Abstracts that will be presented in Annual Meeting of American Academy of Neurology, Seattle, April-25-May-2, 2009

[P06.105] Reduction of TGF Beta 1 Using an Oral TNF Alpha Inhibitor in mdx Dystrophic Mice

Leonardo Horacio Brito, Lucas Scatino Saad, Vinicius Oliveira, Eduardo Augusto Correa Barros, Rosangela Aparecida Dos Santos, Lucas Guimaraes Ferreira, Maria Helena Catelli Carvalho, Maria Tereza Nunes, Szulim Ber Zyngier, Alzira Alves Siqueira Carvalho,  DAVID FEDER,  Sao Paulo, Brazil

OBJECTIVE: To evaluate the effect of Bkt-104, an oral inhibitor of TNF- in mdx mouse BACKGROUND: Tumor necrosis factor (TNF)- is elevated in inflammatory myopathies as well in DMD. The precise role of TNF- is unknown, and it is possible involved in the pathogenesis of fibrosis in dystrophic muscle. Blunting TNF pharmacologically may be useful in preventing muscle fibrosis in dystrophic muscles. DESIGN/METHODS: Long term effects of oral BKT-104 was analysed on mdx mice, male, 8 weeks old; five mice received BKT-104 30mcg/Kg 3 times a week and six received saline (double blinded, placebo). Mice were submitted to exercise in ergometer 5 days/week, at a speed of 20 cm/s, 10 min/day. The muscle strength was measured weekly for 12 weeks. After 12 weeks, all mdx mice undergone to muscle biopsy and right quadriceps and gastrocnemius were collected and stained with acid phosfatase and alkaline for histological evaluation. Left gastrocnemius was dissected, frozen in liquid nitrogen, and stored at -80 C. The tissue was used for Real-time PCR and mRNA of cytocines were measured. RESULTS: The results of this study didn't show any improvement on muscular strength or histological alterations in BKT-104 tretment of mdx mice. There were no difference in genic expression of myostatin but a significant reduction of TNF- and TGF beta 1 in treated mice. CONCLUSIONS/RELEVANCE: Presently, TNF inhibitors are only available as an injectable or infusion. BKT104 is a small molecule (sulfated disaccharide), an oral inhibitor of TNF- (and consequential IL-8 production). BKT104 have show to be highly effective in modulating inflammatory disease in animal models, especially those regulated by TNF-. This is a pilot study with the drug Bkt-104 in muscular dystrophy. The observed reduction in TNF- and TGF beta 1 demonstrate the potential of the drug in reducing the fibrosis seen in muscular dystrophy. Acknowledge: Dr. Orly Eizenberg from Biokine Therapeutics
 

[P06.088] Erythropoietin Application in mdx Mice A Pilot Trial

Alzira Carvalho, Michelle Remiao Ugollini, Augusto Cesar Santomauro, Jr., Leticia Zaccaria Prates Oliveira, Victor Prates Pimentel Lioi, Rosangela Aparecida Dos Santos, Lucas Guimaraes Ferreira, Maria Tereza Nunes, Maria Helena Catteli Carvalho, Szulim Ber Zyngier, DAVID FEDER, Sao Paulo, Brazil

OBJECTIVE: To evaluate the effect of Erythropoietin application in an experimental model of dystrophy, the mdx mouse BACKGROUND: Apart from its erythropoietin function, erythropoietin (Epo) exerts also non-erythroid but tissue protective functions. The potential physiological role of the erythropoietin receptor (Epo-R) in skeletal muscle seems to be the control of proliferation and differentiation of myoblasts and satellites cells. Also, it has been suggested an important role of Epo in response to injury. DESIGN/METHODS: The long term effects of intraperitoneal(IP) Epo injection on mdx mice, male, were analysed. Seven mdx mice received Epo (1000 IU/kg/IP) and six received saline. All animals were submitted to exercise in ergometer 5 days/week, at a speed of 20 cm/s, 10 min/day. The muscle strength was measured weekly for 12 weeks. After 12 weeks of treatment, all mdx mice were undergone to muscle biopsy and muscle samples were collected from right quadriceps and gastrocnemius and stained for histological evaluation. Left gastrocnemius was dissected, frozen in liquid nitrogen, and stored at -80 C. The tissue was used for Real-time PCR and cytocines were measured. RESULTS: The results didm't show that 12 weeks of Epo treatment was beneficial for improving muscular strength in mdx mice but improved muscular strength during one week (week seven) in the treated group. The histological analysis did not detect differences in necrosis and regeneration between both groups. There were no difference in genic expression of TNF alpha and TGF beta 1 but a significant reduction of myostatin in treated mice. CONCLUSIONS/RELEVANCE: Myostatin reduction in erythropoietin treatment in dystrophic mice could explain some effects of erythropoietin in skeletal muscles. The lack of improvement in muscle strength for more than one week may reflect the amount of Epo dose used or the period of treatment. A higher dose and early use of Epo could be a potential application in our next trial.

[P06.097] Behavioral Outcomes of a CINRG Multi-Center, Double-Blind, Controlled Study Comparing High Weekly Versus Low Daily Dose Prednisone Therapy in Boys with Duchenne Muscular Dystrophy

Yaron Fridman, Adrienne Arrieta, Fengming Hu, Lauren P. Hache, Jason M. Weimer, Hoda Z. Abdel-Hamid, Diana Escolar, Avital Cnaan, Paula R. Clemens, Washington, DC, Pittsburgh, PA

OBJECTIVE: To compare the behavioral effects of prednisone dosing 0.75mg/kg daily (daily) with dosing 10mg/kg weekly split over two consecutive days (weekend) for Duchenne muscular dystrophy (DMD) using the Child Behavior Checklist (CBCL). BACKGROUND: Prednisone prolongs ambulation of patients with DMD,but may cause adverse behavioral effects that could be alleviated by alternate dosing regimens. DESIGN/METHODS: We performed a secondary analysis of the behavioral outcomes in a double-blind, multi-center, placebo-controlled equivalence study of daily and weekend prednisone dosing. The CBCL was administered at screening and months 1, 3, 6, 9, and 12. Group comparisons were analyzed with paired t-tests, two-sample t-tests, and statistical significance was defined at p=0.05. RESULTS: Fifty-six (28 in each group) of 64 randomized participants completed the CBCL. The measures for the majority of participants had CBCL scores in the subclinical range throughout the study. Baseline comparison between groups showed slightly more problems in externalizing (p=0.05) and aggressive (p=0.02) behaviors in the daily prednisone group. Total problems at baseline were similar between treatment groups (Weekend=52.18 sd=7.44, Daily=55.19 sd=11.00, p=0.24). Comparison of baseline to one month showed no changes in behavior within weekend prednisone and an improvement in total problems (p=0.029) and attention (p=0.017) with daily prednisone. Following 12 months of treatment there were no significant differences within the weekend group. There were significant decreases in total problems, externalizing, attention, and aggressive behaviors (p<0.001, p=0.001, p<0.001, p<0.001) with daily prednisone. There were also significant differences between the daily and weekend groups (p=0.005, p=0.008, p=0.010, p=0.001), respectively. CONCLUSIONS/RELEVANCE: This study demonstrated significant improvement in total and externalizing behavioral measures determined by the CBCL with daily prednisone dosing over 12 months, and no change in the weekend group. Therefore, from a behavioral standpoint, daily dosing is preferred over weekend dosing for those DMD patients having or at high risk of behavioral side effects from prednisone therapy.

[P06.054] Expected Transaminase Levels in Muscular Dystrophy with Chronically Elevated Creatine Kinase

Erika L. F. Hedderick, Carmen M. Coombs, Richard E. Thompson, Thomas O. Crawford, Baltimore, MD

OBJECTIVE: To define the range of expected transaminase levels in patients with chronically elevated creatine kinase levels due to muscular dystrophy. BACKGROUND: Creatine kinase is prominently elevated in many forms of muscular dystrophy, but serum transaminases ordinarily associated with liver disease are elevated as well due to ongoing leakage of each from muscle. Defining the relationship between serum levels of these enzymes is important to both clinical trial design and clinical care in order to identify patients who may have liver disease in addition to their muscle disorder. DESIGN/METHODS: This retrospective review identified 195 patients with Duchenne, Becker, or limb girdle forms of muscular dystrophy seen by the senior author between 1975 and 2008. All patients who had baseline laboratory measurements of serum CK, AST, and/or ALT on the same day, in the absence of acute illness, were included. RESULTS: Forty-seven patients had at least one assessment that met these criteria. Nineteen of these patients had more than one set of laboratory tests over the course over many years, yielding a total of 122 data sets for AST and 105 for ALT. There were 44 males and 3 females with the following diagnoses: Duchenne (32), Becker (7), congenital muscular dystrophy (2), limb-girdle muscular dystrophy (4) and manifesting carriers of dystrophinopathy (2). Median age is 10.7, range 2.1 and 36.3 years. CK ranges between 41 and 29,595, and naturally declines with age and loss of muscle mass. For AST, upper limit of the 95 percentile confidence interval for a given CK value is 199 + 0.022*CK. For ALT, this value is 313 + 0.027*CK. CONCLUSIONS/RELEVANCE: These values are important to identify potential treatment complications in pending clinical trials.

[P06.089] Cell Therapy for Muscular Dystrophy: CD34 Negative Muscle Derived Cells Present High Myogenic and No Adipogenic Potential

Sabrina Sacconi, Didier Pisani, Noemi Clement, Claude Dechesne, Christian Dani, Claude Desnuelle, Nice, France

OBJECTIVE: to identify, within myogenic muscle precursors, a cell population with a high myogenic and low adipogenic potential in the perspective of cell therapy for muscular dystrophy. BACKGROUND: Myoblast transplantation in clinical trials is based on intramuscular injection of a population of CD56 positive myogenic muscle precursors (MMP). This population contains multipotent stem cells able to differentiate into distinct mesenchymal lineages including adipocytes. Adipocyte accumulation is observed in human dystrophic muscular diseases and stem cells transplanted in a muscle environment permissive to fat development, may be committed towards adipogenesis at the expense of myogenesis. DESIGN/METHODS: After Cytofluorimetric analysis of cell surface markers, distinct MMP populations were sorted using specific magnetic microbead associated antibodies and their ability to differentiate in vitro in adypocytes and myotubes was assessed. Populations of interest underwent in vivo studies conducted in immunodeficient Rag2^-/- g_c^-/- mice. RESULTS: The stem cell marker CD34 allowed us to sort two distinct populations from human paediatric and adult muscle biopsies: CD34 positive (CD34+) and CD34 negative cells (CD34-). In vitro, the CD34+ cells were myogenic and adipogenic whereas the CD34- cells were only myogenic. Muscle regeneration potential after transplantation in cryo-injuried muscle of immunodeficient Rag2^-/- g_c^-/- mice was the same for CD34+ and CD34- cells. To our knowledge, there is no mouse model mimicking fibro-adipose degeneration typical of human muscular dystrophies. Using clodronate-containing liposomes we could obtain adipose degeneration in cryo-injured tibialis anterior muscle of Rag2/gc-/- mice. After transplantation in these mice model CD34-cells were shown to participate solely to muscle regeneration while CD34+ cells were driven partially to differentiate into adipocytes. CONCLUSIONS/RELEVANCE: In conclusion, muscle derived CD34 negative population might represent a new tool for cell therapy of muscular dystrophy because it is committed toward myogenic differentiation even in micro- environment permissive for fat development

[P06.099] Volumeteric Radial and Tibial Bone Mineral Density in Boys with Duchenne Muscular Dystrophy

Wendy King, John Landoll, Velimir Matkovic, John T. Kissel, Columbus, OH

OBJECTIVE: To examine volumetric bone mineral density (BoMD) in boys with Duchenne muscular dystrophy (DMD). BACKGROUND: Whether patients with DMD require bone modifying medications (e.g., bisphosphonates) to mitigate an apparent decreased BoMD is uncertain. Patients with DMD do have lower areal BoMD by DEXA compared to controls, but whether this reflects a true deficit in BoMD, or rather is due to geometric parameters inherent in areal bone density analyses is unclear. DESIGN/METHODS: We examined volumetric BoMD of the non-dominant radius and tibia at distal (4%) and more proximal (33%) sites in DMD boys and age-matched healthy male controls (n=8 pairs, age 10-20 yr, average 15.2) using a Stratec XCT-2000 peripheral quantitiative CT (pQCT) densitometer. Results from DMD boys were compared to controls. RESULTS: Average total cross-sectional area of the radius and tibia was significantly lower in DMD boys compared to controls at both the distal (p=0.03) and proximal (p<0.001) sites. The average total BoMD at the distal radius (p=0.004) and tibia (p<0.001) as well as trabecular BoMD at the distal radius and tibia in DMD was also significantly lower (p<0.001) than in controls. In both radius and tibia, average cortical density at the proximal site was higher than that for controls and was significant for the tibia (p<0.001). The relationships in volumetric density between DMD and controls were consistent for both the radius and the tibia. CONCLUSIONS/RELEVANCE: Boys with DMD have smaller bones than age-matched controls. There is a real deficit in volumetric BoMD of the distal forearm and tibia (trabecular bone) in boys with DMD. However, at more proximal sites (representing cortical bone) BoMD is actually normal. These results suggest that the relationship between BoMD, bone fragility and fracture risk in boys with DMD may be more complex than previously appreciated, and that fracture preventive measures may require more than simple bisphosphonate therapy.

[P06.098] A Comparison of the Six-Minute Walk Test to Commonly Used Outcome Measures in Duchenne Muscular Dystrophy

Cassie J. Spalding, Erik K. Henricson, Craig M. McDonald, Richard T. Abresch, Jay Han, Alina Nicorici, Leone Atkinson, Allen Reha, Gary Elfring, Langdon Miller, Sacramento, CA, South Plainfield, NJ

OBJECTIVE: To evaluate the correlations between the 6-minute walk distance (6MWD) and other commonly used measures of disease severity in Duchenne Muscular Dystrophy (DMD). BACKGROUND: The lack of functional outcome measures is an obstacle to the clinical development of new treatments for DMD. The 6MWT has been used extensively in other disease states to measure global physical functioning and is being used in DMD clinical trials. DESIGN/METHODS: We enrolled ambulatory boys with DMD (n= 17) and able bodied control boys (CTRL) (n=34) ages 4-12 years old. Outcome measures included a modified 6MWT, timed motor function tests, Brooke & Vignos motor function scales, and isometric quantitative strength testing. RESULTS: There was a significant difference in outcome measure values between DMD and CTRL groups (p<0.001). In DMD, 6MWD had a strong negative correlation with time to climb 4 stairs, time to run/walk 10 meters, time to run/walk 25 meters (r=-0.7 to -0.9), and was strongly correlated with Vignos lower extremity functional scale (r= -0.72). 6MWD had a moderate negative correlation (r=-0.45-0.69) with time to stand from supine position, and a moderate correlation with the Brooke upper extremity functional scale, and quantitative knee extensor strength. The 6MWD did not correlate with quantitative knee flexor strength. Correlations between 6MWD and other measures were not as strong in the CTRL group. CONCLUSIONS/RELEVANCE: 6MWD shows moderate to strong correlation with timed measures of physical function in DMD. 6MWD shows a strong correlation with the Vignos lower extremity motor function scale and a moderate correlation with the Brooke upper extremity motor function scale. The 6MWD correlates moderately with isometric knee extensor strength but not with knee flexor strength. The 6MWD measures global physical function and endurance in DMD and provides a quantitative outcome measure that is related to but different from commonly used measures of strength and functional ability.

[P06.093] Oral Protein Supplementation Improves Protein Metabolism after Acute Exercise in Patients with Fascio-Scapulo-Humeral Muscular Dystrophy

Mette Cathrine Orngreen, Nicolai Preisler, Grete Andersen, Tina D. Jeppesen, Gerrit Van Hall, John Vissing, Copenhagen, Denmark

OBJECTIVE: Investigating the effect of an acute bout of exercise on skeletal muscle protein metabolism with and without protein supplementation in patients with Fascioscapulohumeral muscular dystrophy (FSHD). BACKGROUND: It has been shown that patients with FSHD can benefit from a 12-week, low-intensity aerobic exercise program. However, it is unknown how skeletal muscle protein metabolism of FSHD patients responds to an acute exercise bout, and whether the FSHD patients can benefit from oral protein supplementation immediately after exercise, by causing a net muscle protein gain as shown in healthy controls. DESIGN/METHODS: Protein metabolism at rest, during 40 min of submaximal exercise, and three hours after exercise was studied in 3 patients with FSHD and 3 healthy subjects, using arterio-venous differences across the leg, and L-[ring-²H5]phenylalanine dilution methodology. All subjects received a protein drink right after the exercise bout in the second trial. RESULTS: Protein supplementation induced a net protein uptake (synthesis) in the resting period after exercise in FSHD and healthy subjects, as compared to a net protein release (breakdown) without protein supplementation (FSHD: -10437 vs.6717 and CTR: -12728 vs.281180 mmol/min). In accordance with this, arterial phenylalanine concentrations were higher at rest after protein supplementation in both groups. CONCLUSIONS/RELEVANCE: This study shows that net protein balance after an acute exercise bout is comparable to that in healthy subjects, and shows that protein supplementation boosts protein build-up after exercise. This indicates that the known beneficial effect of aerobic training in FSHD, can potentially be enhanced by protein supplementation after exercise training. Future controlled trials are warranted to demonstrate this.
 

[S25.002] Pulmonary Function Characteristics of 255 Boys with Becker and Duchenne Muscular Dystrophy by Age Groups, Ambulatory Status and Steroid Use: Baseline Data from the CINRG Longitudinal Study Project

Richard Abresch, Craig M. McDonald, Erik K. Henricson, Jay Han, Robert Leshner, Diana Escolar, Eric Hoffman, Avital Cnaan, Adrienne Arrieta, Fengming Hu, Angela Zimmerman, Tina Duong, Jill Mayhew, Julaine Florence, Davis, CA, Sacramento, CA, Mc Lean, VA, Washington, DC, Saint Louis, MO

OBJECTIVE: To describe the pulmonary function characteristics of ambulatory and non-ambulatory subjects with Becker and Duchenne Muscular Dystrophy (B/DMD) and to determine whether steroid therapy is associated with improvements in respiratory function. BACKGROUND: Several recent conferences have reported that the lack of adequate natural history data and well-characterized outcome measures limits the ability to conduct therapeutic clinical trials in B/DMD. DESIGN/METHODS: Baseline pulmonary function tests (PFTs) were performed in 255 subjects with confirmed B/DMD (aged 6 to 28 years) in 20 centers from 10 countries of the Cooperative International Neuromuscular Research Group (CINRG). Pulmonary function measures included forced vital capacity (FVC, %FVC), forced expiratory volume in 1 second (FEV1, %FEV1), peak expiratory flow rate (PEFR, %PEFR), maximum inspiratory pressure (MIP), maximum expiratory pressure (MEP), and peak cough flow (PCF). The PFT measures were evaluated by current corticosteroid use, age groups (<10, 10-14, >15), and ambulatory status (ambulatory, part-time wheelchair user, full-time wheelchair user). RESULTS: All PFT measures except PCF were significantly different in the 3 age groups (p<0.02). All PFT measures were significantly different when compared to ambulatory status (p<0.01). All PFT measures except FVC, FEV1, and PEFR were significantly higher in steroid users than non-users (p< 0.01). Overall, all measures are most highly correlated in the oldest age group and least correlated in the youngest group. FEV1 and FVC are strongly correlated across age and ambulatory status (r = 0.9 0.99). PEFR is also fairly strongly correlated with FEV1 (r = 0.770.92). PCEF is the least correlated with the other outcomes (r= 0.16- 0.71). CONCLUSIONS/RELEVANCE: Pulmonary function measures clearly differentiate between D/BMD patients of different ages and ambulatory abilities. Steroid use significantly affects performance on pulmonary function measures suggesting that PFTs are a suitable outcome measure for clinical trials and will help determine the sample sizes needed.
 

[P07.098] Clinical Outcomes in Patients with Duchenne Muscular Dystrophy and Role of Multidisciplinary Care

Muddasir Qureshi, Laurie Bliss, Brian Tseng, Charlestown, MA

OBJECTIVE: We sought to review clinical data in patients with duchenne muscular dytrophy (DMD) seen at the Massachusetts General Hospital (MGH) and assess role of multi-disciplinary care. Multidisciplinary care has been initiated at the Pediatric Neuromuscular Unit at the MGH in June 2007. BACKGROUND: Duchenne muscular dystrophy is a rare, progressive neuromuscular disorder in boys leading to severe disability and death in adulthood. DESIGN/METHODS: A retrospective study of people with DMD seen at the MGH was performed. All participants were identified randomly using the MGH Research Patient Data Registry (RPDR). Participants were selected if they had a confirmed diagnosis of DMD. The data obtained included factors that may affect outcomes and clinical care in patients with DMD including outpatient and emergency consult services, hospitalizations, medication usage, pertinent laboratory values and incidence of important co-morbidities. A univariate analysis was used to analyze the data (Fisher's Exact Test, two-tailed, significance of p<0.05). RESULTS: A total of 43 patients with DMD were identified; 23 patients prior to and 20 patients after the inauguration of multidisciplinary clinic at MGH in June 2007. Patients who were seen after the start of multidisciplinary Pediatric Neuromuscular Clinic at the MGH were more likely to attend an outpatient consult for cardiology (p=0.0002), pulmonology (p=0.0001), endocrinology (p=0.0001), nutrition (p=0.0001), physical therapy (p=0.03) and social work (p=0.0001). Prior to the start of multidisciplinary care, approximately 25% of patients with DMD presented to the emergency room with respiratory complaints without a previous outpatient pulmonology consult. The incidence of cardiomyopathy (p=0.33) was same in both groups however patients seen prior to June 2007 were more likely to have scoliosis (p=0.01). Patients treated at the multidisciplinary clinic were more likely to receive steroid therapy (p=0.03). CONCLUSIONS/RELEVANCE: Multidisciplinary care in boys with DMD is more likely to prevent the various complications that may arise in this patient population.
 

P06.104] An Immortalized Fibroblast Model for Exon-Skipping Studies in Duchenne Muscular Dystrophy (DMD)

Ling Zhao, Gillian Butler-Browne, Vincent Mouly, Kamel Mamchaoui, Steve Wilton, Kevin M. Flanigan, Paris, France, Nedlands, Western Australia, Australia, Salt Lake City, UT

OBJECTIVE: To develop a novel immortalized cell line derived from a patient with a duplication of exons 8 and 9 in the DMD gene as a tool for exon skipping studies. BACKGROUND: AON-mediated exon skipping demonstrates significant potential as a therapy for DMD. Fibroblasts require MyoD induced transdifferentiation in order to express significant amounts of DMD mRNA. DESIGN/METHODS: A primary fibroblast cell line was immortalized via lentiviral transfection with the human telomerase gene, and subsequently transfected with a lentivirus carrying the MyoD gene under the control of a tetracycline-inducible promoter. After growth to near confluence, cells were switched from growth to differentiation medium containing doxycyline (2 ug/ml), and treated with AONs directed toward exon 8, exon 9, or both exons 8 & 9 in both time course and dose escalation experiments. Dystrophin expression was analyzed by immunofluorescence (days 1, 3, 5, and 7) and immunoblot (days 5 and 7). RESULTS: tet-MyoD fibroblast line successfully transdifferentiates to a myoblast lineage as judged by expression of desmin and alpha-actinin. RT-PCR studies from untreated cells reveal some baseline exon skipping, resulting in both wild-type and duplication-containing mRNA. AONs directed toward exons 8 and 9, or exon 9 alone, significantly increase the degree of skipping, whereas treatment with the AON directed toward exon 8 alone induces no significant skipping. Treatment at 100mM results in an increased dystrophin expression, and immunoblot reveals expression of dystrophin protein of apparently normal size. CONCLUSIONS/RELEVANCE: A tet-MyoD fibroblast line is a valid model for AON-mediated exon skipping studies. Duplication of exons 8 and 9 may be amenable to therapy with AONs, resulting in wild type dystrophin protein expression.

P06.090] Cystatin C Permits Assessment of Renal Glomerular Filtration Independant of Muscle Mass in Duchenne Muscular Dystrophy (DMD)

Susan Gailey, Cheryl Wall, Laurence Viollet, David Thornton, Jerry R. Mendell, Columbus, OH

OBJECTIVE: Establish a laboratory test for assessment of renal function in DMD independent of muscle mass BACKGROUND: Assessing renal function using standard laboratory tests represents a major challenge in DMD and other dystrophies where muscle mass is severely reduced. Serum creatinine, a derivative of skeletal muscle creatine, is inadequate for monitoring renal function. Creatinine clearance (CrCL), often considered the gold standard for renal glomerular filtration suffers the same limitations being similarly dependent on muscle derived creatine. Iohexol infusion has been suggested as an alternative to CrCL, but measurement requires prolonged infusion with multiple blood draws, and results are not available for 4-6 weeks. Cystatin C is a nonglycosylated protein that is unrelated to muscle mass making it potentially an ideal gauge of renal function for DMD patients. Testing new therapeutic agents mandates validating an alternative practical approach for measuring renal function. DESIGN/METHODS: Ninety subjects were recruited for this study including three cohorts of 30 subjects each, 5-20 years of age. Thirty subjects had mutation proven DMD, 30 subjects were healthy controls, and 30 subjects had known renal disease. The DMD population was balanced for corticosteroid use and ambulation. RESULTS: Cystatin C levels in the DMD population were within established normal reference range and varied from 0.5-0.9 mg/L. The mean (+ SD) cystatin C for 30 DMD patients was 0.693 + 0.122 compared to normal controls 0.693 + 0.083 and subjects affected with renal disease 3.517 + 2.142. There was no difference between DMD and normals, while the renal disease patients differed from both of the others (p<0.001). Neither corticosteroid use, ambulation or age affected Cystatin C in the DMD population. CONCLUSIONS/RELEVANCE: This is the first study validating cystatin C as an alternative and important means of monitoring renal function in DMD. Cystatin C has particular applicability for testing novel agents with potential renal toxicity.
 

[P06.100] Newborn Screening (NBS) in Duchenne Muscular Dystrophy: A Practical, Comprehensive and Affordable Approach

Roula Al-Dahhak, Susan Gailey, Kandice Roush, Lauren Schueller, Joni Beauman, Heather Lilly, Sharon Linard, Ram Chandrasekar, Diane Dunn, Deborah Cragun, Christopher Shilling, Patrick Wall, Nancy Leslie, Natalie Street, Robert Weiss, Jerry Mendell, Reynoldsburg, OH, Cincinnati, OH, Atlanta, GA, Salt Lake City, UT, Columbus, OH

OBJECTIVE: To establish a comprehensive, practical, affordable approach to NBS for DMD BACKGROUND: DMD, caused by mutations of the dystrophin (DYS) gene, is the most common severe form of childhood MD. DYS, the largest gene identified to date, exhibits a very high mutation rate, estimated to occur in 1/10,000 germ cells. This means that the disease can never be eliminated, emphasizing two important issues related to NBS. First, considering that most DMD cases go undiagnosed until age 3 or later, the possibility of additional affected offspring is very high unless there is detection of the first born male. Secondly, translational research efforts emphasize the value of early intervention, that is best ensured by early detection. In this study, we developed an approach to NBS not previously used, permitting creatine kinase (CK) screening and DNA analysis on the same dried blood spot (DBS) set taken at birth. DESIGN/METHODS: CKs were measured on one of the DBS obtained at birth. Based on sampling of 25,000 anonymous newborn males, CKs >600U/L prompted DNA testing on a second DBS using SCAIP, single condition amplification/internal primer sequencing. RESULTS: To date 7000 newborns have had CK screening (95 fell between 600-999 U/L, 7 between 1000-1499 U/L, 1 between 1500-1999 U/L). Two were >2000 U/L (2461 and 2675 U/L) and DYS mutations were found in both cases. The project is ongoing throughout the State of Ohio and will provide additional results at the time of presentation. CONCLUSIONS/RELEVANCE: This study demonstrates a practical, affordable, and comprehensive approach to conducting NBS for DMD. DNA testing can be limited to DBS exhibiting the highest level CKs, at a cost of $1.00/sample. Of interest, our findings in this outbred population continue to support an incidence of DMD in 1:3500 newborns. Further data obtained through expansion to the statewide program will add to the accuracy of these estimates.

[P06.103] Abundance of Circulating Progenitors with Myo-Endothelial Potential Correlates with a Mild Phenotype in Patients Affected by Duchenne Muscular Dystrophy

Marzia Belicchi, Andrea Farini, Mirella Meregalli, Daniele Parolini, Paola Razini, Raffaella Lopa, Laura Porretti, Simona Maciotta, Claire Navarro, Giulio Cossu, Nereo Bresolin, Yvan Torrente, Milan, Italy

OBJECTIVE: We hypothesized that the levels of circulating stem cells expressing the CD133 antigen which possess myo/endothelial potential would predict the progression of DMD. We tried to find a subpopulation of CD133+ stem cells significantly higher in DMD patients compared with healthy controls and positively correlated with the clinical score. BACKGROUND: We hypothesized that the levels of circulating stem cells expressing the CD133 antigen which possess myo/endothelial potential would predict the progression of DMD. We tried to find a subpopulation of CD133+ stem cells significantly higher in DMD patients compared with healthy controls and positively correlated with the clinical score. DESIGN/METHODS: The levels of several subpopulations of circulating stem cells expressing the CD133 antigen were determined by flow cytometry in 70 DMD patients. The median (6SD) age of the population was 10.6663.81 (range 3 to 20 years). The levels of the subpopulation of the CD133+ stem cells were correlated with the clinical condition. Samples were obtained from routine blood tests performed in healthy and DMD subjects. RESULTS: The levels of CD133+CXCR4+CD34- stem cells were significantly higher in DMD patients compared to healthy controls with a tendency towards decreased levels in older patients. In a subgroup of 19 DMD patients after 24 months of follow-up, increased levels of CD133+CXCR4+CD34- cells was shown to be associated with a phenotype characterised by slower disease progression. CONCLUSIONS/RELEVANCE: Linear regression analysis showed a direct correlation between the levels of circulating CD133+CXCR4+CD34- and the clinical condition of the DMD patients. Based on these data we believe that the levels of a subpopulation of circulating CD133+ stem cells in DMD patients may be a promising new prognostic clinical marker of the progression of the disease with practical significance to allow any beneficial effect in future clinical trials.

[P06.102] Characterization of DMD Mutations in Novel Dog Models of Dystrophinopathy

Kevin M. Flanigan, Aaron Larsen, Laura Taylor, Janet Bogan, Daniel Bogan, Joe Kornegay, Salt Lake City, UT, Chapel Hill, NC

OBJECTIVE: To characterize DMD mutations in novel dog models of Duchenne muscular dystrophy. BACKGROUND: Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are X-linked disorders caused by mutations in the DMD gene. Several dystrophic dogs have been identified in clinical veterinary practice by the presence of variable weakness and altered or absent dystrophin expression. Genomic studies to define causative mutations have not been done in most of these dogs. Here, we describe mutations in a Labrador retriever with severe weakness and a cocker spaniel with milder weakness. Carrier status was also documented in a Yorkshire terrier related to a dog known to have a deletion of exons 3-7. DESIGN/METHODS: Immunofluorescent and immunoblot analysis of skeletal muscle was performed using a panel of anti-dystrophin antibodies. For novel mutations, sequencing of the complete DMD cDNA (generated via reverse transcriptase-PCR from muscle-derived mRNA), and mutations confirmed by sequence analysis from genomic DNA. For detection of deletion 3-7 carrier status, quantitative real-time PCR (qRT-PCR) was performed on an ABI 7500 HT, comparing the amplification curve for exon 3 to that of exon 49 via standard methods. RESULTS: Two novel mutations were found. In the Labrador retriever, we identified a LINE element insertion, resulting in the inclusion of a pseudoexon within the mRNA. In the cocker spaniel, we identified a 4 nucleotide frameshifting deletion in exon 65, resulting in a premature stop codon at the site of the frameshift. In the Yorkshire terrier, qRT-PCR robustly identified the carrier status. CONCLUSIONS/RELEVANCE: Dystrophic dogs are important large animal models of human DMD that can be used to study research questions not addressable in the standard mdx mouse model. Identification of these novel mutations could facilitate preclinical studies directed toward these specific mutation classes.
 

[P06.087] Reduced Necrosis and Inflammatory Change in Muscle of SJL/J Mice by Blocking TNFa Function with Etanercept

Hiroshi Nemoto, Shingo Konno, Hideki Sugimoto, Nobuatsu Nomoto, Hiroshi Nakazora, Mayumi Murata, Toshiki Fujioka, Tokyo, Japan

OBJECTIVE: In SJL/J mice the damage of sarcolemma may relate the inflammatory change, especially cytokines. We test the hypothesis that TNF alpha contribute the myofibril necrosis. In this study, we examine the histological and immunohistological changes of the muscle of SJL/J mice by the treatment of etanercept. BACKGROUND: Dysferlin has the function to repair the muscle membrane from mechanical stress. SJL/J mice show the defect of dysferlin, it believes that SJL mice are an animal model of Limb-girdle type muscular dystrophy 2B and Miyoshi myopathy. Also older SJL/J mice show the inflammatory changes in skeletal muscle. The mechanism of inflammation in muscle may be related immunological abnormality. Anti TNF alpha therapy for mdx mice that are the model mice of Duchenne type muscular dystrophy is effective. DESIGN/METHODS: Sixty-day old mice are injected 30ug/kg of etanercept subcutaneously twice per week until sacrificed. Control mice are injected 0.1% of manitol solution subcutaneously. Mice are sacrificed at day 120, 240, 360(n=5) and 420 (n=3) in each group. Muscle samples were collected from biceps and quadriceps femoris. Histlogical and immunohistlogical study (CD11b, CD4, CD8) are performed RESULTS: Sixty-day old mice are injected 30ug/kg of etanercept subcutaneously twice per week until sacrificed. Control mice are injected 0.1% of manitol solution subcutaneously. Mice are sacrificed at day 120, 240, 360(n=5) and 420 (n=3) in each group. Muscle samples were collected from biceps and quadriceps femoris. Histlogical and immunohistlogical study (CD11b, CD4, CD8) are performed. CONCLUSIONS/RELEVANCE: As a result of blocking TNF a by entercept, it makes reduction of the activity of macrophage. It makes the suppression of the activity of inflammatory changes and also the damage of muscle. In this study we present the suppression of degenerative and inflammatory changes of older SJL/J mice treated by etanercept. TNF a may contribute to the breakdown of dysferinopathy muscle.
 

[P06.094] Partial Response of Dysferlinopathy Patients to Low Dose Dantrolene Sodium Treatment

Itzik Wirguin, Beer-Sheva, Israel

OBJECTIVE: To describe a modest ameliorating effect of Dantrolene Sodium (DS) on the progression of Limb Girdle Muscular Dystrophy 2B (LGMD2b) in 2 siblings. BACKGROUND: LGMD2b, Miyoshy myopathy (MM) and distal anterior compartment myopathy are autosomal recessive progressive muscular disorders caused by mutations in the Dysferlin gene. Dysferlin protein is involved in the calcium dependent membrane fusion step of the muscle fiber repair process. Two clusters of dysferlinopathy were described in Israel, of which one is caused by a frameshift mutation of G deletion at codon 927 (2779delG, Neuromuscular Disorders 2007:17:950-954). DS is an agent used in the treatment of malignant hyperthermia and to relieve spasticity, probably through its ability to block calcium release from the sarcoplasmic reticulum. A recent report suggested its effect in a compound heterozygous variant of MM in Japan. DESIGN/METHODS: Two siblings, homozygous for the 2779delG mutation presented with progressive weakness (MM phenotype) around the age of 15. CK levels were around 14000 in the older male and around 7000 in the younger female. Both patients were treated with DS, 25 mg once daily and monitored for systemic toxicity, clinical status and muscle power. RESULTS: Both patients reported significant improvement in exercise tolerance, ability to work and self perceived life quality, within 1 week of treatment initiation. CK levels declined by 20-25% and compound muscle chart scores rose by about 3 points. Blood tests showed no change in liver enzyme levels. At the time of this report, treatment has been given for 6 months during which no clinical deterioration has occurred. A short term attempt to raise the dose to 25 mg BID brought no additional benefit. CONCLUSIONS/RELEVANCE: DS has a discernible positive effect in patients with MM type dysferlinopathy. These results warrant a randomized clinical trial of DS in a wider range of patients with Dysferlin mutations.

[SC02.003] Uptake and Outcome of Genetic Testing among Participants of the DuchenneConnect Registry

Vanessa Rangel Miller, Giovanna Spinella, Ken Loud, W. Andrew Faucett, Madhuri Hegde, Pat Furlong, Falls Church, VA, Atlanta, GA, Middletown, OH

OBJECTIVE: DuchenneConnect is a self-report patient registry developed to connect patients, providers, and research communities. The program promotes access to genetic testing, genetic counseling, and resources for clinical trials, research studies, clinical care, and management. This report describes the frequency of genetic testing, informative test results, and further needs among participants. BACKGROUND: In Duchenne/Becker muscular dystrophy (DBMD), well-characterized mutations are necessary to evaluate eligibility for therapeutic trials using molecular genetic corrective approaches. Despite advances in genetic testing, many patients do not have results that provide well-characterized mutation information. DESIGN/METHODS: In November 2007, we developed a web-based registry to capture patients' clinical presentation and DMD mutation. Design of the survey and review of the data follows guidelines employed by other North American DBMD registries and the TREAT-NMD Neuromuscular Network. A printable report tool enables participants to have their medical care provider review and verify the registry information. RESULTS: As of October 2008, 918 of 1,234 (74%) registrants completed a survey: 501(55%) had genetic testing, 267(29%) had a muscle biopsy plus genetic testing, 65(7%) had a muscle biopsy alone, 53(5.5%) did not have testing, and 16(1.7%) responded 'I don't know'. From review of 146 of 293 genetic test results: 66(45%) were informative, 31(21%) warranted additional testing, 31(21%) used methods not recommended by the international registry, and 17(12%) were research results needing clinical laboratory confirmation. 812 requests were made to speak with a genetic counselor, including: 291(36%) to discuss genetic testing and/or results, 145(21%) to discuss the registration process, and 77(9.5%) to arrange genetic testing. CONCLUSIONS/RELEVANCE: A substantial number of participants require further genetic testing due to insufficient mutation characterization. Full mutation characterization requires identification of the boundaries of a deletion/duplication or a deleterious point mutation. Comprehensive dystrophin testing provides information essential for participation in clinical trials, therapeutic decision-making, accurate risk counseling, and testing for family members.

[P08.117] Phase I Gene Transfer Clinical Trial in Alpha-Sarcoglycan Deficiency (LGMD2D)

Jerry R. Mendell, Louise R. Rodino-Klapac, Xiomara Rosales-Quintero, Brian D. Coley, Zarife Sahenk, Vinod Malik, Jo Craenen, Sarah E. Lewis, Brent Yetter, Katie J. Campbell, William G. Bremer, Barry J. Byrne, Christopher Walker, K. Reed Clark, Gainesville, FL, Columbus, OH

OBJECTIVE: Alpha-SG gene (SCGA) replacement is a potential treatment strategy for limb girdle muscular dystrophy (LGMD)2D. BACKGROUND: There is no treatment for LGMD2D caused by SCGA deficiency. Adeno-associated virus (AAV) mediated gene replacement appears to be a viable option based on pre-clinical studies in knock out mice. AAV1, using a muscle specific promoter to avoid off target effects, mediates persistent gene expression with a favorable safety profile. DESIGN/METHODS: Under conscious sedation, LGMD2D patients (ages 12 and 10) underwent gene transfer using a double-blind protocol with injections bilaterally to the extensor digitorum brevis (EDB) muscles. One side received only saline while the other received AAV1.tMCK.SGCA, 3.25 X10¹¹ vg in 1.5 ml. Intramuscular injections were guided by ultrasound and EMG to ensure proper needle placement for gene transfer. RESULTS: Thus far, two LGMD2D subjects have received gene transfer without adverse effects. There was no swelling or erythema at the site of injection and no systemic reaction following gene transfer. In one subject the EDB muscles on both sides were removed at day 43 post gene transfer and analyzed without breaking the code. Muscle from left and right EDBs were compared for gene expression using western blots and direct immunofluorescence (IF). A three fold increase in gene expression was seen on the left side. A second case underwent a similar procedure and reached higher levels of gene expression on the right side at three months post gene transfer. Presence of transgene DNA was detected by PCR on the side corresponding to gene expression in both patients. There were no clearly defined immune responses to AAV1 or transgene product. CONCLUSIONS/RELEVANCE: Gene transfer is possible in the injected muscle of LGMD2D patients but the clinical significance and long-term safety require further study. Additional patients will be enrolled (goal= 6 subjects) and will include careful scrutiny of mononuclear cells in muscle.

[P06.101] Sonoporation of Human Biceps Muscle as Preparation for Plasmid-Based Dystrophin Gene Transfer

Maria J. Molnar, Renald Gilbert, Aniko Gal, George Karpati, Budapest, Hungary, Montreal, QC, Canada

OBJECTIVE: To determine the feasibility and safety of sonoporation (SP) in biceps muscles of 5 healthy volunteers in Phase I/A clinical trial. in preparation for plasmid-mediated dystrophin gene transfer in Duchenne muscular dystrophy (DMD) BACKGROUND: Gene transfer is a proven therapeutic modality in genetic diseases such as DMD. Gene transfer using plasmids as vectors is much less problematic than viral vectors. In mdx mice we have demonstrated that plasmid containing a full length dystrophin cDNA was efficient as long as it was coupled with SP (Mol Ther 6:687-693, 2002). SP consists of injection into muscle a suspension of microspheres filled with an inert gas which will burst from the heat generated by SP. This, in turn, creates reversible microgaps at the surface membrane of the muscle fibers through which the plasmid particles can stream into the muscle fibers. DESIGN/METHODS: After signing informed consents, five healthy volunteers (4 males/1 female. aged 25-35 years) had their left biceps muscles injected with 1.5 ml of microsphere solution (DEFINITY^R). The right biceps was injected with saline. Immediately after, ultrasound was delivered percutaneously over a 2 minute period to both muscles from a custom-designed sonoporator (1 MHz, 2-3 w/cm/2, 30% duty cycle).Thirty six hours later a muscle biopsy was studied with histochemistry, confocal and electron microscopy. RESULTS: In 2 subjects, transient reddening of the skin at the sonoporation site was present. There were a few scattered endomysial mononuclear inflammatory cell infiltrates, but no partial invasion or necrosis of muscle fibers or apoptotic myonuclei were observed. Subsarcolemmal vesicular profiles were conspicuous. A few short sarclolemmal gaps were still present. CONCLUSIONS/RELEVANCE: Sonoporation of human muscles is feasible and safe as it produced no significant muscle damage. It is expected to highly facilitate the efficiency of plasmid-mediated therapeutic gene transfer in DMD.
 

[P06.092] LGMD 2I in Twin Brothers: Response to Prednisone

Peter I. Karachunski, Marcia Margolis, Joline Dalton, John W. Day, Minneapolis, MN

OBJECTIVE: We studied monozygotic twins with limb-girdle muscular dystrophy type 2I (LGMD-2I) who were haploinsufficient for fukitin-related protein (FKRP) containing the L276I mutation. They had episodic weakness that became progressive and unremitting. We used quantitative motor function to determine the effects of steroids. BACKGROUND: LGMD-2I is caused by mutations in FKRP, a putative glycosyltransferase that is involved in a-dystroglycan glycosylation. LGMD-2I can present with various phenotypes based on the age of onset. A form of LGMD-2I has been described in children developing weakness subacutely, in a time frame comparable to viral myositis. Anecdotal reports have suggested LGMD-2I responds to steroids. DESIGN/METHODS: We assessed 9 year old twin brothers every 4 to 8 weeks over 18 months in a cross-over study. We measured motor function using handheld dynamometry, manual muscle testing, a Step Watch Activity Monitor, timed function tests (walking 10 m, climbing stairs, and standing from supine) and pulmonary function testing. Twins were assessed prior to treating first one, then the other, with prednisone (0.75mg/kg daily), for 6 months. RESULTS: Episodes of severe weakness did not occur during period of observation in either subject. Muscle strength and function improved while each boy was on prednisone, and declined when prednisone was discontinued in twin A. Forced vital capacity (FVC) improved from 80% to 100% of predicted in twin A but returned to baseline when prednisone was discontinued. FVC remained stable in twin B on steroids. Manual motor testing and handheld dynamometry showed improvement on prednisone but with greater inter-test variability. Weight gain and cushingoid appearance developed on steroids, without correlation with decline in performance. CONCLUSIONS/RELEVANCE: This study expanded description the recognized phenotype of LGMD-2I including infantile onset of episodic weakness, and demonstrated a response to short term prednisone. A larger study will be required to determine whether this treatment ameliorates weakness in all LGMD-2I patients.
 

[P06.106] High-Resolution, Three-Dimensional, In Vivo Bioimaging of Perivascular Stem Cell Homing

Chad D. Markert, Jennifer K. Cann, Martin K. Childers, Winston Salem, NC

OBJECTIVE: To track labelled perivascular progenitor cells following intramuscular or intra-arterial injection. BACKGROUND: Duchenne muscular dystrophy (DMD) is a progressive, lethal, X-linked disease of skeletal and cardiac muscle affecting nearly 1 in 3500 newborn males annually. DMD is caused by mutation of the dystrophin gene, a vulnerable target for new mutations. The cardiac and skeletal muscles of DMD patients are devoid of the dystrophin gene product, a 427-kD protein called dystrophin. To address the progressive and ultimately fatal degeneration of DMD muscles, intense research efforts are aimed at tilting the balance in favor of regeneration. Stem cell transplantation is one therapeutic approach. DESIGN/METHODS: Here we provide data using several tracking methods (intravital microscopy, MRI, in vivo fluorescent imaging, and immunohistochemistry) which allow us to follow the migration of intra-arterially injected perivascular progenitor cells (pericytes) from the point of their systemic injection to the point of their engraftment at a site of skeletal muscle injury, where they are needed for muscle repair. RESULTS: MRI scans of cardiotoxin-injured mice infused (femoral artery) with labeled pericytes show 1) pericyte localization at areas of skeletal muscle injury and 2) accumulation of injected pericytes inside long bones of the hindlimb, indicating that exogenous pericytes home to the bone marrow, a canonical mesenchymal stem cell niche. CONCLUSIONS/RELEVANCE: Isolation of perivascular stem cells allows us to provocatively speculate that these cells embody regenerative promise for the following reasons: first, they are readily available in virtually every vascularized tissue, including term placenta, which is a high-throughput, easily accessible source; second, they have myogenic potential; third, they are conducive to systemic delivery; fourth, they home to sites of injury efficiently, and fifth, experiments are underway to characterize them based on other key properties making them appropriate for cellular therapy of degenerative muscle disease. Importantly, unlike embryonic stem cells, they do not engender ethical debate.

[P08.118] Restoration of Normal Function by Combinational Gene Replacement Using Follistatin and Micro-Dystrophin in Mdx Mice

Louise R. Rodino-Klapac, Paul M. L. Janssen, Kim M. Shontz, Amy Eagle, Chrystal L. Montgomery, K. Reed Clark, Jerry Mendell, Brian Kaspar, Columbus, OH

OBJECTIVE: To normalize force generation and protect against eccentric contraction by combinational gene replacement with follistatin and dystrophin mini-genes. BACKGROUND: Duchenne muscular dystrophy is a devastating disease characterized by severe muscle weakness due to lack of dystrophin. Gene replacement using AAV, with its small insert capacity, forces truncation of the dystrophin gene (mini-gene), resulting in a product that incompletely restores muscle function. AAV delivery of follistatin, a known myostatin inhibitor, increases muscle size and strength in dystrophic mdx mice (Haidet et al. 2008) but leaves the sarcolemma vulnerable. Follistatin combined with micro-DYS is strategically positioned to achieve functional restoration, both protecting the fragile dystrophin-deficient sarcolemma and at the same time, normalizing force generation. DESIGN/METHODS: Six-month old mdx mice were treated with combinatorial gene therapy using rAAV.micro-DYS and FS344 (an isoform sparing activin binding to protect the pituitary gonadal axis) by direct muscle injection into the tibialis anterior/extensor digitorum (EDL) complex. At 1 year these mice were studied for force generation and protection against eccentric contraction and correlative morphology. RESULTS: rAAV.micro-DYS incompletely improves force generation and partially protects against eccentric contractions compared to untreated mdx, but not to wild-type levels. In contrast, simultaneous delivery of AAV1.FS344/micro-DYS reaches wild-type maximum force generation and resistance to damage by repeated eccentric contractions. Of interest, FS344 can accomplish this task with treatment 3 months before or after micro-DYS delivery. These findings correlated with improved hindlimb grip strength and reversal of dystrophic features by histopathology. Complete necropsies of mice undergoing gene therapy demonstrated no evidence of off target affects. CONCLUSIONS/RELEVANCE: Although AAV gene therapy treatment for DMD is limited by the inability to deliver full-length dystrophin, combinational therapy with follistatin augments function, compensating for the limitations of small dystrophin gene replacement. This approach expands translational options.

[P09.092] 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: Methods and Baseline Characteristics

Erik K. Henricson, Craig M. McDonald, Richard T. Abresch, Jay J. Han, Robert Leshner, Eric P. Hoffman, Diana Escolar, Avital Cnaan, Fengming Hu, Angela Zimmerman, Tina Duong, Jill Mayhew, Julaine Florence, Adrienne Arrieta, Sacramento, CA, Mc Lean, VA, Saint Louis, MO, Washington, DC

OBJECTIVE: To conduct the first large-scale longitudinal investigation of severe Becker and Duchenne muscular dystrophy (B/DMD) that uses the World Health Organization's International Classification of Functioning, Health and Disability. BACKGROUND: Investigators studying therapeutics in B/DMD face increasing pressure from regulatory authorities to develop sensitive, reliable and meaningful outcome measures of strength, function, and person-reported health-related quality of life (HRQoL). DESIGN/METHODS: We enrolled males with confirmed B/DMD from 20 participating centers from 10 countries of the Cooperative International Neuromuscular Research Group (CINRG). Following consent and review of diagnostic testing, participants undergo standardized assessments at baseline, months 3, 6, 9, 12, 18, 24, 36, 48 and 60. DNA is collected for banking. Study teams collect age-appropriate measures of clinical history (medical events, medications, durable medical equipment, supportive services), anthropometrics, goniometry (wrists, elbows, knees, ankles), Medical Research Council manual muscle strength, quantitative knee and elbow flexor/extensor and hand grip strength using the CINRG Quantitative Measurement System, timed function (stand from supine, stair climb, 10M walk/run), Brooke and Vignos functional scales, pulmonary function (FVC, FEV¹, MIP, MEP) and health-related Quality of Life (including PedsQL, SF-36, POSNA). RESULTS: Two hundred ninety males with B/DMD aged 2 to 28 years of age were enrolled at 20 CINRG centers worldwide between Fall 2005 and Fall 2008. Participants were well-represented from the youngest ages to the mid-teens, with fewer individuals in their late teens to early twenties. At enrollment, 65% were current corticosteroid users, 63% were fully or partially ambulatory and 13% required some ventilatory assistance. We will present baseline assessment data relative to age, ambulatory status and corticosteroid use. CONCLUSIONS/RELEVANCE: This study provides investigators with data on age-related patterns of clinical, functional and person-reported outcomes in individuals with B/DMD. This will contribute to optimal design of future clinical trials of emerging therapeutics.

[P06.096] Creatine Kinase Elevation: Evaluation of Pediatric Population

Sebastien Perreault, Ala Birca, Dominique Piper, France Gauvin, Michel Vanasse, Montreal, QC, Canada

OBJECTIVE: To determine the etiologies and the clinical evolution of children with creatine kinase (CK) elevation. BACKGROUND: In children, CK elevation is caused by multiple diseases. In some cases, early identification of underlying etiology can improve management DESIGN/METHODS: Retrospective study conducted in a tertiary care university-affiliated pediatric hospital. All consecutive patients from 2001 to 2002 with CK levels 1000 IU/L were included. Using a standardized form, we collected data from each patient's charts. RESULTS: 143 patients were included in the study (males 57%; mean age: 7,5 years 5,9). The median elevation of CK was 2337 (range:1003-811428 IU/L). The most frequent diagnosis were (n;%) : viral myositis (29;20%), trauma (24;17%), surgery (24;17%), anoxia (12;8%), drug reaction (6;4%) and metabolic myopathies (2 ;1,4%). In 17 patients (12%) no formal diagnosis could be made with certainty. Few patients developed complications secondary to rhabdomyolisis. Two patients had acute renal failure due to rhabdomyolisis. For one of them, delay in diagnosing rhabdomyolisis probably contributed to the severity of the acute renal failure. Chronic CK elevation was present in 13 patients, of which 3 had dermatomyositis and 8 dystrophynopathies.In 4 cases of dystrophynopathy, the CK elevation was identified incidentally, prior to clinical suspicion of muscle disorder. Those cases were initially investigated for mental retardation or hepatic enzyme elevation. CONCLUSIONS/RELEVANCE: Etiologies of CK elevation in children are various and differ from those reported in adults. In most patient, CK elevation is benign and presents no recurrency. In some cases, rhabdomyolisis is the initial sign of a metabolic myopathy. For others cases like dystrophynopathy, CK elevation is present even when there is no obvious clinical manifestations.
 

[S25.003] Combining Stem Cells and Exon Skipping and Approach To Treat Muscular Dystrophies

Mirella Meregalli, Andrea Farini, Marzia Belicchi, Rachid Benchaouir, Giuseppe D'Antona, Daniele Parolini, Simona Maciotta, Paola Razini, Roberto Bottinelli, Luis Garcia, Nereo Bresolin, Yvan Torrente, Pavia, Italy, Paris, France, Milan, Italy

OBJECTIVE: The main goal of this work would be to combine gene modification strategies with cell-mediated therapies. This approach could permit the autologous transplantation of cells, minimizing the risk of implant rejection BACKGROUND: Mutations in the dystrophin gene cause hereditary muscular diseases as BMD and DMD. The characterization of dystrophin gene and the evidence that different types of adult stem cells are capable of muscle regeneration has lead to the development of potential gene therapy and stem cell treatments for DMD. In some cases, forced exclusion (skipping) of a single or multiple exons can restore the reading frame, giving rise to a shorter, but still functional protein DESIGN/METHODS: Blood and muscle CD133+ were collected from peripheral blood of normal or DMD patients and from skeletal muscle biopsies respectively. The cells were transduced with lentiviral vectors constructed to convey specific antisense oligonucleotides able to induce an efficient exon-skipping and to correct the initial frameshift caused by the DMD deletion. The skipped stem cells were injected in scid/mdx mice to verify the human dystrophin expression and the capacity of the skipped cells to fuse with regenerating muscle fibers. RESULTS: The skipped blood and muscle-derived stem cells were able to fuse in vivo with scid/mdx mice regenerative fibers and to perform expression of a functional human dystrophin and restructure the dystrophin-associated complex. Muscle function was evaluated in vitro by measuring tetanic force of isolated injected TA and EDL muscles and in vivo by using treadmill exhaustion tests. CONCLUSIONS/RELEVANCE: Autologous engrafting of blood or muscle-derived CD133+ cells, preliminary genetically modified to re-express a functional dystrophin, seems to represent a promising approach for DMD. This approach should offer the chance to distribute the autologous corrected stem cells to the whole body musculature with an intra-arterial injection providing a clinical benefit for the dystrophic patients.