The Concept of Muscular Dystrophy in Europe before and after 19th Century Corrado Angelini

PAPERS THAT WILL BE PRESENTED IN 59TH ANNUAL MEETING OF AMERICAN ACADEMY OF NEUROLOGY, BOSTON, 2007

The Concept of Muscular Dystrophy in Europe before and after 19th Century

Corrado Angelini, Padova, Italy

OBJECTIVE: To present history of muscular dystrophies. Although the first anathomical studies were done in humans in 15th century by Vesalius and Canali, the first clinical entities were only identified in 19th century. BACKGROUND: Early degeneration of muscle and fatty infiltration was described independently in UK by Meryon in 1851 in few cases of progressive severe muscular dystrophy. Clinical features of early muscular dystrophy in two male siblings were described by Seminola in Naples in 1834 and further reported in 1836 by Conte and Gioia. They identified therefore what is nowadays called Duchenne muscular dystrophy. DESIGN/METHODS: We reviewed as primary sources the documents including 19th century medical journal articles and text books. Original articles were reviewed in various european journal in their native languages. Muscle specific entities were classified according to their relevance to nowaday myology. RESULTS: The first full description of progressive muscular dystrophies were given in 1861 by Duchenne in France who called this case progressive muscular atrophy and in 1868 he did the first muscle biopsy. Three Heidelberg Myological clinicians (Friedreich, Erb and Hoffmann) named muscle disorders as muscular dystrophies. Erb in 1884 described a juvenile muscular dystrophy, probably corresponding to LGMD2A. Erb criteria for progressive muscular dystrophy were the presence of weakness and wasting, increased lordosis, peculiar gait, scapular winging. Hoffmann established various categories of Muscular Dystrophy: Duchenne (DMD), limb-girdle (LGMD), facio-scapulo-humeral (FSHD) dystrophy. Further progress was done by Becker and Kiener in 1926 who identified an allelic benign variant of DMD. CONCLUSIONS/RELEVANCE: The concept of muscular dystrophy was pioneered by different clinicians: it is likely that several investigators observed and reported independently similar clinical entities in different European countries. Language barriers and the poor diffusion of medical journals precluded an immediate comparison.The development of clinco-pathological method and of genetics allowed close collaboration and more precise diagnoses.

Integrated DNA, CDNA and Protein Studies in Becker Muscular Dystrophy by Multiple Ligation Probe Amplification

Laura N. Pirra, Akanchha Kesari, Orinthal McIntyre, Lakshmi Bremadesam, Erynn Gordon, Alberto Dubrovsky, Buenos Aires, Argentina, V. Viswanathan, Chennai, India, Eric Hoffman, Washington, DC

OBJECTIVE: Identify mutations in Becker muscular dystrophy (BMD) using the Multiple Ligation Probe Amplification method (MLPA). BACKGROUND: Becker muscular dystrophy is a milder form of X-linked Duchenne muscular dystrophy, where Becker dystrophy typically shows in-frame mutations, and Duchene dystrophy out-of-frame (nonsense) mutations. However, there have been relatively few clinical and molecular studies of Becker dystrophy. Here, we report a study of 76 males with immunoblot and/or immunostaining findings of abnormal but present dystrophin, and thus a biochemical diagnosis of Becker muscular dystrophy. DESIGN/METHODS: We utilized the recently described MLPA method on genomic DNA to screen all 79 exons for both large and small deletions and duplications; mutation negative patients were also tested for mRNA splicing abnormalities using MLPA on biopsy cDNA. RESULTS: We identified disease-causing mutations in 60 (79%) of the Becker dystrophy patients. Of the mutation-positive patients, 42 (70%) showed deletions of exons, 14 (23%) showed duplications, and four (7%) showed mRNA splicing abnormalities. The majority of mutations were novel, and there were a higher proportion of mutations in the initial 30 exons relative to previous reports. CONCLUSIONS/RELEVANCE: The findings from this new series of Becker subjects show that in frame duplications are more common than previously thought, and that these would likely be missed by many commonly used molecular diagnostic methods (immunohistochemistry, sequencing, PCR). We also show that MLPA on muscle biopsy cDNA is a rapid and efficient method for identification of splicing mutations.

Compartmentalized Vascular Delivery of Micro-dystrophin as a Gene Therapy Treatment for Duchenne Muscular Dystrophy

Louise R. Rodino-Klapac, Chrystal L. Montgomery, K. Reed Clark, Brian Coley, Ryan Jensen, Louis G. Chicoine, Jerry Mendell, Columbus, OH

OBJECTIVE: Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder with monogenic mutations setting the stage for successful gene therapy treatment for patients. Long-term therapeutic goals include systemic delivery of a small dystrophin transgene, micro-dystrophin, delivered by adeno-associated virus (AAV) via the vasculature. For safety reasons, we have proceeded in a stepwise fashion, where the transgene is delivered via the femoral artery to an isolated lower limb or muscle thereof. BACKGROUND: Isolated limb perfusion (ILP) is more likely to the be next step in gene therapy for the following reasons: 1) delivery of vector to lower limb muscles can produce clinically meaningful results; 2) the lower limb can be compartmentalized to prevent spread of virus to other organ systems providing an important measure of safety; 3) delivery of virus in a compartmentalized system provides safe passage for the virus since pre-existing immunity to AAV precludes muscle transduction. DESIGN/METHODS: We first tested ILP of micro-dystrophin in the mdx mouse model of DMD with the goal of comparing the efficiency of AAV serotypes [AAV1, 6, or AAV8] in crossing the vascular barrier leading to widespread gene expression in muscle. For this study we used a micro-dystrophin construct which deletes the untranslated regions and C-terminus, while retaining spectrin repeats 1-3 and 24, and hinges 1, 2, and 4 of full-length dystrophin. RESULTS: Comparative studies suggest that AAV6 and AAV8 deliver and transduce micro-dystrophin by ILP more efficiently than AAV1. In addition, we have been successful in translating the ILP vascular delivery approach to a larger animal model by specifically targeting the gastrocnemius muscle with a fluoroscopy guided catheter to deliver AAV8.GFP (green fluorescent protein). CONCLUSIONS/RELEVANCE: These findings and ongoing studies set the stage for a future clinical trial in DMD patients with vascular delivery of the micro-dystrophin transgene.

Complement-inhibition in Dysferlin-deficient Muscular Dystrophy

Simone Spuler, Semjon Taubert, Antje Vieweger, Joanna Zabojsczca, Berlin, Germany

OBJECTIVE: To test whether complement-inhibition in beneficial in dysferlin-deficient muscular dystrophy. BACKGROUND: Complement attack contributes to muscle fiber injury in dysferlin-deficient mice and in patients with limb girdle muscular dystrophy 2B due to downregulation of complement inhibitory factor CD55. Consequently, C5b9 membrane attack complex is upregulated on non-necrotic muscle fibers in dysferlin-deficiency. Complement-inhibition might be beneficial. SJL/J mice that carry a mutation in dysferlin are suitable to test the hypothesis. DESIGN/METHODS: Female SJL/J mice (N=54) age 22 weeks were treated with anti-mouse C5 mab (gift from Alexion Pharmaceuticals, Cheshire CT) (N=21), mouse IgG1 control ab (N=21), albumin (N=6) or sham-injected (N=6) at 40 mg/kg bodyweight every 3 days for 4 weeks. The number of necrotic fibers in the quadriceps muscle was determined at the end of treatment. 300 fibers/section were analyzed by two independent observers. < 1% necrotic fibers was interpreted as mildy abnormal, > 1% was considered dystrophic. Data were analyzed by Chi-square test. RESULTS: After four weeks of treatment with anti-C5 ab, muscle histology in SJL/J mice revealed significantly less necrotic fibers than control animals treated with albumin or sham-treated mice. The comparison between anti-C5 mab and isotype control showed a tendency to more improvement with anti-C5 mab, but did not reach statistical significance. CONCLUSIONS/RELEVANCE: Complement-inhibition improves muscle histology in dysferlin-deficient SJL/J mice.

Clinical Description of Infantile Facioscapulohumeral Muscular Dystrophy (FSHD)

James E. Hilbert, William Martens, Alexis Smirnow, Alrabi Tawil, Richard Moxley, Registry Scientific Advisory Committee, Rochester, NY

OBJECTIVE: To study infantile FSHD in a large, national cohort. BACKGROUND: FSHD, the second most common adult muscular dystrophy, results from a deletion of critical repeat elements (D4Z4) in the telomeric region of chromosome 4q35. The exact pathomechanisms remain unknown. Adult FSHD ranges from asymptomatic patients to progressive weakness of facial, scapular, and truncal muscles. There is limited information about infantile FSHD. DESIGN/METHODS: We analyzed the clinical and molecular diagnostic data of FSHD patients in the NIH sponsored National Registry of Myotonic Dystrophy and FSHD Patients and Family Members. Infantile cases were defined as patients with facial weakness 5 yrs of age and/or proximal weakness 10 yrs old (Klinge et al., 2006). RESULTS: The Registry currently contains 50 infantile FSHD patients (72% female). Average age at onset was 5.2 yrs 3.44. Mean diagnostic age was 19.8 yrs 14.61, and patients were enrolled in the Registry at 41.7 yrs 19.53. Fifty-six percent of patients had molecular testing with an average deletion size of 22.8 8.46. The majority of patients (58%) reported facial weakness as their first symptom followed by proximal upper body weakness (38%). Muscle pain occurred in 78% of patients and was most frequent in the back/hips (64%) and shoulder/arms (58%). The majority of patients reported asymmetric muscle weakness of the arms (70%) and legs (70%). Eight patients (12%) used wheelchairs by age 18. Patients reported hearing loss (24%), respiratory effects due to FSHD (12%), and no retinal vascular disorders. CONCLUSIONS/RELEVANCE: Our report, one of the largest samples of infantile FSHD, helps confirm a more consistent and severe phenotype compared to adult FSHD. Contrasting with previous studies, our infantile patients reported greater asymmetric muscle weakness, greater respiratory effects, and less wheelchair dependency. We will also present our review of medical records available in the Registry database.

Diagnostic Delay in Duchenne and Becker Muscular Dystrophy (DBMD): Data from the MDSTARnet Cohort

Emma Ciafaloni, Shree Pandya, Rochester, NY, Deborah Fox, Troy, NY, Dennis Matthews, Denver, CO, Katherine Mathews, Iowa City, IA, Timothy Miller, Tucson, AZ

OBJECTIVE: To describe the diagnostic delay in Duchenne and Becker muscular dystrophy (DBMD) using data from a large population-based surveillance cohort. BACKGROUND: Delayed diagnosis in DBMD has been reported since the 1980s but it is uncertain whether such delay still persists. Early diagnosis is crucial for timely genetic counseling and clinical management. DESIGN/METHODS: The Muscular Dystrophy Surveillance Tracking and Research Network (MD STARnet) is a surveillance system funded by the Centers for Disease Control to identify all cases of DBMD born since 1982 and/or residing within Arizona, Colorado, Iowa and Western New York State. A pooled database containing clinical information abstracted from medical records on 388 individuals was analyzed with SAS software using non-parametric approaches to investigate the age at first concern, time to first serum CK test and definite diagnosis in boys with and without family history (FH) of DBMD. RESULTS: Complete data for all variables was available for 270 boys, 81 with and 189 without FH. Median ageof first concern was 1.6 years (range 0-7.9 years) for those with FH and 4.5 years(range0-14.6 years) for those without FH (p<.0001).Time between first concern and first CK was 3.4 months (range 0-18months) and 10 months (range0-48 months) for those with and without FH (p<.005). Time between first concern and definite diagnosis was 7.4 months (range0-24months) and 13.5 months (range 0-48 months) for those with and without FH (p<.0001). CONCLUSIONS/RELEVANCE: Our findings suggest that diagnostic delay in DBMD still exists. Until newborn screening is available for DBMD, steps should be taken to educate pediatricians and primary care providers on the common DBMD presentations, the importance of early CK testing and the diagnostic strategies for boys presenting with problems of motor function.

Long Term Follow up of Homozygotes with Oculopharyngeal Muscular Dystrophy.

Sergiu Blumen, Itzhak Braverman, Nava Blumen, Kfar Saba, Israel, Ralph L. Carasso, Hadera, Israel

OBJECTIVE: To evaluate the clinical features, evolution and life expectancy in patients with Oculopharyngeal Muscular Dystrophy (OPMD) who are homozygotes for the (GCG)9 expansion in the PABPN1 encoding gene. BACKGROUND: OPMD is frequent among Bukharan Jews in Israel, who share the (GCG)9 founder mutation in the PABPN1 gene. The high rate of consanguinity in this population, together with late onset and normal fertility in OPMD patients, resulted in homozygous cases. DESIGN/METHODS: For 18 years we followed six OPMD homozygotes and compared them to their heterozygous parents and siblings. In addition to clinical evaluation, electrodiagnostic and psychological tests were performed. RESULTS: In all (GCG)9-(GCG)9 patients OPMD started before the age of 30 with bilateral ptosis and dyspagia; external ophthalmoparesis and dysphonia followed in a few years as well as weakness in the proximal limb muscles. All patients had recurrent aspirations and lost weight; five required feeding gastrostomies. All patients are followed by psychiatrists due to cognitive decline and/or psychotic productivity. Two patients died at the ages of 50 and 51. The eldest patient is now 57 years old: he requires assisted ventilation, feeding gastrostomy and psychiatric care for uninhibited behaviour. CONCLUSIONS/RELEVANCE: OPMD is very severe in homozygous patients. It is not restricted to the muscles, but also involves the central nervous system with cognitive and psychotic manifestations. It may reduce life expectancy.

Clinical, Molecular and Protein Correlations in a Large Sample of Genetically Diagnosed Limb Girdle Muscular Dystrophy Patients

Michela Guglieri, Francesca Magri, Maria Grazia D'Angelo, Alessandro Prelle, Rachele Cagliani, Bosisio Parini, Lecco, Italy, Francesco Fortunato, Andreina Bordoni, Roberto Del Bo, Serena Ghezzi, Sabrina Lucchiari, Sabrina Salani, Chiara Zecca, Costanza Lamperti, Dario Ronchi, Patrizia Ciscato, Lucia Morandi, Marina Mora, Isabella Moroni, Carmelo Rodolico, Antonio Toscano, Messina, Italy, Maurizio Moggio, Nereo Bresolin, Giacomo Pietro Comi, Milan, Italy

OBJECTIVE: To evaluate the relative proportion of Limb Girdle Muscular Dystrophies (LGMD), to describe the clinical pattern of the different forms and to identify correlations between genotype, phenotype and protein expression levels, as prognostic factors. BACKGROUND: LGMD are genetically heterogeneous disorders characterised by progressive muscle weakness affecting shoulder and pelvic girdle muscles. Seven autosomal dominant and eleven recessive loci have been so far identified. DESIGN/METHODS: We selected 153 patients with a progressive LGMD phenotype and a dystrophic pattern at muscle biopsy. Age of onset, rate of progression, heart and respiratory function were evaluated. LGMD proteins were studied by immunohistochemical and Western Blot analysis. Molecular analysis of LGMD genes were performed in keeping with protein screening results. RESULTS: Calpain-3 deficiency is present in the majority of our probands (70 patients), followed by dysferlin (35), sarcoglycans (21) and caveolin-3 (10) deficiencies. Genetic confirmation was obtained in 90/131 probands.The identified 98 different mutations include 2 CAV3, 45 CAPN3, 31 DYSF, 5 -SG, 5 -SG, 3 -SG, 1 -SG and 5 FKRP nucleotide changes. The relative frequency was: 1C 1.5%, 2A 31.3%, 2B 20.6%, 2C 2.3%; 2D 4.6%, 2E 3.8%, 2F 0.7%, 2I 3.8%, undetermined: 31.3%. The order of decreasing clinical severity was as follows: sarcoglycanopathy, calpainopathy, dysferlinopathy, caveolinopathy and 2I. Both genotype (null versus missense mutations) and protein defects (absence versus residual amount) correlated with clinical parameters in LGMD2B. This correlation was less striking for sarcoglycanopathy and calpainopathy. CONCLUSIONS/RELEVANCE: Compared to North-European populations, Italian LGMD patients are more affected by calpain and dysferlin deficiency. Conversely LGMD2I has a lower frequency. Protein studies maintain a central role in LGMD characterisation to address genetic diagnosis and to identify prognostic factors. In this regard, LGMD2B patients with partial dysferlin deficiency caused by missense mutations present a more benign clinical phenotype when compared to patients with complete deficiency and null mutations.

MDSTARnet: Case Definition in Surveillance for Childhood Onset Duchenne and Becker Muscular Dystrophy (DBMD)

Katherine Mathews, Emma Ciafaloni, Rochester, NY, Timothy Miller, Dennis Matthews, Valerie Cwik, Paul Romitti, Iowa City, IA, Chris Cunniff, Tuscon, AZ, Charlotte Druschel, Troy, NY, Lisa Miller, Denver, CO, John Meaney, Tucson, AZ

OBJECTIVE: Define diagnostic categories for surveillance of DBMD. BACKGROUND: The Muscular Dystrophy Surveillance Tracking and Research Network (MDSTARnet) is an ongoing multistate consortium to define the epidemiology of childhood onset DBMD. MDSTARnet uses active surveillance to identify all cases of DBMD born after January 1, 1982 who resided in the participating regions. The value of MDSTARnet data is dependent on accurate and consistent DBMD case definition. DESIGN/METHODS: Potential cases were identified through a variety of case finding techniques (e.g., review of ICD9 codes, neuromuscular clinic rosters). Medical records of potential cases were abstracted by trained abstractors. Critical diagnostic elements of each abstract was reviewed independently by 4 clinicians and assigned to a diagnostic category (definite, probable, possible, suspected, female, asymptomatic, or not DBMD) using defined criteria. Assignments were compared and cases without consensus were discussed by participating clinicians to arrive at a consensus case definition. RESULTS: As of January 31, 2006, 481 cases were reviewed for case assignment. Five were returned for additional abstraction and 3 were not DBMD. The remaining 474 cases were assigned as follows: 349 (73%) definite, 56 (12%) probable, 52 (11%) possible, 12 (3%) asymptomatic and 4 (1%) female. All reviewers agreed on initial review in 66% of the cases, the remainder went to conference call. On blinded re-review, 92% of cases were assigned the same diagnostic category. Diagnosis is dependent on muscle biopsy in 25%. CONCLUSIONS/RELEVANCE: MDSTARnet is a unique population-based dataset. Its value is dependent on accuracy of DBMD case classification. The MDSTARnet case definitions and the review approach allow a high degree of confidence in the results. It is of interest that approximately 25% of patients lack diagnostic genetic information. Supported by: Centers for Disease Control and Prevention.

Interim Analysis of a Phase 2 Study of PTC124 for Nonsense Mutation Suppression Therapy of Duchenne Muscular Dystrophy (DMD)

Richard Finkel, Samit Hirawat, Brenda Wong, Cincinnati, OH, Kevin Flanigan, Carsten Bonnemann, H. Lee Sweeney, Elisabeth R. Barton, Philadelphia, PA, Michael Howard, Salt Lake City, UT, Valerie J. Northcut, Gary L. Elfring, Langdon L. Miller, S. Plainfield, NJ

OBJECTIVE: To evaluate the safety, compliance, pharmacokinetics (PK) and pharmacological activity of PTC124 in patients with DMD due to a nonsense mutation (premature stop codon). BACKGROUND: PTC124 is a novel, oral, nonantibiotic, small molecule that promotes ribosomal readthrough of nonsense mutations. PTC124 induces production of full-length dystrophin protein and decreases muscle injury in mdx mice, which have a nonsense mutation in the dystrophin encoding DMD gene. DESIGN/METHODS: 26 patients, ages 5 to 13 years, each received PTC124 for 28 days at a low-dose, 16-mg/kg/day [n=6] or a high-dose 40-mg/kg/day [n=20]. Blood PK samples, extensor digitorum brevis muscle biopsies, serum muscle enzymes, and myometry and timed function test data were obtained at baseline and on Day 27. Immunofluorescence expression of dystrophin is being evaluated using a C-terminal antibody. RESULTS: PTC124 was well tolerated with few potentially drug-related adverse events (mild, transient, abdominal pain/diarrhea in 4 patients), no clinically relevant drug-related laboratory abnormalities, and excellent compliance (>98%). PK assessments indicated stable drug exposures over time, although plasma trough concentrations were lower than predicted from PTC124 studies in adult subjects. In vitro assessments demonstrated dose-dependent PTC124-induced dystrophin expression by immunofluorescence in biopsy-derived cultured myotubes. In vivo assessments suggested qualitative increases in muscle biopsy dystrophin protein by immunofluorescence in a number of patients over the 28 days of PTC124 treatment; more definitive quantitative measurements are underway. Serum muscle enzyme levels declined significantly during treatment. Evaluations of dystrophin mRNA expression by polymerase chain reaction and analyses of myometry and functional test data are in progress. CONCLUSIONS/RELEVANCE: These interim data indicate that PTC124 nonsense-suppression therapy is safe and suggest evidence of pharmacodynamic activity over a 28-day treatment. Because the doses tested did not achieve the target plasma concentrations associated with maximal preclinical activity, further dose escalation in an additional cohort of boys with DMD is warranted.

Neoplasms in Myotonic Dystrophy Type 1 (DM1): Observations in the NIH Sponsored National Registry and Preliminary Hypothesis for Excess Pilomatricomas.

James E. Hilbert, William Martens, Christine Mueller, Mark H. Greene, Rockville, MD, Richard Moxley, Rochester, NY, Registry Scientific Advisory Committee, Rochester

OBJECTIVE: To analyze the prevalence of neoplasms in DM1. BACKGROUND: DM1 is the most common adult muscular dystrophy and results from an unstable trinucleotide repeat expansion (CTG) in the dystrophia myotonica-protein kinase gene located on chromosome 19q13.3. It causes progressive muscle weakness, myotonia, and affects the eyes, heart, endocrine and central nervous systems. Several tumors have been shown to be associated with DM1, mostly benign calcifying cutaneous tumors known as pilomatricomas. Overall, there is limited information on the prevalence of neoplasms in DM1 and how the genetic instability of the disease and new therapeutics may influence neoplasia. DESIGN/METHODS: We have reviewed published articles about neoplasms in DM1. We have also analyzed tumors reported by DM1 patients enrolled in the NIH sponsored National Registry of DM and Facioscapulohumeral Muscular Dystrophy Patients and Family Members. RESULTS: Pilomatricomas were the most prevalent neoplasms in the literature (n=30 patients). The other most common tumors published were thymoma, multiple basal cell carcinoma, and adenomas of the parotid, pituitary, parathyroid, pancreas and thyroid glands. Analysis of the Registry database revealed that 52 of the first 441 enrolled DM1 patients reported neoplasms. Data indicated that 16 patients had skin tumors with 4 basal cell carcinomas and 2 melanomas. The other most commonly reported sites of tumors were breast (n=7); cervix, colon, and thyroid (all n=4). CONCLUSIONS/RELEVANCE: DM1 patients may have an increased frequency of pilomatricomas and other neoplasms. We hypothesize that altered RNA binding protein activity in DM1 may lead to tumor progression through the up-regulation of -catenin via the WNT signaling pathway. Alterations in this pathway have been implicated in the development of pilomatricomas in other disorders and in various neoplasms reported by DM1 patients.

BMD Phenotypes from DMD Mutations: Molecular Mechanisms of Phenotypic Amelioration of Mutations in the DMD Gene

Kevin Flanigan, Olga Gurvich, Chris Anderson, Kim Hart, Robert Weiss, Michael T. Howard, Salt Lake City, UT

OBJECTIVE: To describe mechanisms of phenotypic amelioration in DMD mutations predicting a null or truncated dystrophin protein. BACKGROUND: Both Duchenne Muscular Dystrophy (DMD) and Becker Muscular Dystrophy (BMD) are due to mutations in the DMD gene, encoding dystrophin. In most (90%) cases, the determinant of DMD vs. BMD is whether the mutations preserve an open reading frame for the residual DMD mRNA. However, certain frameshifting point mutations, premature stop codon mutations, and out-of-frame pseudoexon insertions are associated with BMD, rather than the more severe DMD. DESIGN/METHODS: From a large database of dystrophinopathy patients, we identified genotype/phenotype discordant subjects for further molecular characterization, including immunofluorescent and immunoblot analysis and mRNA analysis (including quantitative real-time PCR), and compared to DMD and wild-type controls. Selected mutations were further analyzed using in vitro transcription/translation assays. RESULTS: We have identified three classes of molecular rescue. One is inefficient splicing induced by mutations within native splice sites, or by intronic mutations activating cryptic splice sites. In the latter, case efficiency of splicing is inversely proportional to wild type message levels, and to severity of disease. A second mechanism is seen with point mutations early in the DMD gene: targeted mutational analysis of in vitro constructs demonstrates that two methionine codons in exon 6 may serve as alternate inititiation codons, and both contribute to phenotypic rescue. The third class is point mutations which affect exonic splice enhancer elements, resulting in exon skipping of point-mutation containing exons. Our results confirm recent reports that such exon skipping may result in in-frame mRNA transcripts associatated with BMD. CONCLUSIONS/RELEVANCE: These examples of post-transcriptional molecular mechanisms which significantly reduce disease severity accentuate the difficulty in providing prognostic information from mutation analysis alone, and suggest potential alternate pathways of treatment.

Ribonuclear Inclusions and Muscleblind-like Protein 1 Nuclear Sequestration Do Not Affect Myoblast Differentiation and Splicing Pattern in Myotonic Dystrophy Type 2

Giovanni Meola, San Donato Milanese, Milan, Italy, Rosanna Cardani, Enzo Mancinelli, Milan, Italy, Annalisa Botta, Fabrizio Rinaldi, Giuseppe Novelli, Rome, Italy

OBJECTIVE: We have evaluated if in vitro differentiating myoblasts obtained from Myotonic Dystrophy type 2 (DM2) patients might be a powerful experimental model to characterize the molecular defect in DM2. BACKGROUND: DM2 is an autosomal dominant multisystemic disorder caused by a CCTG expansion in intron 1 of the ZNF9 gene. The mutant transcripts aggregate in multiple ribonuclear inclusions (RNI), which cause trans-dominant effects such as aberrant RNA processing by interacting with RNA binding proteins, as muscle blind proteins (MBNLs). The nuclear sequestration of MBNLs appears to be involved in splicing defects of genes directly related to the DM phenotypes. Many evidences suggest that RNI and MBNL1 sequestration are strongly involved in DM2 pathogenesis. DESIGN/METHODS: The presence of RNI and MBNL1 nuclear sequestration in DM2 myoblast cultures (6) as compared to sister control cultures (5) at different times of differentiation by FISH (CAGG)₅ probe in combination with MBNL1 immunofluorescence, have been analyzed . The degree of differentiation of DM2 myoblasts as compared to controls has been also determined by assessing the expression of several markers of skeletal muscle differentiation (Myogenin-myoD-Myosin slow and fast). Moreover the splicing pattern of genes directly related to the DM2 phenotype (insulin receptor and chloride channel 1) have been examined. RESULTS: In DM2 cell cultures, a clear colocalization between RNI and MBNL1 foci is demonstrable in myonuclei both at myoblast and myotube stages. All the markers examined are expressed at comparable levels in DM2 and normal muscle cell cultures. Our data indicate that in in vitro differentiating DM2 myoblasts and myotubes, the splicing patterns of insulin receptor and chloride channel 1 appear to be similar to those observed in controls. CONCLUSIONS/RELEVANCE: Our findings indicate that neither alteration in myogenic maturation nor splicing defects of chloride channel 1 and insulin receptor occur in DM2 diffentiating myoblasts but DM2 possesses its own molecular features.

The United Dystrophinopathy Project: Design and Current Enrollment in a Large Genotype/Phenotype Database and Patient Registry

Kevin Flanigan, Kim Hart, Eduard Gappmaier, Wendy King, Cheryl Wall, Columbus, OH, Alan Pestronk, Julaine Florence, Saint Louis, MO, Katherine Mathews, Iowa City, IA, Richard Finkel, Carsten Bonnemann, Philadelphia, PA, Brenda Wong, Cincinnati, OH, John Day, Minneapolis, MN, Michael T. Howard, Robert B. Weiss, Salt Lake City, UT

OBJECTIVE: To describe the design and organization of the United Dystrophinopathy Project, a large multicenter consortium which has established a genotype/phenotype database and dystrophinopathy patient registry. BACKGROUND: Mutations in the DMD gene, encoding the dystrophin protein, are responsible for Duchenne and Becker muscular dystrophies (DMD and BMD), X-linked dilated cardiomyopathy, and symptomatic carrier states. Improvements in methods of molecular diagnosis of DMD mutations allows us to determine the influence of dystrophin sequence variation on disease phenotype, identify novel dystrophin mutations and mutation mechanisms associated with disease, and identify cohorts of patients for clinical trials. DESIGN/METHODS: Dystrophinopathy patients seen by investigators at seven large muscular dystrophy centers are enrolled in the UDP. All subjects undergo a yearly standardized prospective clinical examination by a trained clinical evaluator. Clinical history, disease course, diagnostic testing results, medications, and clinical laboratory testing are updated yearly. Blood samples are obtained for DNA banking, and all participants undergo mutation analysis using a combination of direct sequencing and quantitative gene dosage methodologies. Archived muscle specimens from selected patients are analyzed by dystrophin immunoblot, immunofluorescence, RT-PCR, or other studies. RESULTS: There are currently 737 subjects enrolled. Prospective phenotypic evaluation has been performed on 332 subjects, and genotypic evaluation alone has been performed on the remaining 405. Among prospectively studied subjects are 236 DMD patients (mean age = 12.4 years; range 2.6 33.1 years) and 30 BMD patients (mean age = 26.0 y; range 4.75 73.4 years). CONCLUSIONS/RELEVANCE: The UDP represents a valuable tool for the study of natural history of the dystrophinopathies in the setting of modern clinical practice. The presence of a catalogue of patients with complete genotypic and phenotypic characterization will facilitate the performance of clinical trials directed at gene correction therapies.

Correction of Chloride Channelopathy and Myotonia in a Transgenic Mouse Model of DM1 by Oligonucleotide-mediated Skipping of ClC-1 Exon 7a

Thurman M. Wheeler, John D. Lueck, Matthew C. Krym, Robert T. Dirksen, Charles A. Thornton, Rochester, NY

OBJECTIVE: Elucidate the physiological basis of myotonia in myotonic dystrophy type 1 (DM1). BACKGROUND: The mechanism of myotonia in DM1 is controversial. Expression of chloride channel 1 (ClC-1) is reduced in DM1 muscle. It is unknown, however, whether chloride channelopathy is a unitary explanation for the myotonia. Reduced expression of ClC-1 may result either from misregulated alternative splicing (Mankodi, 2002) or reduced transcription (Ebralidze, 2004). DM1 is associated with inappropriate inclusion of ClC-1 exon 7a, which leads to truncated ClC-1 protein. Like DM1, the HSALR transgenic mouse model displays myotonia, reduced ClC-1 expression, and spliceopathy. DESIGN/METHODS: The goal was to induce skipping of ClC-1 exon 7a using antisense oligonucleotides. We designed a morpholino oligonucleotide targeting this exon. Antisense or control morpholino was injected into tibialis anterior and flexor digitorum brevis muscles of HSALR mice. Entry of morpholino into muscle fibers was enhanced by electroporation in vivo. Splicing of exon 7a was determined by RT-PCR. Expression of ClC-1 protein was assessed by immunofluorescence. ClC-1 function was examined by patch clamp analysis of single muscle fibers. Electromyographic myotonia was assessed by a blinded examiner. RESULTS: Antisense morpholino had the intended effect of inducing skipping of ClC-1 exon 7a. Correction of ClC-1 spliceopathy was associated with (1) restoration of ClC-1 protein to the surface membrane; (2) normalization of chloride current density; and (3) reversal of myotonia. Effects persisted up to 8 weeks after a single injection. Injection of control morpholino had no effect. CONCLUSIONS/RELEVANCE: These results show that myotonia in a transgenic mouse model of DM1 results from chloride channelopathy, that loss of ClC-1 function results from spliceopathy rather than reduced ClC-1 transcription, and that antisense-mediated exon skipping is a viable therapeutic strategy in DM1

A Long-term Blinded Controlled Efficacy Study of SNT-MC17/Idebenone in the Dystrophin-Deficient MDX Mouse

Gunnar M. Buyse, Leuven, Belgium, Gerry Van der Mieren, Michael Erb, Jan D'Hooge, Paul Herijgers, Eric Verbeken, Alejandro Jara, Luc Mertens, Leuven, Belgium, Isabelle Courdier-Fruh, Patrizia Barzaghi, Thomas Meier, Liestal, Switzerland

BACKGROUND: The identification of a disease-modifying or protective compound for a certain disease may require early-started and long-term administration, but such type of therapeutic trial is not evident in human patients. We have performed such a trial of SNT-MC17/idebenone in the mdx mouse model of Duchenne muscular dystrophy (DMD), based on the drugs potential to improve mitochondrial respiratory chain function and reduce oxidative stress. DESIGN/METHODS: 200 mg/kg bodyweight of either SNT-MC17/idebenone (ide) or vehicle/placebo (veh) was given from the age of 4 weeks until the age of 10 months in mdx mice (mdx-ide, n=18; mdx-veh, n=13) and wild-type mice (WT-veh, n=9). In vivo cardiac assessments at the age of 10 months included transthoracic echocardiography and strain rate imaging, and haemodynamic pressure-volume loop measurements for determination of intrinsic contractile properties in basal state and during dobutamine stress. Cardiac morphology was evaluated for degree of inflammation and fibrosis. Voluntary wheel running performance was recorded from week 5 to week 12. All evaluators were blinded to mouse type and treatment groups. RESULTS: 10 month old mdx mice showed cardiac hypertrophy and diastolic dysfunction, the latter significantly improving with SNT-MC17/idebenone treatment. During physiological low dose dobutamine stress, placebo-treated mdx mice showed a 58% mortality due to systolic contractile failure, which was significantly reduced to 19% in the SNT-MC17/idebenone treated mdx group. SNT-MC17/idebenone treatment significantly improved long-term voluntary wheel running performance in the mdx mouse. CONCLUSIONS/RELEVANCE: We show that the mdx animal model provides opportunities for long-term controlled preclinical therapeutic studies. We have identified a novel potential therapeutic strategy for DMD, as early-started and long-term treatment with SNT-MC17/idebenone was cardioprotective and improved exercise performance in the dystrophin-deficient mdx mouse. Encouraged by these animal data, SNT-MC17/idebenone is currently under investigation in a randomised controlled trial in DMD patients.

Molecular Diagnosis Yield Is Higher in Severe Than in Milder LGMD Phenotypes.

Corrado Angelini, Marina Fanin, Anna C. Nascimbeni, Elena Pegoraro, Padova, PD, Italy

OBJECTIVE: Limb Girdle Muscular Dystrophies (LGMD) include heterogeneous clinical presentation, ranging from severe to pre-symptomatic. We determined the distribution of LGMD subtypes in a unique referral center. BACKGROUND: LGMDs map to 19 different loci; 7 autosomal dominant (1A-1G) and 12 autosomal recessive (2A-2L). In LGMDs type 2, LGMD2A, LGMD2B and LGMD2I are the most frequent, although variably distributed in different countries. DESIGN/METHODS: We investigated over 1000 patients with severe LGMD (weakness before 12 years, Gowers sign, difficulty climbing stairs, dystrophic biopsy), classical LGMD (weakness after 12 years, difficulty climbing stairs, dystrophic biopsy), proximal/distal myopathy (myopathic biopsy, onset of weakness after 12 years) or idiopathic hyperCKemia (CK over 500 U/L). Dystrophinopathy, congenital dystrophy, dominant myopathies (DM1, FSHD), inflammatory, metabolic, congenital myopaties were excluded. We selected 873 biopsies for a multiple protein screening to identify muscle specific defects: calpain-3, dysferlin, alpha-sarcoglycan (by immunoblotting), caveolin-3 and emerin (by immunohistochemistry). 148 patients with all normal proteins were further investigated for calpain-3 autolytic activity. Gene mutation analysis was conducted to confirm molecular diagnosis. RESULTS: Among 351 cases with the severe or classic LGMD phenotype, 46% were molecularly defined (24% LGMD2A and 12% sarcoglycanopathies). In 179 cases with proximal/distal myopathy only 20% were molecularly identified (14.% LGMD2B and 3% LGMD1C) and in 253 cases with hyperCKemia 11% were characterized (5% LGMD2A and 3% LGMD2D). The most frequent form was LGMD2A (101 patients), followed by LGMD2B (41 cases), LGMD2D (33 cases), LGMD2I (14 cases) and LGMD1C (11 cases). CONCLUSIONS/RELEVANCE: Our data demonstrate that severe phenotypes have higher yield of molecular diagnosis: this is not likely to br due to a methodological bias since all patients underwent an extensive protein/gene analysis. A large group of cases (about 50%) still have no molecular diagnosis: since most are sporadic it is difficult to identify novel candidate genes by linkage analysis.

Development of a Potent Viral Based Therapeutic for Alpha-Sarcoglycan Deficiencies

Louise R. Rodino-Klapac, Chris J. Shilling, Zarife Sahenk, K. Reed Clark, Jerry Mendell, Columbus, OH

OBJECTIVE: Limb-girdle muscular dystrophy (LGMD) type 2D is characterized by skeletal muscle weakness and results from mutations occurring in the -sarcoglycan gene. Localized in the sarcolemma, the sarcoglycans (,,, and ) are a subcomplex of the dystrophin-associated proteins (DAP). Alpha-sarcoglycan (-SG) deficiency is the most common form of sarcoglycan-LGMD and no therapeutic treatments are currently available. BACKGROUND: The -SG mouse model provides an opportunity to test translational treatment approaches. Prior studies have suggested that AAV-mediated -SG gene transfer could not sustain expression because of transgene toxicity, potentially precluding this approach for LGMD2D. DESIGN/METHODS: Herein we describe our in vivo work comparing -SG gene expression from either the ubiquitously expressed cytomegalovirus (CMV) promoter or the muscle specific promoter, muscle creatine kinase (MCK) in the -SG KO mouse in the context of rAAV gene delivery. We injected the tibialis anterior (TA) muscle of 4-6 week old -SG KO mice with rAAV1.-SG (CMV or MCK promoter) at low (3 x 10⁹vg) and high (3 x 10¹⁰ vg) dose. RESULTS: Sustained gene expression was observed irrespective of promoters at six weeks post gene transfer. Quantitation of -SG gene expression by fiber counts yielded similar levels of myofiber transduction (CMV 69%; MCK 63% using high doses). Our studies show sustained and robust gene expression beyond the time at which transgene cytotoxic effects were previously reported using rAAV2. CMV.-SG. Mononuclear cell analysis in our studies showed no evidence of infiltrating B or T cell subsets or macrophages. Differences in findings could relate to AAV serotype. CONCLUSIONS/RELEVANCE: In summary, our data demonstrate equally robust -SG gene expression driven by either the CMV or MCK promoter using AAV1. These findings enhance the possibility for gene therapy as a potential treatment option for LGMD2D

The Expression Profile of Myostatin (MST) in Biopsies of Normal and Pathological Human Muscles.

Amelie Nadeau, George Karpati, Montreal, QC, Canada

OBJECTIVE: To determine the expression profile of MST in biopsied normal and pathological human muscle with the specific aim of studying its possible role in the pathogenesis of muscle fiber atrophy. BACKGROUND: MST is a recently discovered molecule of the TGF super-family with a proven negative role in influencing muscle fibre volume . Eliminating or reducing MST leads to substantial increase of muscle mass in several species. We wondered if upregulation of MST expression, on the other hand, have a role in the pathogenesis of muscle fibre atrophy in various neuromuscular diseases. DESIGN/METHODS: Cryostat sections of routine muscle biopsies were studied: normals (3), Duchenne muscular dystrophy (3), chronic partial denervation(3), spinal muscular atrophy (3), type 2 fibre atrophy (3), dermatomyositis (3), sporadic inclusion body myositis, sIBM (3). For immunostaining, a rabbit polyclonal antiMST antibody was used and the bound antibody was ultimately displayed by Cy-3 fluorescence. Muscle homogenates were studied with immunoblots with the same antibody. RESULTS: In normal muscle, diffuse cytoplasmic immunostaining, with Z disc predilection, was noted mainly in type 2 fibres. Atrophic fibres in the pathological muscles showed no increased immunostaining except in sIBM, where about 30% of atrophic fibres showed increased immunostaining. Immunoblots showed strong bands at 55 kb (precursor) and 28 kb (active dimer) in normal and pathological muscles. In sIBM, the 55kb band was markedly increased while the 28 kb band was substantially reduced. CONCLUSIONS/RELEVANCE: MST upregulation does not appear to be a part of the pathogenetic events in muscle fibres atrophy in the diseases studied. Even in sIBM, only the precursor moiety is unregulated, while the active moiety is significantly reduced suggesting that conversion from the former to the latter is compromised.

Crosstalk between IGF-1 and Myostatin Signaling Pathways in Skeletal Muscle

Yanlei Hao, Kathryn Wagner, Baltimore, MD

OBJECTIVE: Our present knowledge suggests that the signaling pathways for myostatin and IGF-1 are independent. We hypothesize that integration of signals may occur through interaction of downstream signaling components or at the level of transcription. BACKGROUND: Increasing muscle growth and regeneration is an important therapeutic goal for the treatment of muscular dystrophies, acquired myopathies and sarcopenia. Myostatin and IGF-1 are potent negative and positive regulators of muscle growth respectively. There is a variety of data that suggests that myostatin signals through phosphorylation of Smad2 and Smad3, while IGF-1 through AKT. DESIGN/METHODS: The activity of IGF-1-Akt and myostatin-Smad signaling pathways were evaluated in myogenic cell cultures using luciferase assay and western blot treated with IGF-1and/or myostatin and in quadriceps muscles from IGF-1 transgenic and myostatin knockout mice. RESULTS: When we treated RD myogenic cells transfected with a pGL3-(CAGA)12 smad luciferase reporter gene with myostatin and/or IGF-1, luciferase activity increased in the myostatin treated cells in dose dependent manner but was not affected by IGF-1 in the presence or absence of myostatin. Meanwhile when we treated C2C12 cells with IGF-1, the expression of phospho-Akt and its downstream substrate phospho-mTOR increased, but phospho-smad2 did not change. However, C2C12 cells treated with myostatin, displayed decreased phospho-Akt and phospho-mTOR as well as increased phospho-smad2 as would be predicted. In IGF-1 transgenic mice, the immunoactivity of smad2 did not change compared to wild-type littermates while in myostatin knockout mice, the immunoactivity of both phospho-Akt and phospho-mTOR increased. CONCLUSIONS/RELEVANCE: Our data is consistent with the hypothesis that IGF-1 does not directly influence the myostatin-smad signaling pathway, but myostatin directly inhibits the Akt signaling pathway. To further evaluate this hypothesis, we will determine at what step in the Akt signaling pathway does myostatin interact, and whether the IGF-1 and myostatin inhibition can have synergistic effects in vivo as this model would suggest.

Therapeutic Poisoning: Immunosuppresive Therapy for Non-neoplastic Neurological Disease

Michael Glantz, MD, FAAN

Program Description:
Since the introduction of nitrogen mustard for the treatment of solid tumors more than 60 years ago, immunosuppressive and myelosuppressive agents have become increasingly important components of the neurologists therapeutic armamentarium. Initially limited to the use of cytotoxic drugs for nervous system malignancies and corticosteroids for inflammatory disorders affecting the nervous system, therapeutic poisoning is now routinely applied to patients with non-neoplastic central (e.g., multiple sclerosis) and peripheral (e.g., paraproteinemic neuropathies; acute inflammatory demyelinating polyneuropathy and chronic inflammatory demyelinating polyneuropathy) nervous system and neuromuscular (e.g., myasthenia gravis, polymyositis, inclusion body myositis) disorders. Increasingly, novel techniques (e.g., bone marrow transplantation) and novel disease targets for immune system modulation (e.g., Alzheimers disease, Parkinsons disease, Duchenne muscular dystrophy) are being incorporated into the routine care of neurology patients. As a consequence, neurologists must be familiar not only with the neurological toxicities of these agents, but with the practical details of their administration and the spectrum of disorders in which myelosuppressive and immunosuppressive therapy are appropriate.
Upon Completion:
Participants should be able to describe the currently available classes of immunosuppressive and myelosuppressive agents, their neurological and non-neurological toxicities, and the practical aspects of their administration; explain the rationale and optimum strategies for the use of these agents in a wide spectrum of primary and secondary nervous system disorders including demyelinating, neuromuscular, and paraneoplastic diseases; and establish a safe and efficient office-based program for the use of immuno-suppressive and myelosuppressive therapies in patients with nervous system diseases, develop algorithms for treatment and monitoring, and apply standardized metrics for assessing outcome.

The Longterm Fate of Murine Embryonic Stem Cells (MESC) Injected into Skeletal Muscles of Isogenic or Non-isogenic Immuno-suppressed Mice.

Chai Tian, George Karpati, Montreal, QC, Canada

OBJECTIVE: To determine the longterm fate of MESC after injection into the subcutaneous tissue or skeletal muscles of isogenic 129/5v mice or non-isogenic{plusmn]immuno-suppressed mdx mice. BACKGROUND: Cell therapy is a potential tool for the management of muscle diseases either as means of gene transfer or tissue replacement. ESC, on account of their pluripotentiality and immortality, are candidates for this role. However, scanty information is available in this regard. DESIGN/METHODS: CCE MESC derived from 129/5v mouse strain were used. When cultured in maintenance medium, the majority of the MESC were labellable with SSEA-1 and Oct-4 Suspension of MESC (10^{6 or 10}7 cells) or embryoid bodies(100 or 1000) were injected into either the subcutaneous tissue or the anterior tibialis muscles (ATM) of the following strains of 6-week-old animals: 129/Sv mice (isogenic) and mdx mice(nonisogenic) In a cohort of mdx mice, daily injection of tacrolimus (2.5 mg/kg) was performed. Thirty days post injection of MESC, the injected tissues were removed and studied with histology and histochemistry. RESULTS: In the subcutaneous tissue and in the ATM of the 129/Sv, large teratomas developed that contained scanty skeletal muscle cells identified by immunostaining for myosin heavy chain and dystrophin. The same was observed in the subcutaneous tissue and ATM of immunosuppressed mdx mice. In the non-immunosuppressed mdx mice, MESC were not detectable. CONCLUSIONS/RELEVANCE: 1. MESC are immunogenic and will survive only in isogenic hosts or immunosuppressed non-isogenic hosts. 2. MESC form large teratomas indicating their in-vivo pluripotentiality and immortality in skeletal muscle. 3. MESC will not differentiate preferentially into skeletal muscle cells in postnatal skeletal muscles. 4. Thus, MESC differ from myoblasts injected into isogenic skeletal muscles as myoblasts do not form teratomas and they show myogenicity. 5. MESC seem to require molecular modifications to secure preferential myogenecity and suppress tumorigenicity when injected into skeletal muscles.

Vascular Endothelial Growth Factor Gene Transfer Using Adeno-Associated Viral Vectors Stimulates Skeletal Muscle Regeneration and Enhances Muscle Function in mdx Mice

Sonia Messina, Mohammed Aguennouz, Anna Mazzeo, Alessandra Bitto, Mauro Giacca, Trieste, Italy, Francesco Squadrito, Messina, Italy, Giuseppe Vita, Messina, Italy

OBJECTIVE: Vascular endothelial growth factor (VEGF) is a major regulator of physiological and pathological angiogenesis. We tested VEGF effect on muscle function, histopathology, immunohistochemistry and biochemical parameters in mdx and wild type mice. BACKGROUND: Several studies support its role in myogenesis and in myoblast migration and survival. Recently it has been reported that the delivery of VEGF using adeno-associated-virus (AAV) vectors reduces muscle damage and promotes muscle regeneration in experimental models of muscle necrosis. DESIGN/METHODS: We performed intramuscular administration into the biceps and tibialis anterior muscles of AAV-VEGF or AAV-LacZ as controls. RESULTS: One month after injection, AAV-VEGF treated muscles showed augmented expression of VEGF (p<0.05) and VEGFR-2 (p<0.01) compared to controls. VEGF treated mdx mice showed higher forelimb strength (+19,5%, p<0.05) as well as higher strength normalised to weight (+ 14,9%, p<0.05). At quantitative morphological evaluation of mdx mice, VEGF-treated muscles showed a reduction of necrotic fibers area (p<0.05) and an increase of small centrally nucleated fibers area (p<0.05). VEGF-treated mdx mice muscle showed an increased number of cells positive for markers of early and late regeneration, respectively myogenin and developmental myosin heavy chain (p<0.05), and also an augmented capillary density in regenerating fibers area (p<0.05). CONCLUSIONS/RELEVANCE: We report the novel observation of a beneficial effect of VEGF in mdx mice exerted mainly by a pro-regenerative and angiogenic effect. Further studies are needed to better clarify the mechanisms of action of VEGF and therefore the possible therapeutic implications in Duchenne muscular dystrophy.

T-Cap Knockdown by RNA Interference Inhibits Myogenic Regulatory Factors in C2C12 Cells

Chad D. Markert, Martin K. Childers, Winston-Salem, NC

OBJECTIVE: To test the hypothesis that T-Cap knockdown with RNA interference will lead to differential expression of genes involved in muscle proliferation and differentiation, leading to impaired muscle cell growth. BACKGROUND: Null mutation of titin-cap (T-Cap) causes limb-girdle muscular dystrophy type 2G (LGMD2G). Patients with LGMD2G develop muscle wasting and weakness, and lose the ability to walk by the third decade. Previous findings suggest that T-Cap regulates myostatin, a key regulator of muscle growth. It has been shown that T-cap overexpression decreases myostatin. DESIGN/METHODS: C2C12 myoblasts were grown to confluence in growth medium (0 D) and then shifted to differentiation medium for a period of 5 Days. Cells were treated with T-Cap siRNA duplex constructs and analyzed for RNA and protein expression using Northern blots, real-time RT-PCR, and Western immunoblots. Additionally, cDNA from T-Cap knockdown was compared with control in a multi-well PCR-based array designed to provide pathway-focused gene expression profiling. RESULTS: SiRNA treatment decreased T-Cap RNA and protein expression in differentiating muscle cells. Levels of myostatin were subsequently increased, while decreases were observed in the myogenic regulatory factors MyoD and myogenin. SiRNA treatment also prevented development of the normal phenotype of muscle cells in a dose-dependent manner. Finally, statistically significant (p0.05) differential expression of key genes controlling cell cycle was observed. CONCLUSIONS/RELEVANCE: Our findings support the hypothesis that T-Cap knockdown with RNA interference increases myostatin and impairs normal muscle cell differentiation. This process likely occurs downstream of myostatin via repression of myogenic regulatory factors, and cyclin-dependent kinases.

Myostatin Null Mice Have Less Muscle Atrophy in Response to Denervation Than Wild-Type Mice

Brett M. Morrison, Reena S. Shetty, Kathryn R. Wagner, Baltimore, MD

OBJECTIVE: We hypothesize that myostatin, which is a negative regulator of muscle growth, contributes to muscle atrophy following denervation and that myostatin null mice will have less atrophy than wild-type mice following denervation. BACKGROUND: Myostatin is a member of the TGF-beta superfamily that functions as a negative regulator of muscle growth. It is expressed almost exclusively in developing and mature skeletal muscle. Overexpression of myostatin in transgenic mice causes profound muscle atrophy, and myostatin null mice have dramatically increased skeletal muscle mass. In response to denervation, myostatin expression in wild-type mice is increased within a few hours, and this increase may be partly responsible for muscle atrophy following denervation. The role of myostatin in denervation atrophy will be investigated in myostatin null mice. DESIGN/METHODS: We performed sciatic nerve transections in wild-type and myostatin null mice and measured wet weight and myofiber diameter in denervated and non-denervated muscle. RESULTS: At 7 days post-transection, denervated muscle weights and myofiber diameters from wild-type mice were reduced 22% and 36%, respectively. In contrast, muscle weights and myofiber diameters in denervated myostatin null mice were reduced only 7% and 13%, respectively. CONCLUSIONS/RELEVANCE: The mechanism underlying the attenuated muscle atrophy in myostatin null mice is unclear, but may involve reduced apoptosis of myogenic precursors or decreased muscle protein breakdown by either the ubiquitin-proteasome or caspase degradation pathways. Given the profound atrophy found in many neuromuscular diseases, including neuropathies and amyotrophic lateral sclerosis, these results provide important pre-clinical data for the rational development of new therapies.

Physical and Mental Perception of Quality of Life in Patients with Myotonic Dystrophies: Correlation between Objective and Functional Measures of Muscle Disability, Cognitive, Behavioral and Educational Aspects

Valeria Sansone, Sandra Gandossini, Marta Panzeri, Corrado Angelini, Arianna Palmieri, Padua, Italy, Siciliano Gabriele, Leda Volpi, Pisa, Italy, Tiziana Mongini, Torino, Italy, Luisa Politano, Naples, Italy, Roberto Massa, MariaBeatrice Panico, Vittorio Pisani, Rome, Italy, Maurizio Grandi, Costa Masnaga Como, Italy, Antonio Toscano, Olimpia Musumeci, Carmelo Rodolico, Messina, Italy, Enzo Mancinelli, Michael R. Rose, London, United Kingdom, Giovanni Meola, Milan, Italy

OBJECTIVE: (i) To assess health-related Quality of Life (QoL) in patients with myotonic dystrophy type 1 (DM1) and type 2 (DM2) using an individualized quality of life questionnaire for neuromuscular disorders (INQoL); (ii) To correlate QoL perception with muscle weakness and disability, cognitive, behavioural and educational aspects. BACKGROUND: Whether it is the muscle symptoms or the multiorgan involvement that most affect quality of life in DM patients and which life domains are most affected is yet to be determined. DESIGN/METHODS: We administered INQoL Italia to 86 patients with moderately severe DM1 (CTG range 100-650, n = 73) and genetically confirmed DM2 (n = 33) and determined: (i) a total QoL index resulting from the impact of 4 muscle symptoms (weakness, myotonia, pain, fatigue) on 5 life domain subscores (activities, independence, relationships, emotions, body image); (ii) muscle strength (modified 5-point MRC scale, MMRC); (iii) muscle impairment (Muscular Impairment Rating Scale, MIRS); (iv) Mini Mental State Examination, MMSE; (v) WAIS-R profile, IQ; (v) apathy (apathy scale). RESULTS: (i) All muscle symptoms correlate significantly to INQoL total scores, muscle weakness and fatigue (R =0.8, p < 0.0001) more than myotonia ( R=0.7, p <0.0001) and pain (R = 0.32, p<0.05); (ii) Patients with DM2 have a significantly worst perception of muscle pain compared to patients with DM1 (p <0.0001); (iii) Perceived severity of muscle symptoms in DM1 does not correlate with (CTG)n expansion; (iv) INQoL muscle weakness and fatigue perception significantly correlated with MMRC and MIRS (R=0,5, p< 0.0001); (v) There is no correlation between INQoL total score and years of education, MMSE, apathy and IQ scores. CONCLUSIONS/RELEVANCE: Weakness and fatigue are the most important symptoms affecting QoL perception in our DM study population. This information is crucial to target potential treatment trials and to direct future human and economic resources.