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Uso da melatonina em pacientes com distrofia muscular de Duchenne (13/03/10)

Espanha - melatonina é um hormônio produzido pelo organismo e que atua na regulação do sono e que apresenta propriedades anti-inflamatórias. No presente estudo 10 pacientes com Duchenne, em média com 13 anos, que foram tratados com melatonina (60mg a noite e 10 mg pela manhã). Os pesquisadores observaram uma significativa redução das citoninas inflamatória no sangue dos pacientes além da redução do stress oxidativo. O seguimento foi realizado por 9 meses e houve também redução das enzimas no sangue (CK, TGO, TGP, DHL). O estudo sugere que a melatonina pode contribuir para redução da degeneração dos músculos em Duchenne.

Estudo de um inibidor da desacetilação da histona nas arritmias dos camundongos com distrofia muscular (13/03/10)

Itália - inibidores da desacetilação da histona já demonstraram efeitos positivos em camundongos com distrofia muscular; neste estudo a avaliação foi feita sobre as arritmias em repouso, no stress e induzidas por drogas. Redução significativa das arritmias foi observada nos animais tratados; sugere-se que este efeito possa se relacionar com a ação da droga sobre canais de sódio. Esta classe de drogas necessita ser melhor estudada em modelos de distrofia muscular. O resumo em inglês pode ser lido abaixo:

(Cardiovasc Res, Mar 2010) The histone deacetylase inhibitor suberoylanilide hydroxamic acid reduces cardiac arrhythmias in dystrophic mice

Claudia Colussi, Roberta Berni, Jessica Rosati, Stefania Straino, Serena Vitale, Francesco Spallotta, Silvana Baruffi, Leonardo Bocchi, Francesca Delucchi, Stefano Rossi, Monia Savi, Dante Rotili, Federico Quaini, Emilio Macchi, Donatella Stilli, Ezio Musso, Antonello Mai, Carlo Gaetano, and Maurizio C. Capogrossi - Italy

Aims The effect of histone deacetylase inhibitors on dystrophic heart function is not established. To investigate this aspect, dystrophic mdx mice and wild-type (WT) animals were treated 90 days either with suberoylanilide hydroxamic acid (SAHA, 5 mg/kg/day) or with an equivalent amount of vehicle.

Methods and results The following parameters were evaluated: (i) number of ventricular arrhythmias in resting and stress conditions (restraint test) or after aconitine administration; (ii) cardiac excitability, conduction velocity, and refractoriness; (iii) expression and distribution of connexins (Cxs) and Na_v1.5 sodium channel. Ventricular arrhythmias were negligible in all resting animals. During restraint, however, an increase in the number of arrhythmias was detected in vehicle-treated mdx mice (mdx-V) when compared with SAHA-treated mdx (mdx-SAHA) mice or normal control (WT-V). Interestingly, aconitine, a sodium channel pharmacologic opener, induced ventricular arrhythmias in 83% of WT-V mice, 11% of mdx-V, and in 57% of mdx-SAHA. Epicardial multiple lead recording revealed a prolongation of the QRS complex in mdx-V mice in comparison to WT-V and WT-SAHA mice, paralleled by a significant reduction in impulse propagation velocity. These alterations were efficiently counteracted by SAHA. Molecular analyses revealed that in mdx mice, SAHA determined Cx remodelling of Cx40, Cx37 and Cx32, whereas expression levels of Cx43 and Cx45 were unaltered. Remarkably, Cx43 lateralization observed in mdx control animals was reversed by SAHA treatment which also re-induced Na_v1.5 expression.

Conclusion SAHA attenuates arrhythmias in mdx mice by a mechanism in which Cx remodelling and sodium channel re-expression could play an important role.

Estudo populacional sobre o uso de corticóides na distrofia muscular de Duchenne e Becker (06/03/10)

USA - os autores fazem uma revisão do uso de corticóides na prática clínica diária no período de 1991 a 2005 para tratamento de Duchenne e Becker nos Estados Unidos. O uso de corticoide aumentou nos últimos anos, chegando a 44% do total de pacientes. O uso se iniciou em média aos 6,9 anos. Os motivos para suspensão da medicação foram: ganho de peso, alterações comportamentais, parada da deambulação com o uso de cadeira de rodas continuadamente. O resumo em inglês pode ser lido abaixo:

(Journal of Child Neurology, 2010) Use of Corticosteroids in a Population-Based Cohort of Boys With Duchenne and Becker Muscular Dystrophy

Dennis J. Matthews, Katherine A. James, Lisa A. Miller, Shree Pandya, Kimberly A. Campbell, Emma Ciafaloni, Katherine D. Mathews, Timothy M. Miller, Christopher Cunniff, MD, F. John Meaney, Charlotte M. Druschel, Paul A. Romitti, P, and Deborah J. Fox - USA.

The use of corticosteroids for treatment of Duchenne and Beckermuscular dystrophy in clinical practice from 1991 through 2005was reviewed in a large population-based cohort (MD STARnet)of boys in 4 regional sites and 6 clinics of the United States.Corticosteroid use increased from 20% (11 of 56 individuals)in 1991 to 44% (93 of 218 individuals) in 2005. Average use varied by site and ranged from 15% to 49%. The median age of corticosteroid initiation was 6.9 years (range, 3.7-17.4 years). Dosage and growth information was available for 102 participantsand showed a median dose as 0.729 mg/kg for prednisone and 0.831mg/kg for deflazacort. T. The most common reasons that corticosteroidswere discontinued included weight gain, behavioral side effects,and loss of ambulation, resulting in full-time wheelchair use.Substantial variations in clinical practice were identifiedamong study sites.

Estudos clínicos com a droga Ataluren foram suspensos (05/03/2010)

USA - todos os estudos clínicos com a droga ataluren na distrofia muscular de Duchenne foram suspensos; pacientes que estavam recebendo a droga após os estudos prévios não receberão mais o medicamento até a conclusão final dos resultados. Novas informações serão divulgadas nos próximos dias.

Resultados iniciais da fase 2B com ataluren foram desanimadores (03/03/2010)

USA - hoje as empresas PTCTherapeutics and Genzyme divulgaram os primeiros resultados da fase 2B com a droga ataluren (PTC-124). A droga seria útil na mutação sem sentido (mutação de ponto) que está presente em menos de 15% dos pacientes. O objetivo primário era avaliar o efeito da droga sobre a capacidade de caminhar 6 minutos; não houve diferença entre os tratados e os controles. A droga foi bem tolerada, sem efeitos colaterais significativos e sem abandono do tratamento. As outras avaliações laboratoriais, biópsias, etc serão divulgadas nos próximos meses.

Homeopatia não tem comprovação, diz estudo britânico (27/02/2010)
Pesquisas que serão apresentadas no Congresso Anual da Academia Americana de Neurologia, em abril de 2010 (21/02/2010)

Canadá - neste ano o congresso realizar-se-á em Toronto e haverá a apresentação de 21 trabalhos relacionados com distrofia muscular e cujos resumos estão abaixo com uma pequena explicação sobre cada um deles:

1) Preliminary Results with AVI-4658 of Dystrophin Expression, Safety and Pharmacokinetics from the First Systemic Administration Study in Boys with Duchene Muscular Dystrophy (DMD), with a Phosphorodiamidate Morpholino Oligomer (PMO) to Skip Exon 51

Francesco Muntoni, London, United Kingdom, Kate Bushby, Newcastle upon Tyne, United Kingdom, Cirak Sebahattin, London, United Kingdom, Michela Guglieri, Newcastle upon Tyne, United Kingdom, Shirley Leow, Stephen B. Shrewsbury, Bothell, WA

OBJECTIVE: We have previously identified a PMO to skip exon 51 in DMD patients, restore the reading frame and enable dystrophin expression. The current study, on repeated IV treatment to select an effective, tolerated dose, should complete in February 2010. BACKGROUND: DMD is the commonest inherited muscular dystrophy, affecting 1 in every 3,500 boys. DMD boys present with leg weakness by age 5, wheelchair confinement by age 10-12; respiratory insufficiency requiring mechanical ventilation by late teens, and cardiomyopathy, placing a huge burden on patient and parents/caregivers. Out of frame deletions abolishing the production of the muscle protein dystrophin are the commonest mutations. DESIGN/METHODS: Open label, dose escalation study in ambulant DMD boys aged 5-15 years with relevant deletions, of 12 weekly administrations of AVI-4658; muscle biopsy to assess dystrophin expression at baseline and 14 weeks. Clinical parameters are followed for 26 weeks, consisting of safety (adverse events, physical examinations, laboratory tests), skeletal muscle, pulmonary and cardiac function, and pharmacokinetics at 1^st, 6^th and 12^th doses. A DSMB guides dose escalation decisions (across the doses 0.5, 1.0, 2.0, 4.0, 10.0 and 20.0 mg/kg). RESULTS: Cohorts 1, 2, 3 and 4 completed 12 weeks of dosing (October 2009). Cohort 1 has completed follow up to 26 weeks. Cohorts 5 and 6 are proceeding with dosing. No drug related SAEs or severe drug related AEs have been reported so far. To date, maximum cumulative PMO dose approaches 3,000mg. Preliminary analysis of the exon skipping, RNA and dystrophin protein expression will be presented and safety data updated. CONCLUSIONS/RELEVANCE: The first PMO for DMD has been well tolerated at all doses to date. Preliminary data is expected to show dystrophin expression at one or more doses and will allow dose selection for confirmatory clinical studies and extended (compassionate) use Supported by: Medical Research Council, UK and AVI Biopharma.

Pesquisa que descreverá os resultados do uso de oligonucleotídeos para pacientes com Duchenne e mutação do exon 51. Estudo ainda em andamento.

2) Preclinical Safety of AVI-4658, a Phosphorodiamidate Morpholino Oligomer (PMO) Being Developed To Skip Exon 51 in Duchenne Muscular Dystrophy

Peter Sazani, Stephen Shrewsbury, Bothell, WA

OBJECTIVE: The current studies were designed to assess the safety of AVI-4658 (and PMO in general). BACKGROUND: AVI-4658 is a PMO that skips dystrophin exon 51, restores the reading frame and enable dystrophin expression in selected DMD boys, proven by our recent single IM dose study in the UK. To enable clinical trials in the US, three 12-week GLP studies in animals were performed. Published data suggests the older phosphorothioate antisense oligonucleotides have dose limiting toxicities. DESIGN/METHODS: (1) mdx mice were dosed IV with 0, 12, 120 or 960mg/kg (the maximum feasible dose (MFD)), or subcutaneously at 960mg/kg; wild type C57 mice at 0 and 960 mg/kg (i.e., 7 groups) with AVI-4658. (2) A second identical study with AVI-4225, the PMO to skip exon 23 of in the dystrophic mouse and restore dystrophin, was also performed. (3) Cynomolgus monkeys were dosed IV with 0, 5, 40 or 320mg/kg (MFD) and 320mg/kg subcutaneously. A 28 day recovery period was included in all studies. RESULTS: In mice, both AVI-4658 and AVI-4225 were well tolerated at doses including 960 mg/kg/injection, with no adverse effects. Findings were generally limited to the kidney, and were generally reversible, as shown in the 28 day recovery groups. No evidence of kidney function change was detected. In cynomolgus monkeys, AVI-4658 was also well tolerated at all doses including 320 mg/kg/injection, with no adverse effects. Findings were similar to those seen in the mouse studies. CONCLUSIONS/RELEVANCE: AVI-4658, the first PMO for DMD, was extremely well tolerated at all doses in dystrophic mice, normal mice and primates. In addition, AVI-4225, which restores dystrophin in mdx mice, also led to no adverse effects. Based on this preclinical package, and encouraging safety and dystrophin expression results from a concurrent UK clinical study, US clinical studies are anticipated.

Pesquisa que descreve o uso de oligonucleotídeos em estudos experimentais para tratamento da mutação do exon 51 em portadores de distrofia muscular de Duchenne.

3) A Phase I/IIa Systemic Study on Antisense Oligonucleotide Compound PRO051 in Patients with Duchenne Muscular Dystrophy

Nathalie M. Goemans, Leuven, Belgium, Mar Tulinius, Gothenburg, Sweden, Gunnar Buyse, Leuven, Belgium, Sjef J. de Kimpe, Judith C. van Deutekom, Leiden, The Netherlands

OBJECTIVE: In this phase I/IIa open label study we evaluated the systemic delivery of the antisense oligonucleotide compound PRO051 in Duchenne Muscular Dystrophy (DMD) patients. BACKGROUND: DMD patients suffer from progressive muscle degeneration due to mutations in the DMD gene and the resulting absence of dystrophin at the muscle fiber membrane. PRO051, an antisense oligonucleotide compound, induces specific exon 51 skipping during pre-mRNA splicing, and can induce novel dystrophin expression in a subpopulation of DMD patients (Van Deutekom N Eng J Med 2007;357(26):2719-2722). DESIGN/METHODS: Twelve DMD patients received 5 weekly subcutaneous injections. Four dosing cohorts were applied (0.5 mg/kg, 2 mg/kg, 4 mg/kg, 6 mg/kg), three patients each. Muscle biopsies were taken at baseline for patients in cohort 1, and at two and seven weeks after the last administration for all patients. Adverse events were recorded and safety assessments (laboratory analysis and ECG) were performed at regular intervals. RESULTS: PRO051 induced specific exon 51 skipping in cohorts 2, 3 and 4, and novel dystrophin expression in a dose related manner in all cohorts. The treatment was well tolerated, none of the patients discontinued. A review of the safety data revealed no clinically significant changes in laboratory values and ECG. Antibodies against dystrophin were not detected. CONCLUSIONS/RELEVANCE: This is the first study showing successful systemic administration of an antisense oligonucleotide compound in DMD patients. An extension study is ongoing to collect at least 6 months safety data in all 12 patients at 6 mg/kg. A phase 3 study is in preparation. Supported by: Prosensa Therapeutics BV, the Netherlands.

Pesquisa que mostra os resultados iniciais do uso de oligonulceotídeos para tratamento da mutação do exon 51 em pacientes com Duchenne, demonstrando segurança do procedimento, sem efeitos colaterais.

4) Gentamicin Treatment of Duchenne Muscular Dystrophy Reinforces the Potential for Mutation Suppression Therapy

Vinod Malik, Louise R. Rodino-Klapac, Laurence Viollet, OH, Cheryl Wall, Wendy King, Roula Al-Dahhak, Sarah Lewis, Christopher J. Shilling, Janaiah Kota, Columbus, OH, John Hayes, Forest Grove, OR, John D. Mahan, Katherine J. Campbell, Columbus, OH, Brenda Banwell, Toronto, ON, Canada, Majed Dasouki, Victoria Watts, Kansas City, KS , Kumaraswamy Sivakumar, Ricardo Bien-Willner, Scottsdale, AZ, Kevin M. Flanigan, Zarife Sahenk, Columbus, OH, Richard J. Barohn, Kansas City, KS, Christopher M. Walker, Jerry R. Mendell, Columbus, OH

OBJECTIVE: Establish if the biopotency of gentamicin demonstrated in the mdx mouse can be confirmed in a clinical setting and if so, how could it be administered considering that readthrough would be an ongoing requirement. Address the percent increase in dystrophin expression required to provide clinically meaningful outcomes, and the potential immunogenicity of newly expressed dystrophin epitopes following treatment. BACKGROUND: Mutation suppression, also referred to as readthrough of stop codons, to restore the dystrophin gene is undergoing clinical trials in Duchenne muscular dystrophy (DMD). DESIGN/METHODS: Duchenne muscular dystrophy (DMD) subjects included: 1) Cohort 1 (n = 10) stop codon patients and Cohort 2 (n=8) frameshift controls receiving 14-days of gentamicin (7.5 mg/day). 2) Cohort 3 (n =12) and Cohort 4 (n=4) received an unprecedented six month delivery of weekly or twice-weekly gentamicin permitting an accumulating dystrophin pool to reach potential therapeutic levels. Readthrough was assessed in pre-and post-treatment biopsies and by clinical outcomes. RESULTS: In the 14-day biopotency study serum CK dropped by 50%, not seen in frameshift DMD controls. After 6-months of gentamicin, dystrophin levels significantly increased (p = 0.027) reaching levels 13% to 15% of normal accompanied by a drop in serum CK, stabilization of strength by manual muscle testing and a slight increase in forced vital capacity without adverse events. Stable transcripts that escaped nonsense mediated decay predicted the greatest increase of dystrophin following gentamicin. The efficiency of readthrough was not affected by either the stop codon or the fourth nucleotide surrounding the stop. Novel immunogenic epitopes were found in post-treatment biopsies by antigen specific IFN-g ELISpots. CONCLUSIONS/RELEVANCE: The results support on-going efforts to achieve drug-induced mutation suppression of stop codons. Immunogenic epitopes resulting from readthrough emphasize the importance of monitoring T cell immunity during clinical gene manipulation trials including mutation suppression, exon skipping and gene therapy. Supported by: NIH, NINDS, MDA, Jesse's Journey, The University of Kansas Medical Center GCRC Grant

Pesquisa que descreve os resultados do uso da gentamicina para tratamento da mutação sem sentido (mutação de ponto) em Duchenne. Houve uma redução da CK no sangue, aumento discreto da expressão da distrofina e discreto aumento da capacidade pulmonar.

5) Transient Expression of a Therapeutic Dystrophin Transgene in Duchenne Muscular Dystrophy Revealed by T Cell Mediated Immunity

Jerry Mendell, Katherine Campbell, Louise Rodino-Klapac, Zarife Sahenk, Christopher Shilling, Sarah Lewis, Columbus, OH, Dawn Bowles, Steven Gray, Chengwen Li, Chapel Hill, NC, Gloria Galloway, New Albany, OH, Vinod Malik, Brian Coley, Reed Clark, Columbus, OH, Juan Li, Xiao Xiao , Jade Samulski, Scott McPhee, R. Samulski, Chapel Hill, NC, Christopher Walker, Columbus, OH

OBJECTIVE: Describe the immune response following gene therapy for Duchenne muscular dystrophy (DMD) using adeno-associated virus (AAV) to transfer the mini-dystrophin gene. BACKGROUND: Gene therapy for DMD is a potentially promising means of gene replacement to restore dystrophin expression. This study reports the first clinical trial of viral mediated gene transfer in DMD. DESIGN/METHODS: Six DMD subjects were enrolled in a double-blind gene transfer study to the biceps muscle. Four were receiving glucocorticoid therapy at the time of gene transfer. The mini-dystrophin transgene used in this study encoded the actin binding domain, 5 rod spectrin repeats (R1, R2, R22, R23, and R24), 3 hinge domains (H1, H3 and H4), and the cysteine-rich domain. Expression was under control of the human cytomegalovirus immediate early promoter. Vector genomes were packaged in a hybrid AAV 2 capsid with 6 amino acid substitutions designed to minimize recognition by serum neutralizing antibodies. RESULTS: Four of six subjects with frame-shifting deletions in the DMD gene had detectable T cell responses to mini-dystrophin. Most of the targeted epitopes were non-self, located in sequences unique to the therapeutic dystrophin protein. However in at least one instance the dystrophin-specific T cells were present at low frequency before vector treatment and expanded rapidly after mini-dystrophin expression. The target of this unexpected memory T cell response was a self-epitope expressed from the defective dystrophin gene in revertant muscle fibers. CONCLUSIONS/RELEVANCE: This study illustrates the potential for a host response to foreign transgene products that are caused by large deletions or frame-shifting mutations. Recall of auto-reactive T cells has important clinical significance beyond gene therapy for DMD. Similar cell-mediated immune responses could be elicited by any strategy that increases expression of functional dystrophin in subjects with DMD and should be considered in the design and monitoring of experimental therapies for this disease. Supported by: The MDA and Jesse's Journey.

Pesquisa que descreve a reação imunológica que ocorre com a terapia gênica na distrofia muscular de Duchenne; crianças tratadas com vetor viral e o mini-gene da distrofina apresentaram linfócitos contra o gene da distrofina.

6) Initial Efficacy and Safety Evaluation in Cynomolgus Monkeys of AVI-5038, a Peptide Conjugated Phosphorodiamidate Morpholino Oligomer (PPMO) Being Developed To Skip Exon 50 in Duchenne Muscular Dystrophy

Peter Sazani, Stephen Shrewsbury, Bothell, WA

OBJECTIVE: To evaluate the efficacy of AVI-5038 at inducing skipping of dystrophin exon 50, as determined by RT-PCR, and also perform an initial toxicology assessment. BACKGROUND: AVI-5038 is a PPMO that induces dystrophin exon 50, and is designed to restore the reading frame and enable dystrophin expression in DMD patients. DESIGN/METHODS: Cynomolgus monkeys were dosed IV with 0, 3, or 9 mg/kg with AVI-5038, once weekly for 4 weeks. Following a 21 day recovery period, animals were sacrificed and a toxicological evaluation was performed. Selected muscle tissues were also evaluated by RT-PCR for evidence of skipping of dystrophin exon 50. RESULTS: AVI-5038 was well tolerated, with no adverse effects detected at doses up to 9 mg/kg. Toxicological findings were generally limited to the kidney, and included basophilic granules and instances of tubular degeneration / regeneration that was dose dependant. No clear evidence of kidney function change was detected, as shown by clinical chemistry and urinalysis evaluations. Significant levels of exon skipping were detected by RT-PCR in all major muscle groups evaluated, including diaphragm, heart, and quadriceps, at 9 mg/kg. CONCLUSIONS/RELEVANCE: AVI-5038, the first PPMO for DMD, was extremely well tolerated at all tested doses in primates. In addition, the safety data indicate that higher doses could be used to produce greater efficacy. Exon skipping was induced by the PPMO in healthy primate's muscles following systemic administration.

Estudo em macacos com o uso de oligonucleotídeos para tratamento da mutação do exon 51 na distrofia muscular de Duchenne.

7) Outcome Measures Validation Study for Mesoangioblasts Transplantation in Children Affected by Duchenne Muscular Dystrophy

Serena Bonfiglio, Alberto Lerario, Andrea Tettamanti, Sarah Marktel, Sara Napolitano, Stefano Previtali, Marina Scarlato, Maria Grazia Natali Sora, Nereo Bresolin, Giancarlo Comi, Roberto Gatti, Fabio Ciceri, Giulio Cossu, Yvan Torrente, Milano, Italy

OBJECTIVE: The aim of this study is to establish a reliable tool of reproducible assessment of muscle strength in children affected by Duchenne muscular dystrophy (DMD) which will be selected for mesoangioblasts transplantation. BACKGROUND: We have developed a potential treatment for DMD based on infusion of cells (mesoangioblasts) from a healthy donor capable. The results of the current functional study will hopefully establish reliable qualitative and quantitative tool to assess results of a future cell therapy clinical trial with mesoangioblasts. DESIGN/METHODS: This is a single centre, prospective, non-randomised, study of validation of outcome measures on 30 ambulant patients aged 5 to 12 years old affected by DMD including a cohort of 15 healthy aged matched males. We perform two days evaluation each three month for one year. During each assessment the following outcome measures are applied to DMD subjects: North Star Scale and 6 minute walking test during the first day; quantitative assessment using the Kin Com 125 machine during the second day. The controls subjects will perform quantitative assessment twice in a year. Twice during this evaluation year patients perform spyrometry, cardiac assessment and lower limb MRI. RESULTS: We divided the patients into 3 subgroups of age (5-7 years, 8-9 years, 10-12 years). The results of this preliminary part of the study show specific correlation between functional and quantitative tests in stronger children. Kin Com measurements correlate appropriately with functional tests for 10-12 years old DMD boys, while show a major variability in muscle strength for 8-9 years old DMD boys. The comparison with healthy subjects showed a difference of muscle strength that increases with age. CONCLUSIONS/RELEVANCE: This preliminary study demonstrates that our assessment may represent a useful tool to monitor the progress of DMD in ambulant children to determine the pre-transplantation story of the children who will be later treated with mesoangioblasts.

Pesquisa preliminar demonstrando como será realizada a avaliação dos pacientes que participarão do estudo clínico com o uso de mesangioblastos no tratamento da distrofia muscular de Duchenne.

8) The 6-Minute Walk Test in Duchenne Muscular Dystrophy: Longitudinal Observations

Craig McDonald, Erik Henricson, Sacramento, CA, Richard Abresch, Davis, CA, Jay Han, Alina Nicorici, Erica Goude, Sacramento, CA, Gary Elfring, Allen Reha, Samit Hirawat, Langdon Miller, S. Plainfield, NJ

OBJECTIVE: To develop the six-minute walk test (6MWT) as a clinically-valid outcome measure for ambulatory boys with Duchenne muscular dystrophy (DMD). BACKGROUND: We evaluated longitudinal changes in the ambulation of boys with (DMD) and age-matched controls, ages 4-13 years, using a 6MWT modified for use in DMD therapeutic trials. DESIGN/METHODS: We tested 18 boys with confirmed DMD and 22 healthy boys, ages 4-13 years using a our previously reported 6MWT methodology. Boys were tested at baseline and after 1 year. RESULTS: The median [range] test-retest interval was 58 [35-84] weeks in boys with DMD and 66 [51-114] weeks in healthy boys. At baseline, 13/18 (72%) boys with DMD were using corticosteroids. The groups were similar in age, height, and weight. Mean six-minute walk distance (6MWD) declined (p=0.054) in boys with DMD (mean [SD] change = -54 [100] m) but increased in healthy boys (mean [SD] change = 16 [54] m). Mean stride length decreased in boys with DMD (mean [SD] change = -0.03 [0.18] m) but increased in healthy boys (mean [SD] change = 0.03 [0.18] m). Cadence decreased in boys with DMD (mean [SD] change = -9 [15] strides/min) but was stable in healthy boys (mean [SD] change = 0.02 [9] strides/min). In boys with DMD, change in 6MWD correlated strongly with change in stride length (r=0.94, p<0.0001) and change in cadence (0.71, p=0.0045). In healthy boys, change in 6MWD correlated strongly with change in stride length (r=0.73, p=0.0001) but not at all with change in cadence (r=0.002). CONCLUSIONS/RELEVANCE: This modified 6MWT shows clinically important changes over an 1-year period. These changes are age-dependent, consistent with the known natural history of DMD. Improvement or stabilization of 6MWD during the course of a long-term therapeutic trial would represent a clinically meaningful benefit for boys with DMD. Supported by: PTC Therapeutics, Inc.

Descreve a validade do uso do teste de caminhar 6 minutos na avaliação de pacientes com distrofia muscular de Duchenne.

9) Risk Factors for Fractures in the Muscular Dystrophy Surveillance, Tracking and Research Network Cohort

Lisa A. Miller, Kathy James, Denver, CO, Katherine Mathews, Iowa City, IA, Shree Pandya, Rochester, NY, Susan Apkon, Seattle, WA, Chris Cunniff, Tucson, AZ

OBJECTIVE: To determine the occurrence of fractures and assess risk factors for fractures among the Muscular Dystrophy Surveillance, Tracking and Research Network (MD STARnet) cohort, a large population-based cohort of individuals with well-characterized dystrophinopathy. BACKGROUND: Fracture occurrence is a significant problem in Duchenne muscular dystrophy, with reported rates between 21% and 44%. DESIGN/METHODS: All males with definite or probable dystrophinopathy identified through MD STARnet who were greater than 3 years of age and had a birth year from 1982 to 2006 were included. Data were abstracted annually from medical records for all individuals with a potential dystrophinopathy in the surveillance regions of Arizona, Colorado, Iowa, Georgia and Western New York. Cox proportional hazard modeling, a complex multivariate survival analysis, was used to assess risk factors for fractures. For each patient, wheelchair use (part-time or full-time); bisphosphonate, steroid, and calcium/vitamin D use prior to fracture; and the duration of each were determined at each month of fracture occurrence. RESULTS: There were 187 first fractures among 578 individuals (32.4 percent). The most common site was the femur (31.4 percent), followed by the tibia/fibula (16.0 percent). The fracture risk for individuals who were in a wheel chair full-time was 3.2 times higher than for individuals who were ambulating, but for every month of full-time wheel chair use, risk decreased by 2.0 percent. For each additional month of steroid use beyond 6 months, fracture risk increased by 1.2 percent. Calcium/vitamin D and bisphosphonate use prior to fracture did not significantly affect risk. CONCLUSIONS/RELEVANCE: Our results confirm the high rate of fractures in individuals with dystrophinopathy and their relation to steroid use. In this population, the risk of fracture is higher during early wheel chair use and decreases over time. Fracture risk in these individuals is likely complex and the result of multiple factors. Supported by: Centers for Disease Control and Prevention Cooperative Agreement (5U01DD000191).

Descrição do risco de fraturas em Duchenne: maior risco com o uso de cortícóides, no começo do uso da cadeira de rodas e redução do risco ao longo do tempo. O risco de fraturas é um resultado de múltiplos fatores.

10) Disparities in the Diagnosis of Duchenne and Becker Muscular Dystrophy: Data from the MDSTARnet, 1999-2007

Christopher Cunniff, Jennifer Andrews, Tucson, AZ, Emma Ciafaloni, Rochester, NY, Deborah Fox, Troy, NY, Caleb Holtzer, Zhenqiang Lu, Tucson, AZ, Lisa Miller, Denver, CO, John Meaney, Tucson, AZ

OBJECTIVE: To characterize the association of sociodemographic factors with delays at specific steps in the diagnosis of Duchenne and Becker Muscular Dystrophy (DBMD). BACKGROUND: Prior studies of DBMD observed diagnostic delays ranging from 1.9 to 4.3 years, with diagnosis occurring around age 5 years. No studies report on sociodemographic disparities in the diagnostic process. DESIGN/METHODS: We analyzed medical records for 593 boys in the MDSTARnet database and assessed mean age differences by sociodemographic group at first sign or symptom, initial medical evaluation, and first creatine kinase (CK) test using generalized linear regression models and T tests. We assessed the uptake of diagnostic testing and mutation analysis using the Cox Proportional Hazard Model. RESULTS: Family history information was available for 549 boys. Of these, 174 (29.3%) had a known family history of DBMD prior to diagnosis. Boys with a family history were diagnosed at 33 months and experienced each step to diagnosis at a younger age than boys without a family history (p<.001). Boys without a family history were diagnosed on average at 66 months. Within this group, age at evaluation was younger for boys with more educated mothers (p<.001). Whites underwent CK and DNA testing earlier than Blacks and Hispanics (p<.005). Mean ages for boys grouped by socioeconomic status or place of residence were not different at any time point. The rate of point mutation analysis following a negative deletion/duplication test was greater among boys with more educated mothers (p<.001). CONCLUSIONS/RELEVANCE: In the MDSTARnet population, children without a family history are diagnosed at age 5 years, 6 months, despite longstanding symptoms. Differences in race/ethnicity and mother education are associated with longer delays at multiple steps in the diagnostic process. The reasons for such delay are not known but may result from lack of awareness of symptoms or decreased access to diagnostic services. Supported by: A cooperative agreement from the Centers for Disease Control and Prevention through the Association of American Medical Colleges, grant number U36/CCU319276, AAMC ID number MM-1064-09/09. Publication and report contents are solely the responsibility of the authors and do not necessarily represent the official views of the AAMC or the CDC. This study was also funded by the Centers for Disease Control and Prevention under the Cooperative Agreement for Surveillance and Epidemiologic Research of Duchenne and Becker Muscular Dystrophy DD000187, DD000189, DD000190, DD000191.

Estudo epidemiológico; diagnóstico é mais precoce de Duchenne e Becker quando já há casos na família; se não o diagnóstico é feito mais tardiamente, após os 5 anos, apesar dos sintomas. Há diferença neste tempo dependendo do nivel cultural das mães e com fatores raciais.

11) Respiratory Care Trends for Duchenne and Becker Muscular Dystrophies (DBMD): Data from the MD STARnet, 2001-2007

Daniel A. Mandel, Atlanta, GA, Daniel W. Sheehan, Buffalo, NY, Shree Pandya, Rochester, NY, Christina P. Westfield, Deborah J. Fox, Troy, NY, Sarah K. Nabukera, Katherine Mathews, Iowa City, IA, Christopher Cunniff, Tucson, AZ, Carolyn M. Constantin, Atlanta, GA, David J. Birnkrant, Cleveland, OH

OBJECTIVE: To profile forced vital capacity (FVC) monitoring for males who have DBMD and to evaluate non-invasive positive pressure ventilation (NPPV) and mechanical insufflator/exsufflator (MI/E) use among males with very low FVC measurements. BACKGROUND: Semiannual FVC measurements are recommended for patients age > 12 who have Duchenne muscular dystrophy. FVC measurements of < 1 L are associated with complications of hypoventilation and early mortality. NPPV and MI/E are used to support patients with poor pulmonary function. DESIGN/METHODS: MD STARnet is a population-based surveillance system that identifies all patients who have DBMD in defined geographic areas. MD STARnet records were analyzed for 132, 177, and 199 males age > 12 in 2001, 2004, and 2007, respectively. Inclusion criteria for FVC < 1L were: > 5 recorded FVC measurements; > 2 consecutive FVC measurements < 1 L; and no subsequent FVC measurements > 1L. Sample sizes for FVC < 1 were 11, 22, and 33 in 2001, 2004, and 2007, respectively. Logistic regression models were used to investigate NPPV and MI/E use over time, clustering on patient. RESULTS: The percentage of males who have DBMD age > 12 years with a recorded FVC measurement ranged from 60-65% in the three study years. The rates of NPPV use significantly increased among males with FVC < 1L (Wald X² = 11.5, p = .003) over the study period. NPPV rates ranged from a low of 27% in 2001 to a high of 85% in 2007. Rates of MI/E use followed a less clear pattern: 18% in 2001; 9% in 2004, 58% in 2007. CONCLUSIONS/RELEVANCE: Data show consistent FVC monitoring for males who have DBMD and increasing use of NPPV for males who have very low FVC measurements between 2001 and 2007. MI/E was relatively underused compared with NPPV. Supported by: CDC cooperative agreements DD000187, DD000189, DD000190, and DD000191.

Estudo da função respiratória na distrofia muscular de Duchenne e o uso da ventilação não invasiva no período de 2001 a 2007.

12) Neuropsychological Profile of Adult Patients with Duchenne Muscular Dystrophy

Natalia Sierra, Lilia Mesa, Alberto Dubrovsky, Pablo Sojo, Teresa Torralva, María Roca, Fernando Chloaca, Laura Pirra, Facundo Manes, Buenos Aires, Argentina

OBJECTIVE: To analyze the cognitive profile of adult patients with normal IQ diagnosed with Duchenne Muscular Dystrophy (DMD). BACKGROUND: Research on muscular dystrophies has focused extensively on the impact of peripheral neural structures affected by the degenerative nature of the disease. However, investigating the cognitive impairment in these patients may contribute to the understanding of the pathophysiological changes occurring on the central nervous system (CNS) and its relationship with peripheral structures. DESIGN/METHODS: Ten patients with diagnosis of DMD aged between 17 and 28 years were assessed with a general comprehensive cognitive battery as well as a specific executive battery. All patients had within normal IQ scores. RESULTS: Tasks associated with a motor component such as the Complex Rey Figure (z= -2,02) and TMT-A (z= -2,31) and TMT-B (z=-2,47) were impaired. Performance deficits were also found on tasks of verbal inhibitory control (z=-3,02) with normal scores on reading speed (z=-0,69) and color naming (z=-0,81) on the Stroop task. Decreased scores were observed for tasks of theory of mind (z=-1,19) and decision-making (Iowa Gambling Task), although performance on the latter did not correlate significantly with theory of mind or verbal inhibitory control scores (both p > .05). CONCLUSIONS/RELEVANCE: These results are in accordance with previous studied (e.g. Hinton et al, 2007) and suggest that normal IQ patients with DMD present theory of mind and verbal inhibitory control deficits. Our results also revealed decision-making impairments, although apparently not associated with theory of mind and executive deficits.

Estudo através de testes das funções cognitivas em pacientes com distrofia muscular de Duchenne e QI normal demonstrando deficit do controle inibitório verbal.

13) Pulmonary Function Characteristics of Boys with Duchenne and Becker Muscular Dystrophy by Age Groups and Steroid Use: One-Year Data from the CINRG Longitudinal Study Project

R. Ted Abresch, Craig M. McDonald, Davis, CA, Jay J. Han, Sacramento, CA, Robert Leshner, Washington, DC, Diana Escolar, McLean, VA, Avital Cnaan, Eric Hoffman, Adrienne Arrieta, Tina Duong, Fenming Hu, Washington, DC, Julaine Florence, Saint Louis, MO, CINRG Investigators, Washington, DC

OBJECTIVE: To describe the pulmonary function characteristics of Duchenne Muscular Dystrophy (DMD) over a one-year time period. BACKGROUND: Lack of well-characterized pulmonary function data inhibits the development of therapeutic clinical trials in DMD. DESIGN/METHODS: Pulmonary function tests (PFTs) were performed in subjects with confirmed DMD at ages <7, 7-12, 13-18 and > 18 in 20 centers from the Cooperative International Neuromuscular Research Group at baseline and 12 months. Fifteen percent of subjects were steroid nave. Pulmonary function measures included absolute and % predicted forced vital capacity (FVC and %FVC), forced expiratory volume in 1 second a (FEV₁, %FEV₁), peak expiratory flow rate (PEFR, %PEFR), maximum inspiratory pressure (MIP, %MIP), and maximum expiratory pressure (MEP, %MEP). Significance was accepted at p < 0.05. RESULTS: There was a significant one-year increase in FVC (0.15l 0.1 [sd]), FEV₁ (0.15l 0.2) and PEFR (0.49 l/s 0.5) in the <7 year age groups (n=10). There was a significant one-year increase in FVC and FEV1 (0.11l 0.2 and 0.13l 0.2, respectively; n=85), as well as MIP and MEP (3.7 cmH₂0 12.5 and 5.7 cmH₂0 10.9, respectively; n = 102) in the 7-12 year age groups. There was a significant one-year decline in %FVC and %PEFR (-5.8% 5.7 and 5.0% 10.3, respectively) in the 13-18 year age groups (n=57). At age 19 (n=23) there was a significant one-year decline in FVC (-0.14l 0.2), %FVC (-3.0% 3.4), FEV₁ (-0.14l 0.2), %FEV₁ (-3.6% 3.9), %PEFR (-3.5% 6.5) and %MIP (-2.7% 2.8). No other PFT measures exhibited significant differences over a one-year period. CONCLUSIONS/RELEVANCE: Pulmonary function testing reflects changes associated with growth at age groups <7 and 7-12. DMD subjects exhibit significant one-year PFT declines in the 13-18 and >18 year age groups. Supported by: National Institute of Disability and Rehabilitation Research Grant H133B980008-03; National Institutes of Health Grant 1U54HD053177-01A1; Department of Defense Grant 0616USAMRAA.

Estudo de um ano da função pulmonar na Distrofia muscular de Duchenne, demonstrando significante declínio da função pulmonar após os 13 anos.

14) Functional Motor Performance Characteristics of Boys with Duchenne Muscular Dystrophy by Age Groups and Steroid Use: One-Year Data from the CINRG Longitudinal Study Project

Craig McDonald, Erik Henricson, Sacramento, CA, Richard T. Abresch, Davis, CA, Jay J. Han, Sacramento, CA, Robert Leshner, Washington, DC, Diana Escolar, McLean, VA, Eric Hoffman, Avital Cnaan, Addrienne Arrietta, Fenming Hu, Angela Zimmerman, Tina Duong, Washington, DC, Julaine Florence, Saint Louis, MO, CINRG Investigators, Washington, DC

OBJECTIVE: To describe the changes in timed function testing (TFT) in Duchenne muscular dystrophy (DMD) over one-year and the predictive value of TFTs for determining loss of ambulation. BACKGROUND: In DMD, TFT measures are used as clinical endpoints for therapeutic clinical trials and may predict loss of ambulation over one-year. DESIGN/METHODS: TFTs were performed in 255 subjects with confirmed DMD at ages <7, 7-12, and >13 in 20 centers from the Cooperative International Neuromuscular Research Group at baseline and 12 months. Fifteen percent of subjects were steroid nave. TFTs (in seconds) included run/walk 10 meters, climb 4 steps, and standing from lying. Significance was accepted at p < 0.05. RESULTS: There was a mean decrease in all TFTs over one-year for the <7 year group (n=56): run/walk 10 meters (-0.46 1.7[sd]), climb 4 steps (-1.83 4.4) and standing from lying (-0.65 2.9). There was a significant one-year increase in all TFTs in 7-12 year olds (n=65): run/walk 10 meters (+1.38 1.8), climb 4 steps (+2.47 4.9) and standing from lying (+3.42 6.1). Those >13 also increased time to run/walk 10 meters (+3.21 5.2, n=10) and climb 4 steps (+1.58 1.3, n=7). Only one subject >13 was able to perform standing from lying. Loss of ambulation over 12 months was compared for three groups: baseline run/walk 10 meters < 6 seconds, 6-12 seconds, and >12 seconds. Survival analysis for the milestone of loss of ambulation showed all three groups to be significantly different using a Log-rank test (p<0.0001). CONCLUSIONS/RELEVANCE: In DMD, TFTs show relative improvement with time in younger subjects <7 years. For those 7 and older TFTs show disease-related progression in both steroid-users and steroid-nave subjects. Timed to run/walk 10 meters is predictive of loss of ambulation over the following 12 months. Supported by: National Institute of Disability and Rehabilitation Research Grant H133B980008-03; National Institutes of Health Grant 1U54HD053177-01A1; Department of Defense Grant 0616USAMRAA

Estudo de um ano da função motora em pacientes com Duchenne. O estudo da marcha em 10 metros foi preditivo da parada de deambulação após 12 meses.

15) A Cooperative International Neuromuscular Research Group (CINRG) Study of the Relationship between Impairment, Activity Limitation, Participation and Quality of Life in Persons with Confirmed Dystrophinopathies: One Year Follow-Up of Skeletal Muscle Strength and Timed Motor Performance

Erik Henricson, Craig McDonald, Sacramento, CA, Richard Abresch, Davis, CA, Jay Han, Sacramento, CA, Robert Leshner, Eric Hoffman, Washington, DC, Diana Escolar, McLean, VA, Avital Cnaan, Fengming Hu, Angela Zimmerman, Tina Duong, Washington, DC, Julaine Florence, Saint Louis, MO, Adrienne Arrieta, CINRG Investigators, Washington, DC

OBJECTIVE: To evaluate 12-month change in skeletal muscle strength and timed motor function tests in individuals with Duchenne muscular dystrophy (DMD) aged 2-28 years. BACKGROUND: Lack of adequate natural history data in steroid-treated DMD and lack of well-characterized outcome measures across age ranges limits the ability to assess therapeutic effectiveness in clinical trials in DMD. DESIGN/METHODS: We enrolled males with confirmed DMD from 20 participating centers from 10 countries of the Cooperative International Neuromuscular Research Group (CINRG). Ambulatory and transitioning participants underwent strength assessment (modified MRC manual muscle test (MMT), quantitative muscle tests (QMT) of grip, elbow flexion/extension, knee flexion/extension) and timed function tests ((TFT) stand from supine, run/walk 10m, climb 4 stairs) at baseline, and months 3, 6, 9, 12. RESULTS: 255/347 males with DMD aged 2 to 28 years of age underwent strength and functional testing, and 15% were glucocorticoid-nave. Significant changes over 12 months included: Children <7 years decreased time to climb 4 steps by 1.83(SD=4.4)s (p<0.0001 N=56) and increased quantitative grip strength by 2.96(SD=2.5)lbs (p<0.0001 N=37) and knee flexors by 1.18(SD=2.6)lbs (p<0.04 N=37). Children aged 7-12 years increased time to run/walk 10m by 1.38(SD=1.8)s (p<0.0001 N=65), time to climb 4 stairs by 2.47(SD=4.9)s (p<0.0001 N=65) and time to stand from supine by 3.42(SD=6.1)s (p<0.0001 N=65). Children aged 13-18 years decreased quantitative elbow extensor strength by 1.23(SD=1.1)lbs (p<0.001 N=61) and elbow flexor strength by 0.98(SD=1.3)lbs (p<0.01 N=61) and decreased manual muscle test score by 0.32(SD=0.4) points (p<0.01 N=61). Adults aged >18 years who were testable decreased quantitative grip strength by 0.6(SD=0.9)lbs (p<0.02 N=31). CONCLUSIONS/RELEVANCE: Few measures of strength and function in steroid-treated boys with DMD show significant disease-related changes over one-year. Relative stability over this time suggests that clinical trials must demonstrate improvement rather than stabilization using these measures. Supported by: National Institute of Disability and Rehabilitation Research Grant H133B980008-03 National Institutes of Health Grant 1U54HD053177-01A1 Department of Defense Grant 0616USAMRAA.

Estudo de um ano das alterações de força muscular em pacientes com Duchenne de 2 a 28 anos, demonstrando poucas alterações em um pequeno intervalo de tempo.

16) Multiple Sclerosis in a Patient with Duchenne Muscular Dystrophy

Adrienne A. Salomon, Negar Sodeifi, Babak Movassaghi, David Libell, Morgantown, WV

OBJECTIVE: To describe a rare association of multiple sclerosis (MS) in a patient with Duchenne Muscular Dystrophy (DMD). BACKGROUND: MS is an autoimmune disorder that most commonly begins between ages 20 and 40, but can be seen at any age. The average age at diagnosis is 31 years in males. DMD is an X-linked disorder which affects the DYS gene. It is the most common muscular dystrophy and carries an average life expectancy ranging from early teens to mid30s. No reported association between these two conditions was found. DESIGN/METHODS: Case report involving a 23-year-old man with known DMD who presented with unilateral left eye blurry vision. The patient admitted to having suffered a similar episode of visual changes approximately one year prior. Examination revealed left eye visual acuity of 20/100 with abnormal enhancement of the left optic nerve on MRI consistent with optic neuritis. MRI of the brain demonstrated multiple periventricular and juxtacortical lesions with post-contrast enhancement. Lumbar puncture was significant for increased oligoclonal bands with elevated IgG index (1.63) and myelin basic protein (3.5g/L). While the patient's diagnosis of DMD had previously been established by neurological examination, family history, elevated creatine kinase levels, and DNA analysis, the patient's new findings were consistent with a diagnosis of MS based on McDonald criteria. RESULTS: The patient received high-dose intravenous solumedrol for three days. The patient improved minimally during his hospital course and was discharged on a steroid taper. Follow-up examination confirmed resolution of his blurry vision with no further symptoms at six months. CONCLUSIONS/RELEVANCE: We report the rare association between DMD and MS. The patient's clinical course suggests that DMD did not impact the clinical course or treatment of his MS. The fact that many DMD patients do not survive long enough to manifest symptoms of MS may partly explain the rare association of these two disorders.

Descrição de um caso raro: paciente com Duchenne e que desenvolve a esclerose múltipla, uma doença auto-imune.

17) Expression of Heat Shock Proteins in Skeletal Muscle from Idiopathic Inflammatory Myopathy and Duchenne Muscular Dystrophy Patients

Jan L. De Bleecker, Kim K. Creus, Ghent, Belgium, Jean-Jacques Martin, Antwerp, Belgium, Joachim Weis, Aachen, Germany, Boel De Paepe, Ghent, Belgium

OBJECTIVE: To investigate heat shock protein families 70 (HSP70) and HSP90 in idiopathic inflammatory myopathies (IIM) and Duchenne Muscular dystrophy (DMD). BACKGROUND: HSP70 and HSP90 chaperones assure proper protein folding and activity. Furthermore, HSP90 enhances the cytotoxic activity of inflammatory cells. In view of current approaches exploring anti-HSP90 therapy in inflammatory diseases, more in-depth knowledge of the individual pros and cons of chaperones could be relevant. DESIGN/METHODS: The expression of HSP70 and HSP90 was investigated in muscle biopsies from controls, and from IIM and DMD patients using immunofluorescence, in situ hybridization and Western blotting. RESULTS: Inflammatory cells in IIM and DMD expressed low levels of HSP70, HSP90 expression was increased in macrophages and cytotoxic T-cells in proximity of invaded nonnecrotic myofibers of PM/IBM. HSP90alpha mRNA was localized in endomysial infiltrates of PM/IBM along with faint HSP90beta expression. Part of the invaded nonnecrotic myofibers showed sarcolemmal staining for HSP70 and HSP90 proteins. The sarcoplasm of most small fibers, and some normal appearing myofibers were strongly HSP70 positive. Double staining showed important overlap between HSP70 and HSP90alpha in small NCAM+ fibers, and rare co-localization with HSP90beta. Western blotting detected HSP70 and HSP90 proteins in all muscle tissues, but protein levels were increased in all (HSP70) or part of (HSP90) IIM/DMD patients. In normal controls that had received glucocorticoids prior to biopsy, nuclear over cytoplasmic protein ratios were increased for HSP70, and decreased for HSP90. CONCLUSIONS/RELEVANCE: Our data appoint pathological and physiological roles for HSP70 and HSP90, ascribing these factors both adverse and beneficiary potential. On the one hand, HSP90 was associated with the active invasion targeting the nonnecrotic myofibers in PM/IBM. On the other hand, a strong expression of HSP70 occured in myofibers at different regeneration-stages, with important but no absolute overlap with HSP90 in the small regenerating muscle fibers, indicating both general and member-specific involvement. Supported by: L'Association Franaise contre les Myopathies (AFM, France) L'Association Belge contre les Maladies neuro-Musculaires (ABMM, Belgium) The Muscular Dystrophy Association (MDA, USA).

Pesquisa que estuda proteínas musculares que podem estar emvolvidas na necrose e regeneração muscular na distrofia muscular de Duchenne.

18) Adult Murine Derived Mesoangioblasts Successfully Recovered Dysferlin Expression in a Murine Model of Dysferlinopathy

Jordi Díaz-Manera, Barcelona, Spain, Thierry Touvier, Rossana Tonlorenzi, Laura Perani, Arianna Dellavalle, Graziella Messina, Patrizia Pessina, Milano, Italy, Eduard Gallardo, Isabel Illa, Barcelona, Spain, Yvan Torrente, Giulio Cossu, Milano, Italy

OBJECTIVE: Our aim was to treat a mouse model of dysferlinopathy with transplantation of adult derived murine mesoangioblasts (mMABs). BACKGROUND: Mutations in dysferlin gene produce a muscular dystrophy characterized by adult onset and progressive weakness leading to a severe phenotype. The A/J mouse is a good model to study cell therapy as it completely lacks dysferlin and develops a slowly progressive muscular dystrophy. Mesangioblasts (MABs) are vessel associated progenitors that has been successfully used in preclinical models for cell therapy for muscular dystrophy including the a-sarcoglycan null mice and the Golden Retriever dogs affected by Duchenne's disease. DESIGN/METHODS: Murine mesoangioblasts were obtained from skeletal muscle of 8 days old wild type mice (C57 strain), and labeled with a lentiviral vector expressing nuclear LacZ. To avoid an immunological response, A/J-SCID mice were generated by crossing A/J and SCID strains. Firstly, we performed a single intramuscular injection of 5x10⁵mMABs both in cardiotoxin treated and untreated muscles from 5 months old mice. Then we proceed with a single injection of 5x10⁵mMABs in the right femoral artery. We analyzed the expression of dysferlin 3 weeks after the injection using immunofluorescence, quantitative RT-PCR and Western-Blot. RESULTS: The first dystrophic features in AJ-SCID mice appeared at 4-5 months of age, without significant differences in the progression and distribution of them compared with control A/J mice. Three weeks after transplantation, multiple areas of injected muscles expressed dysferlin which was absent in non-injected contralateral muscles. Presence of the protein in the membrane of Lac-Z + fibers was also demonstrated in intra-arterially injected animals. The expression of dysferlin was significantly higher in muscles treated with cardiotoxin. CONCLUSIONS/RELEVANCE: Treatment with wild type mMABs successfully recovered the expression of dysferlin in A/J-SCID mice. This fact suggests that adult derived mesoangioblasts may be a promising candidate for future cell-therapy protocols in dysferlinopathy patients. Supported by: European Federation of Neurology grant for young neurologist.

Pesquisa em camundongos deficientes em disferlina e tratados com mesangioblastos, células tronco de vasos. Os resultados foram positivos com expressão significativa da disferlina nos animais.

19) rAAV5 Mediated Delivery of Dysferlin as a Therapeutic Strategy for LGMD2B and Miyoshi Myopathy

Louise R. Rodino-Klapac, Kimberly M. Shontz, Chrystal Montgomery, Vinod Malik, Nancy Davis, Paul M. L. Janssen, K. Reed Clark, Columbus, OH, Robert H. Brown, Worcester, MA, Jerry R. Mendell, Columbus, OH

OBJECTIVE: To develop an adeno-associated virus (AAV) mediated therapeutic transgene to treat dysferlinopathies including limb-girdle muscular dystrophy (LGMD) type 2B and Miyoshi myopathy (MM). No therapeutic treatments are currently available for these disorders. BACKGROUND: The size of the DYSF cDNA (6.5 kb) negates packaging into traditional AAV serotypes (capacity < 4.7 kb) known to express well in muscle (i.e. rAAV1, 2, 6, 8). Potential advantages of a full cDNA versus a truncated transgene include: maintaining structural-functional protein domains, evading protein misfolding, and avoiding novel epitopes that could be immunogenic. This work describes in vivo delivery of AAV5.DYSF to limb muscle and diaphragm of dysferlin deficient (Dysf^-/-) mice by both intramuscular and vascular (femoral artery) approaches. DESIGN/METHODS: A cassette containing the DYSF cDNA driven by the muscle specific MHCK7 promoter was packaged into an AAV2/5 vector. Physiological characterization of three dysferlin deficient mouse strains (AJ, SJL, and 129-Dysf^tm1Kcam/J) revealed functional deficits in the diaphragm but not skeletal muscle. Efficacy of rAAV5.MHCK7.DYSF gene replacement was tested following intramuscular and intravascular delivery to skeletal muscle and diaphragm of 4-6 week old Dysf ^-/- mice. Functional improvement was measured by tetanic force and resistance to fatigue in the diaphragm at 2 months. RESULTS: Robust dysferlin gene expression was achieved in a dose-dependent manner by both intramuscular and vascular approaches. Western blot analysis confirmed the immunostaining results demonstrating a 237kDa band in treated samples that was absent in controls. Band intensity correlated with dose. Gene transfer improved both force generation and resistance to fatigue in the functionally impaired diaphragm in Dysf^-/- mice. CONCLUSIONS/RELEVANCE: These results provide proof of principle that a full-length dysferlin cDNA can be delivered efficiently to muscle using AAV5 leading to physiological improvement. Future studies in a larger animal model using a vascular approach targeting multiple muscles will guide clinical trial design for LGMD2B and MM patients. Supported by: Day Foundation.

Pesquisa experimental que demonstra bons resultados em camundongos deficientes em disferlina e tratados com vetor viral transportando o gene da disferlina.

20) Progressive Dysphagia in Limb Girdle Muscular Dystrophy Type 2B: An Extension of the Clinical Phenotype

Richard A. Walsh, Fiona Hill, Francesca M. Brett, Dublin, Ireland, Richard Charlton, Rita Barresi, United Kingdom, Dominick J. H. McCabe, Dublin, Ireland

OBJECTIVE: Genetically-confirmed autosomal recessive limb girdle muscular dystrophy type 2B (LGMD2B) typically presents in early adulthood with lower more than upper limb-girdle weakness. Facial muscles are usually spared and dysphagia has not been reported as part of the phenotype. BACKGROUND: We present this case report as an important extension of the clinical phenotype of LGMD2B. DESIGN/METHODS: A 40 year-old woman with non-specific generalised clumsiness since age 10 had a 7 year history of progressive proximal lower limb weakness followed by mild upper limb weakness. She also reported progressive subjective dysphagia for solids and liquids resulting in recurrent respiratory infections. Neurological examination revealed mild bilateral facial weakness, neck flexor more than extensor muscle weakness, mild proximal upper limb weakness, severe proximal and mild distal lower limb weakness. Upper limb reflexes were reduced with lower limb areflexia and a myopathic gait. RESULTS: Creatine kinase has varied between 3588 and 8058 IU/L (normal 34170). Quadriceps muscle biopsy showed features of an acute-on-chronic myopathy. Western blot analysis indicated a dysferlinopathy and genetic testing confirmed two pathogenic mutations in the dysferlin gene on chromosome 2p13: (DYSF c.5908 C>T, p.Pro1970Ser and DYSFc353delT). Videoflouroscopy revealed penetration of the airway above the vocal cords. Esophagogastroduodenoscopy identified impaired esophageal relaxation and barium swallow demonstrated significant delay in swallow initiation and tertiary esophageal peristaltic contractions. CONCLUSIONS/RELEVANCE: Dysphagia must now be considered part of the phenotypic spectrum of LGMD2B. Pharyngeal and esophageal involvent may have arisen as a consequence of the particular combinaltion of gene mutations in this patient although no good genotype-phenotype correlation has been descrined in LGMD2B. The case is important in alerting physicians to the need to screen this patient group for symptoms of dysphagia. Also, misdiagnosis of LGMD2B as polymyositis is not uncommon. The presence of dysphagia should not be a further deterrent in considering a dysferlinopathy in the appropriate clinical setting. Supported by: The Diagnostic and Advisory Service for Rare Neuromuscular Disorders is Funded by the NHS National Commissioning Group.

Pesquisa que descreve dificuldades progressivas da deglutição (disfagia) em pacientes com distrofia tipo cinturas; este sintoma não tinha sido descrito com frequência nesta forma de distrofia e que deveria ser pesquisado nestes pacientes.

21) Muscle Imaging in Oculopharyngeal Muscular Dystrophy

Celedonio Marquez, Carmen Paradas, Seville, Spain, Montse Olive, Hospitalet de Llobregat, Barcelona, Spain, Juan Bautista, Jose Manuel Morales, Antonio Cano, Seville, Spain, Fernando Castellanos, Plasencia, Caceres, Spain, Laura Gonzalez, Hospitalet de Llobregat, Barcelona, Spain, Ricard Rojas, Barcelona, Spain, Raul Garcia, Seville, Spain

OBJECTIVE: To describe muscle imaging findings in a cohort of patients with Oculopharyngeal Muscular Dystrophy (OPMD). BACKGROUND: Muscle imaging has become a useful tool in identifying patterns of muscle involvement in neuromuscular disorders. It can be relevant for diagnosis and assessment of progression. DESIGN/METHODS: We obtained lower extremities muscle imaging studies (12 CT scans, 35 MRI) in 47 carriers of a mutation in the PABPN1 gene: 4 asymptomatic carriers of a (GCN)₁₃ repeat (2 women, mean age 37.55.3), and 43 OPMD patients (22 women, mean age 65.410.3): 3 patients with a (GCN)₁₂ repeat, 25 (GCN)₁₃, 1 (GCN)₁₄, 2 (GCN)₁₅, 1 (GCN)₁₆, 1 (GCN) ₁₇. We described the muscle imaging findings and compared the groups with normal and abnormal imaging studies. RESULTS: The four asymptomatic carriers showed no abnormalities on the MRI. Muscle imaging was normal in 10 patients (23.3%) and abnormal in 33 patients (76.7%). The abnormalities were decreased attenuation on CT scans or high signal on T1-weighted MRI consistent with fatty infiltration in gluteus maximus, hip adductors or soleus muscles. The mean evolution of disease was longer in the abnormal imaging group (11,61 8.08 years) than in the normal imaging group (3.52.13 years)(p=0.013). Proximal muscle weakness in lower limbs was present in 18 out of 33 (54.5%) patients with abnormal muscle imaging and in no patients with normal findings. CONCLUSIONS/RELEVANCE: Fatty infiltration of gluteus maximus, hip adductors or soleus muscles represents a characteristic pattern of muscle involvement in OPMD. This radiologic involvement is not a early feature of the disease. Muscle imaging of lower limbs can be useful in diagnosing and monitoring progression of OPMD patients in clinical practice and research. Supported by: FIS 060382

Pesquisa que descreve o uso da ressonância nuclear magnética no estudo dos músculos na distrofia óculo-faríngea.

Resultados positivos com o uso de células placentárias em camundongos com distrofia muscular (19/02/2010)

Japão - a placenta é rica em células que podem originar músculos; os autores utilizaram 6 tipos diferentes de células placentárias para tratamento de camundongos com distrofia muscular; a utilização destas células em camundongos mantidos com imunossupressão resultou em expressão de distrofina humana nestes camundongos. O estudo destas células permitirá escolher aquelas com melhor potencial para tratamento das distrofias musculares.

Cirurgia de escoliose em pacientes com Duchenne e com alto risco de disfunção pulmonar: função pulmonar, qualidade de vida e satisfação (19/02/2010)

Japão - em trabalho anterior os autores já tinham descrito o tratamento de escoliose (desvio lateral da coluna em pacientes com distrofia muscular de Duchenne e função pulmonar muito baixa. Neste trabalho eles relatam o seguimento a longo prazo destes pacientes. Os pacientes relataram satisfação e melhor qualidade de vida após a cirurgia. A capacidade pulmonar continuou a se reduzir em 3,6% ao ano, apesar dos pacientes continuaram com os exercícios respiratórios. A conclusão final foi positiva a longo prazo com este tratamento

Biópsia de pele perioral para estudar a expressão das proteínas musculares (19/02/2010)

Itália - muitas vezes a biópsia muscular é necessária para diagnóstico das distrofias e muitas vezes biópsias repetidas são necessárias para acompanhamento de tratamentos experimentais. Neste estudo pequenas biópsias da pele em volta dos lábios foram realizadas para estudo de pequenos músculos que se localizam em volta da pele. O estudo das proteínas destes músculos se mostrou viável e menos invasiva que a biópsia muscular convencional mostrando-se uma boa alternativa para estudo ou diagnóstico das distrofias musculares.

Resultados apresentados pelo transplante de células tronco de medula óssea e do cordão umbilical em pacientes com distrofia muscular na China (13/02/2010)

China - neste trabalho os autores descrevem os resultados do tratamento com células tronco (de medula óssea e de corfão umbilical) em pacientes com distrofia muscular. Não é possível confiar plenamente nos resultados porque o artigo tem vários pontos não esclarecidos; o texto é publicado em chinês e as poucas informações estão no resumo em inglês; o resumo diz que foram tratados 82 pacientes com distrofia muscular progressiva mas não diz a forma da doença; os resultados foram considerados bons mas os critérios de avaliação não foram objetivos; eles não descrevem efeitos colaterais.

Correção genética completa das células tronco multipotentes induzidas na distrofia muscular de Duchenne (4/02/2010)

Japão - o cromossomo humano artificial (HAC) tem vantagens sobre os vetores virais para tratamento das doenças genéticas. Células tronco multipotentes induzidas podem ser úteis para tratamento por serem são retiradas do próprio paciente Nesta pesquisa HAC foi utilizado para corrigir o defeito genético de células tronco multipotentes induzidas do camundongo com distrofia e das células de pacientes com Duchenne. Os resultados demonstraram uma significativa expressão da distrofina nas células tratadas (em torno de 90%). Ou seja a combinação de terapia gênica com células tronco pode ser um caminho para o tratamento da distrofia muscular de Duchenne. O resumo em inglês pode ser lido abaixo:

(Molecular Therapy 2010;18(2):386–393) Complete Genetic Correction of iPS Cells From Duchenne Muscular Dystrophy

Yasuhiro Kazuki, Masaharu Hiratsuka, Masato Takiguchi, Mitsuhiko Osaki, Naoyo Kajitani, Hidetoshi Hoshiya, Kei Hiramatsu, Toko Yoshino, Kanako Kazuki, Chie Ishihara, Shoko Takehara, Katsumi Higaki, Masato Nakagawa, Kazutoshi Takahashi, Shinya Yamanaka and Mitsuo Oshimura - Japan

Human artificial chromosome (HAC) has several advantages as a gene therapy vector, including stable episomal maintenance that avoids insertional mutations and the ability to carry large gene inserts including the regulatory elements. Induced pluripotent stem (iPS) cells have great potential for gene therapy, as such cells can be generated from the individual's own tissues, and when reintroduced can contribute to the specialized function of any tissue. As a proof of concept, we show herein the complete correction of a genetic deficiency in iPS cells derived from Duchenne muscular dystrophy (DMD) model (mdx) mice and a human DMD patient using a HAC with a complete genomic dystrophin sequence (DYS-HAC). Deletion or mutation of dystrophin in iPS cells was corrected by transferring the DYS-HAC via microcell-mediated chromosome transfer (MMCT). DMD patient- and mdx-specific iPS cells with the DYS-HAC gave rise to differentiation of three germ layers in the teratoma, and human dystrophin expression was detected in muscle-like tissues. Furthermore, chimeric mice from mdx-iPS (DYS-HAC) cells were produced and DYS-HAC was detected in all tissues examined, with tissue-specific expression of dystrophin. Therefore, the combination of patient-specific iPS cells and HAC-containing defective genes represents a powerful tool for gene and cell therapies.

Avaliação da administração crônica de timosina beta-4 na função cardíaca e muscular esquelética de camundongos com distrofia muscular (4/02/2010)

USA - timosina beta 4 é uma proteína envolvida na proliferação e diferenciação celular e que interfere com vários mediadores inflamatórios. Os camundongos portadores de distrofia foram tratados por 6 meses. Os camundongos tratados apresentaram maior número de fibras em regeneração em relação aos não tratados. Os demais parâmetros estudados como força muscular, ecocardiograma e fibrose cardíaca não tiveram alteração com o tratamento.

Pacientes com distrofia muscular deverão ser vacinados a partir de março contra gripe A (26/01/2010)

Brasil - Ministério da Saúde divulgou hoje calendário de vacinação contra a gripe A (H1N1). Pacientes com distrofia muscular serão vacinados juntamente com a população portadora de outras doenças mais propensas a complicações dos quadros gripais como os obesos, cardíacos, etc. A vacinação ocorrerá de 22 de março a 2 de abril. Importante lembrar que os portadores de distrofia muscular deverão receber também vacina habitual contra as outras cepas do vírus da gripe (vacinação normal dos outros invernos).

Doença gordurosa não alcoólica do fígado na distrofia miotônica (16/01/2010)

USA - na distrofia miotônica é frequente a resistência a insulina que pode levar ao diabetes mellitus; pacientes com resistência a insulina são mais predispostos a ter doença gordurosa não alcoólica do fígado. Esta doença não havia sido estudada na distrofia miotônica; 36 pacientes foram estudados e 44% apresentavam testes hepáticos alterados e 87% deles apresentavam doença gordurosa do fígado. Além disso apresentavam triglicérides e colesterol elevados, insulina de jejum elevada, obesidade abdominal entre outras alterações. Sugere-se então o melhor controle destas alterações com dieta e medicação para evitar a doença gordurosa não alcoólica do fígado. O resumo em inglês pode ser lido abaixo:

(Muscle & Nerve, 2009) Frequency and predictors of nonalcoholic fatty liver disease in myotonic dystrophy

Kenneth Shieh, James M. Gilchrist, Kittichai Promrat - USA

Nonalcoholic fatty liver disease (NAFLD) is a common chronic liver disease that is strongly associated with insulin resistance. Myotonic dystrophy (DM1) is the most common form of adult-onset muscular dystrophy, and there is a high frequency of insulin resistance due to insulin receptor mRNA splicing defects in muscle tissue. The frequency and predictors of NAFLD in this population have not been described. Thirty-six patients with DM1 were prospectively assessed for the presence of NAFLD and insulin resistance. NAFLD was defined by abnormal liver chemistry tests with ultrasound or pathologic evidence of steatosis in the absence of other liver disease. Abnormal liver chemistry tests were found in 44% of DM1 patients (mean ALT 73 ± 21 U/L, AST 53 ± 15 U/L), and 87% were attributable to NAFLD. Clinical predictors of NAFLD included increased insulin resistance by the homeostasis model assessment (HOMA) method (9.5 vs. 4.0 U, P = 0.03), elevated fasting insulin (40.4 vs. 16.1 IU/ml, P = 0.03), abdominal obesity (98.6 vs. 90.8 cm, P = 0.03), elevated triglycerides (195.7 vs. 136.8 mg/dl, P = 0.02), and elevated total cholesterol (213.6 vs. 180.6 mg/dl, P = 0.02). NAFLD is very common and should be considered in the management of DM1. It is strongly associated with markers of insulin resistance and features of the metabolic syndrome. These findings support the role of peripheral insulin resistance in the pathogenesis of NAFLD

A empresa Cytokinetics anuncia os resultados positivos com a droga CK-2017357em pessoas normais: aumento significante da força muscular (13/01/2010)

USA - a empresa Cytokinetics tem estudado pequenas moléculas para tratamento dos sintomas das doenças musculares (cardíacas e esqueléticas); esta semana eles divulgaram os resultados do estudo fase 1 (teste da droga em pessoais sem doença alguma) da droga CK-2017357, uma droga que ativa uma proteína muscular relacionada com a função muscular; esta droga já tinha demonstrado resultados positivos em camundongos para melhorar a força, diminuir o cansaço e melhorar os sintomas cardíacos e musculares em diversas doenças. Neste relato a droga testada em humanos causou aumento siginifcativo da força muscular e deverá continuar em testes em pessoas saudáveis até que se determine a segurança e a dose necessária para uso em doenças neuromusculares.

Defeito de canal de cálcio do músculo cardíaco pode ser o desencadeante de arritmias cardíacas graves na distrofia muscular de Duchenne (09/01/2010)

França - em geral as manifestações cardíacas na distrofia muscular de Duchenne são atribuídas a fibrose do músculo cardíaco e podem levar a arritmias graves e fatais. Muitas vezes as alterações elétricas do coração ocorrem sem o apareciemnto da fibrose; neste estudo os autores descrevem um canal de cálcio defeituoso no músculo cardíaco de camundongos com distrofia muscular que poderiam desencadear arritmias ventriculares. O uso de medicações que corrigem o funcionamento deste canal previnem o aparecimento das arritmias. Estas medicações ainda são estudadas somente experimentalmente em camundongos. O resumo em inglês pode ser lido abaixo:

(PNAS, 2010) Leaky RyR2 trigger ventricular arrhythmias in Duchenne muscular dystrophy

Jérémy Fauconnier; Jérôme Thireau; Steven Reiken; Cécile Cassana; Sylvain Richarda; Stefan Matecki; Andrew R. Marks; Alain Lacampagne - France

Patients with Duchenne muscular dystrophy (DMD) have a progressive dilated cardiomyopathy associated with fatal cardiac arrhythmias. Electrical and functional abnormalities have been attributed to cardiac fibrosis; however, electrical abnormalities may occur in the absence of overt cardiac histopathology. Here we show that structural and functional remodeling of the cardiac sarcoplasmic reticulum (SR) Ca²⁺ release channel/ryanodine receptor (RyR2) occurs in the mdx mouse model of DMD. RyR2 from mdx hearts were S-nitrosylated and depleted of calstabin2 (FKBP12.6), resulting in “leaky” RyR2 channels and a diastolic SR Ca²⁺ leak. Inhibiting the depletion of calstabin2 from the RyR2 complex with the Ca²⁺ channel stabilizer S107 (“rycal”) inhibited the SR Ca²⁺ leak, inhibited aberrant depolarization in isolated cardiomyocytes, and prevented arrhythmias in vivo. This suggests that diastolic SR Ca²⁺ leak via RyR2 due to S-nitrosylation of the channel and calstabin2 depletion from the channel complex likely triggers cardiac arrhythmias. Normalization of the RyR2-mediated diastolic SR Ca²⁺ leak prevents fatal sudden cardiac arrhythmias in DMD.