prolongs the lifespan of δ-sarcoglycan-deficient hamsters. Because a defect of dystrophin-associated protein complex causes muscular dystrophies, and δ-sarcoglycan is a component of this complex, we hypothesized that resveratrol might be a new therapeutic tool for muscular dystrophies. Here we examined resveratrol′s effect in mdx mice, an animal model of Duchenne muscular dystrophy. Mdx mice that received resveratrol in the diet for 32 weeks (4 g/kg diet) showed significantly less muscle-mass loss and non-muscle interstitial tissue in the biceps femoris compared with mdx mice fed a control diet. In the muscles of these mice, resveratrol significantly decreased oxidative damage shown by the immunostaining of nitrotyrosine and 8-hydroxy-2′-deoxyguanosine and suppressed the up-regulation of NADPH oxidase subunits Nox4, Duox1, and p47phox. Resveratrol also reduced the number of α-smooth muscle actin (α-SMA)+myofibroblast cells and endomysial fibrosis in the biceps femoris, although the infiltration of CD45+ inflammatory cells and increase in TGF-β1 were still observed. In C2C12 myoblast cells, resveratrol pretreatment suppressed the TGF-β1-induced increase in reactive oxygen species, fibronectin production, and expression of α-SMA, and SIRT1 knockdown blocked these inhibitory effects. SIRT1 siRNA also increased the expression of Nox4, p47phox, and α-SMA in C2C12 cells. Taken together, these findings indicate that SIRT1 activation may be a useful strategy for treating muscular dystrophies.
 

4 - Successful bone marrow transplantation in a patient with Diamond-Blackfan anemia with co-existing Duchenne muscular dystrophy: a case report

MAY

27 - (ENDO 2011, Boston, 6-9 June) Puberty Is Delayed or Absent in Duchenne Muscular Dystrophy Boys on Chronic Glucocorticoid Therapy

MM Rutter, SR Rose, J Collins, H Sucherew, B Wong. Cincinnati Children's Hospital Medical Center and University of Cincinnati, Cincinnati, OH; Cincinnati Children's Hospital Medical Center and University of Cincinnati, Cincinnati, OH; Cincinnati Children's Hospital Medical Center, Cincinnati, OH.

Background: Duchenne Muscular Dystrophy (DMD) is a progressive neuromuscular disorder affecting 1 in 3500 boys. Chronic glucocorticoid (GC) treatment is considered standard therapy, and slows disease progression. However, GCs cause osteoporosis, growth failure, obesity, insulin resistance and pubertal delay, compromising quality of life. Absent or delayed puberty is an important problem which negatively impacts bone health, growth, self esteem and muscle strength. The prevalence in DMD is not known, and it is frequently under-recognized and untreated.
Objective: To determine the prevalence of delayed or absent puberty in DMD on GC therapy.
Methods: Patients aged 13 to 19 years with confirmed DMD on chronic daily GC treated in the Cincinnati Neuromuscular Comprehensive Care Center were studied. Clinical data was obtained from an IRB-approved database and chart review. Pubertal status was assessed annually by measuring early morning total testosterone concentrations and/ or pubertal examination by an endocrinologist.
Results: Of 64 boys aged ≥ 13 years, 56 (88%) had no evidence of puberty. Of 49 boys aged ≥ 14 years, 41(84%) showed no signs of puberty. Overall, 3 boys had pubertal arrest with very low testosterone levels for age or testicular size. Only 5 boys were in puberty; of these, 2 had 4 mL testes at age 14 years and 2 showed no significant progression over 2 years. Of 22 boys aged ≥ 16 years, 21 (95%) still had absent or arrested puberty. 47/59 (80%) boys with absent/ arrested puberty had severe osteoporosis. Absent puberty was distressing to 25 boys who as a result were treated with testosterone, 14 using topical gel.
Conclusions: Most DMD boys on chronic daily GC do not develop spontaneous puberty, or fail to progress. The problem does not tend to improve with age. Primary providers need an increased awareness to facilitate timely referral to an endocrinologist. We recommend clinical assessment and discussion by age 12 and referral by age 14 years. Management should be proactive and interdisciplinary, with attention to related co-morbidities such as osteoporosis.

27 - A dual acting compound releasing nitric oxide (NO) and ibuprofen, NCX 320, shows significant therapeutic effects in a mouse model of muscular dystrophy

27 - Rapamycin ameliorates dystrophic phenotype in mdx mouse skeletal muscle

21 - Membrane Sealant Poloxamer P188 Protects Against Isoproterenol Induced Cardiomyopathy in Dystrophin Deficient Mice

21 - Daily Treatment with SMTC1100, a Novel Small Molecule Utrophin Upregulator, Dramatically Reduces the Dystrophic Symptoms in the mdx Mouse

14 - Interview with Dr. Annemieke Aartsma-Rus   How to find which exon or exons to skip

11 - Chemical treatment enhances skipping of a mutated exon in the dystrophin gene

7 - Effective Drug Delivery System for Duchenne Muscular Dystrophy Using Hybrid Liposomes Including Gentamicin along with Reduced Toxicity

APRIL

22 - Research that will be presented in 14th American Society of Gene Therapy - May, 18-21, 2011, Seattle, USA

19 - (Clinical Biochemistry, 2011)  Melatonin treatment counteracts the hyperoxidative status in erythrocytes of patients suffering from Duchenne muscular dystrophy

Mariam Chahbouni, Germaine Escames, Luis C. López, Belén Sevilla, Carolina Doerrier, Antonio Muñoz-Hoyos, Antonio Molina-Carballo  and Darío Acuña-Castroviejo - Spain

Abstract:

Objectives: To analyzed whether the antioxidant melatonin could reduce the hyperoxidative status in the blood of patients with Duchenne's muscular dystrophy.

Design and methods: Ten patients aged 12.8 ± 0.9 years were treated with melatonin (60 mg at 21:00 h plus 10 mg at 09:00 h) for 9 months, and erythrocyte markers of oxidative stress were determined at 3, 6, and 9 months of treatment. Healthy age- and sex-matched subjects served as controls.

Results: Prior to treatment, the patients had higher glutathione disulfide/glutathione ratio and higher glutathione transferase and superoxide dismutase activities, and lower glutathione reductase activity than controls. After 3 months of melatonin treatment, the hyperoxidative status of these patients was counteracted, being reduced to the normal redox state between 3 and 9 months.

Conclusion: These results, together with the reduction in the inflammatory process and in muscle injury recently reported in the same patients, support the efficacy of melatonin therapy in DMD patients.

12 - THE MUSCLE-HYPERTROPHIC EFFECT OF CLENBUTEROL IS ADDITIVE TO THE HYPERTROPHIC EFFECT OF MYOSTATIN SUPPRESSION

12 - SOLUBLE ACTIVIN RECEPTOR TYPE IIB INCREASES FORWARD PULLING TENSION IN THE mdx MOUSE

9 - Effect of Intravenous Bisphosphonate Therapy among Boys with Duchenne Muscular Dystrophy and Vertebral Fractures due to Osteoporosis

9 - Rescue From Respiratory Dysfunction by Transduction of Full-length Dystrophin to Diaphragm via the Peritoneal Cavity in Utrophin/Dystrophin Double Knockout Mice
 

2 - Researchs that will be presented in "Experimental Biology 2011" in Washington, DC - April 2011

MARCH

27 - Systemic Administration of PRO051 in Duchenne's Muscular Dystrophy

23 - Idebenone as a novel, therapeutic approach for Duchenne muscular dystrophy: Results from a 12 month, double-blind, randomized placebo-controlled trial

9 - (Neuromuscular Disorders, 2011) Pre-clinical study of 21 approved drugs in the mdx mouse

Maïté Carre-Pierrat, Aude Lafoux, Guillaume Tanniou, Lucie Chambonnier, Alexandra Divet, Francoise Fougerousse, Corinne Huchet-Cadiou and Laurent Ségalat - France

Duchenne muscular dystrophy, a genetic disease caused by the absence of functional dystrophin, remains without adequate treatment. Although great hopes are attached to gene and cell therapies, identification of active small molecules remains a valid option for new treatments.We have studied the effect of 20 approved pharmaceutical compounds on the muscles of dystrophin-deficient mdx5Cv mice. These compounds were selected as the result of a prior screen of 800 approved molecules on a dystrophin mutant of the invertebrate animal model Cænorhabditis elegans. Drugs were administered to the mice through maternal feeding since 2 weeks of life and mixed in their food after the 3rd week of life. The effects of the drugs on mice were evaluated both at 6 weeks and 16 weeks. Each drug was tested at two concentrations. Prednisone was added to the molecule list as a positive control. To investigate treatment efficiency, more than 30 histological, biochemical and functional parameters were recorded. This extensive study reveals that tricyclics (Imipramine and Amitriptyline) are beneficial to the fast muscles of mdx mice. It also highlights a great variability of responses according to time, muscles and assays.

 

FEBRUARY

26 - Research that will be presented in American Academy of Neurology Meeting - April 2011

26 - Endocrine Aspects of Duchenne Muscular Dystrophy

22- Evaluation of potential synergistic action of a combined treatment with alpha-methyl-prednisolone
and taurine on the mdx mouse model of Duchenne muscular dystrophy

8 - (Journal of Cardiovascular Pharmacology and Therapeutics, Mar 2011; 16: 87 - 95) Losartan Decreases Cardiac Muscle Fibrosis and Improves Cardiac Function in Dystrophin-Deficient Mdx Mice

Christopher F. Spurney, Arpana Sali, Alfredo D. Guerron, Micaela Iantorno, Qing Yu, Heather Gordish-Dressman, Sree Rayavarapu, Jack van der Meulen, Eric P. Hoffman, and Kanneboyina Nagaraju - USA

Recent studies showed that chronic administration of losartan, an angiotensin II type I receptor antagonist, improved skeletal muscle function in dystrophin-deficient mdx mice. In this study, C57BL/10ScSn-Dmdmdx/J female mice were either untreated or treated with losartan (n = 15) in the drinking water at a dose of 600 mg/L over a 6-month period. Cardiac function was assessed via in vivo high frequency echocardiography and skeletal muscle function was assessed using grip strength testing, Digiscan monitoring, Rotarod timing, and in vitro force testing. Fibrosis was assessed using picrosirius red staining and Image J analysis. Gene expression was evaluated using real-time polymerized chain reaction (RT-PCR). Percentage shortening fraction was significantly decreased in untreated (26.9% ± 3.5%) mice compared to losartan-treated (32.2% ± 4.2%; P < .01) mice. Systolic blood pressure was significantly reduced in losartan-treated mice (56 ± 6 vs 69 ± 7 mm Hg; P < .0005). Percentage cardiac fibrosis was significantly reduced in losartan-treated hearts (P < .05) along with diaphragm (P < .01), extensor digitorum longus (P < .05), and gastrocnemius (P < .05) muscles compared to untreated mdx mice. There were no significant differences in skeletal muscle function between treated and untreated groups. Chronic treatment with losartan decreases cardiac and skeletal muscle fibrosis and improves cardiac systolic function in dystrophin-deficient mdx mice.

JANUARY

26 - (Am J Physiol Cell Physiol, Jan 2011) Nitric-oxide donors improve prednisone effects on muscular dystrophy in the mdx mouse diaphragm

Wataru Mizunoya, Ritika Upadhaya, Frank J. Burczynski, Guqi Wang, and Judy E Anderson

In Duchenne muscular dystrophy, palliative glucocorticoid therapy can produce myopathy or calcification. Since increased nitric oxide (NO) synthase activity in dystrophic mice promotes regeneration, the outcome of two NO-donor drugs, MyoNovin (M) or isosorbide dinitrate (I), on the effectiveness of the anti-inflammatory drug, prednisone (P, in alleviating progression of dystrophy was tested. Dystrophic mdx mice were treated (18 days) as: controls or with an NO-donor ± prednisone. Fiber permeability and DNA synthesis were labeled by Evans blue dye (EBD) and bromodeoxyuridine uptake, respectively. Prednisone decreased body-weight gain; M increased quadriceps mass and I increased heart mass. Prednisone increased fiber permeability (%EBD+ fibers) and calcification in diaphragm. Treatment with NO-donors plus prednisone (M+P, I+P) reduced the %EBD+ fibers and calcification vs. P alone. The %EBD+ fibers in M+P diaphragm did not differ from control. NO-donor treatment reduced proliferation and the population of c-met+ cells, and accelerated fiber regeneration. Concurrent with prednisone, NO-donor treatment suppressed two important detrimental effects of prednisone in mice, possibly by accelerating regeneration, re-balancing satellite cell quiescence and activation in dystrophy, and/or increasing perfusion. Results suggest NO donors could improve current therapy for DMD.

22 -  (Neuromuscular Disorders, 2011) Blockade of TNF in vivo using cV1q antibody reduces contractile dysfunction of skeletal muscle in response to eccentric exercise in dystrophic mdx and normal mice

A.T. Piers, T. Lavin, H.G. Radley-Crabb, A.J. Bakker, M.D. Grounds, G.J. Pinniger - Australia

This study evaluated the contribution of the pro-inflammatory cytokine, tumour necrosis factor (TNF) to the severity of exercise-
induced muscle damage and subsequent myofibre necrosis in mdx mice. Adult mdx and non-dystrophic C57 mice were treated with the mouse-specific TNF antibody cV1q before undergoing a damaging eccentric contraction protocol performed in vivo on a custom built mouse dynamometer. Muscle damage was quantified by (i) contractile dysfunction (initial torque deficit) immediately after the protocol, (ii) subsequent myofibre necrosis 48 h later. Blockade of TNF using cV1q significantly reduced contractile dysfunction in mdx and C57 mice compared with mice injected with the negative control antibody (cVaM) and un-treated mice. Furthermore, cV1q treatment significantly reduced myofibre necrosis in mdx mice. This in vivo evidence that cV1q reduces the TNF-mediated adverse response to exercise-induced muscle damage supports the use of targeted anti-TNF treatments to reduce the severity of the functional deficit and dystropathology in DMD.
 

22 -  (Neuromuscular Disorders, 2011) Bone is functionally impaired in dystrophic mice but less so than skeletal muscle  

Susan A. Novotny, Gordon L. Warren, Angela S. Lin, Robert E. Guldberg, Kristen A. Baltgalvis, Dawn A. Lowe -USA

The primary purpose of this study was to determine if tibial bone strength is compromised in dystrophic mice and if so, what geometric and material properties contribute. Results of three-point bending tests showed that tibia of mdx and dko (dystrophin- and utrophin-deficient) mice had up to 50% lower strength and stiffness compared to wild-type mice. Micro-computed tomography indicated that dystrophic tibia had reductions of 6–57% in cortical cross-sectional moment of inertia and cross-sectional area. Metaphyseal trabecular bone morphometry was also altered up to 78% in dystrophic mice. Bone-to-muscle functional ratios (i.e., three-point bending measures:muscle strength) indicated that bone strength was relatively high in 7-week-old dystrophic mice compared to muscle strength, but ratios were similar to wild-type mice by 24 months of age. Young dystrophic mice have compromised bone strength; these modelsmay be useful for designing therapeutic regimens aimed at improving the skeleton.

17 -  (Pediatrics, 2011) Impact of Bisphosphonates on Survival for Patients With Duchenne Muscular Dystrophy

Kevin E. Gordon,, Joseph M. Dooley, Kayla M. Sheppard, Judith MacSween, Michael J. Esser - Canada  

Objective In this article we describe the association of bisphosphonate therapy on survival within a regional cohort of patients with Duchenne muscular dystrophy (DMD) who received steroid therapy and were managed in a single center.

Patients and Methods The records of all patients with confirmed DMD who were born between 1963 and 2006 and who had received at least 1 year of steroid therapy were reviewed from birth until they reached the study end points (death, loss to follow-up, or the last follow-up was in 2009). A survival analysis was used to account for the variable follow-up duration within this cohort.

Results Forty-four boys from this cohort with DMD were exposed to continuous steroid use. Bisphosphonate therapy was initiated for 16 patients (36%) between 1997 and 2007 at a median age of 12.5 years (range: 7–23 years). At the time of the last follow-up in 2009, 13 patients had died (30%) at a median age of 16 years (range: 14–27 years). Survival curves demonstrate that the prescription of bisphosphonates was associated with a significant improvement in survival rate (P = .005, log-rank test). Furthermore, a possible therapy-duration effect could be shown for bisphosphonate use (P = .007, log-rank test).

Conclusions The treatment of patients with DMD with steroids and bisphosphonates seems to be associated with significantly improved survival compared with treatment with steroids alone.

12 - One-year Treatment of Morpholino Antisense Oligomer Improves Skeletal and Cardiac Muscle Functions in Dystrophic mdx Mice

12 - Biglycan recruits utrophin to the sarcolemma and counters dystrophic pathology in mdx mice

8 - (Neurology,2011) SPP1 genotype is a determinant of disease severity in Duchenne muscular dystrophy

E. Pegoraro, E.P. Hoffman, L. Piva, B.F. Gavassini, S. Cagnin, M. Ermani, L. Bello, G. Soraru, B. Pacchioni, M.D. Bonifati, G. Lanfranchi, C. Angelini, A. Kesari, I. Lee, H. Gordish-Dressman, J.M. Devaney, C.M. McDonald, and On behalf of the Cooperative International Neuromuscular Research Group

Objective: Duchenne muscular dystrophy (DMD) is the most common single-gene lethal disorder. Substantial patient–patient variability in disease onset and progression and response to glucocorticoids is seen, suggesting genetic or environmental modifiers

Methods: Two DMD cohorts were used as test and validation groups to define genetic modifiers: a Padova longitudinal cohort (n = 106) and the Cooperative International Neuromuscular Research Group (CINRG) cross-sectional natural history cohort (n = 156). Single nucleotide polymorphisms to be genotyped were selected from mRNA profiling in patients with severe vs mild DMD, and genome-wide association studies in metabolism and polymorphisms influencing muscle phenotypes in normal volunteers were studied

Results: Effects on both disease progression and response to glucocorticoids were observed with polymorphism rs28357094 in the gene promoter of SPP1 (osteopontin). The G allele (dominant model; 35% of subjects) was associated with more rapid progression (Padova cohort log rank p = 0.003), and 12%–19% less grip strength (CINRG cohort p = 0.0003).

Conclusions: Osteopontin genotype is a genetic modifier of disease severity in Duchenne dystrophy. Inclusion of genotype data as a covariate or in inclusion criteria in DMD clinical trials would reduce intersubject variance, and increase sensitivity of the trials, particularly in older subjects.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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