Abstracts from de Annual Meeting - American Academy of Neurology - april 2004 San Francisco

[P01.139] Bone Mineral Density in Pediatric Neuromuscular Disorders. Are All Patients at Equal Risk of Developing Osteopenia?

Ismail A. Khatri, Susan T. Iannaccone, Mouin G. Seikaly, Dallas, TX

OBJECTIVE: To evaluate bone mineral density (BMD) in different pediatric neuromuscular disorders to identify the high-risk group(s) of patients. BACKGROUND: Pathological fractures are common in pediatric neuromuscular disorders. The relationship of BMD to fracture risk is well established in post-menopausal women. However, this relationship is still controversial in children. Dual energy X-ray absorptiometry (DEXA) has become a standard technique for the measurement of BMD in adults. Relatively limited data is available on BMD in pediatric neuromuscular diseases other than Duchenne muscular dystrophy. DESIGN/METHODS: We reviewed the results of all DEXA scans done in our pediatric neuromuscular clinic during 2002 and 2003. Bone mineral density was performed on spine region L1-4 using dual X-ray absorptiometry (Hologic QDR 4500). Osteopenia was classified as mild if the Z-scores were between 0 and -1.5; moderate if Z-scores were between -1.5 and -2.5; and severe if Z scores were <-2.5. RESULTS: 84 DEXA scans were performed: 3 on patients with Becker muscular dystrophy (BEMD), 55 on patients with Duchenne muscular dystrophy (DMD), 3 on patients with limb girdle muscular dystrophy (LGMD), 2 on patients with myotonic dystrophy (DM), 3 on patients with congenital myopathies (CM), 7 on patients with congenital muscular dystrophies (CMD), and 11 on patients with spinal muscular atrophy (SMA). The mean Z-scores +/- the standard error of the mean (SEM) in each group were as follows: BEMD -1.53+/-0.40 (range -2.20 to -0.82); DMD -1.72+/-0.10 (range -3.91 to -0.11); LGMD -0.37+/-0.64* (range -1.22 to +0.91); DM -0.25+/-0.05 (range -0.31 to -0.21); CM -1.77+/-1.13 (range -4.01 to -0.31); CMD -1.67+/-0.42 (range -4.02 to -0.62); SMA -2.25+/-0.31* (range -3.82 to 0). ANOVA showed that spinal muscular atrophy and limb girdle muscular dystrophy were significantly different from the other disorders. CONCLUSIONS: 1. Patients with spinal muscular atrophy have the lowest Z-scores and therefore may be at high risk for pathologic fractures.
2. Children with limb girdle muscular dystrophies have high Z-scores and perhaps are at minimal risk of pathological fractures.
3. We recommend that bone mineral density should be evaluated in all patients with spinal muscular atrophy. Treatment for low BMD in children remains controversial. Supported by: Muscular Dystrophy Association of America

P02.159] Strength and Fatigue in mdx Mice Treated with Weekly Oral Prednisolone for 52 Weeks

Anne M. Connolly, Elizabeth M. Streif, Richard M. Keeling, St. Louis, MO

OBJECTIVE: 1. To establish an effective in vivo measurement of strength and fatigue in mdx mice. 2.To demonstrate long term effectiveness of weekly oral prednisone in this mouse model. BACKGROUND: We have previously shown weekly oral prednisolone prolongs survival and improves strength in dydy mice(1). This regimen has also been shown to be effective in one pilot study of boys with Duchenne muscular dystrophy (DMD)(2). While there is debate about how well the mdx mouse mimics DMD, the longest in vivo strength analysis was only 24 weeks(3). While corticosteroids have been shown to benefit mdx mice over short periods of time, no long term studies have been performed. Effective therapy of boys with DMD will require lifelong treatment. Here we model treatment over one year in the C57Bl10 mdx mouse. DESIGN/METHODS: We studied forepaw grip strength 5 consecutive times at age 3, 4, 6, 8, 10, 12 and then every 4 weeks until 52 weeks. We calculated fatigue at each time point as previously described(3). Eighteen mice were treated with weekly oral prednisolone (10mg/kg/week in two doses on consecutive days); 18 were untreated. RESULTS: 1. While both untreated mdx and controls peak strength by 8-10 weeks, the mdx is significantly weaker at that time (17g.force/ g.body weight versus 20g.force/g.body weight p<0.05). Thereafter, an average progressive decline in strength of 0.3 g.force/g.body weight occurs weekly (versus <0.1 g.force/g.body weight for controls). 2. Mice treated with weekly oral prednisolone were stronger at each time point and showed a slower average decline in strength (0.2g.force/g.body weight). 3. Fatigue is present in all untreated mdx mice at all time points (25-40% fatigue versus 0-5% for healthy controls p<0.005). 4. Treatment with weekly oral prednisolone improved fatigue in female mdx at ages 8-36 weeks (p<0.05) but does not become normal. Males show no improvement. CONCLUSIONS: We demonstrate effective methods for measuring long term strength and fatigue in the mdx mouse. We show for the first time long term benefit of oral prednisolone in this model. Given that corticosteroids remain the best treatment of boys with DMD and that multiple pharmacologic agents may be necessary to improve outcome, it is critical to be able to demonstrate in an animal model which drugs are acting synergistically or antagonistically over long periods.
1. Connolly AM, Keeling RM, Streif E, Pestronk A, Mehta S. Complement 3 deficiency and oral prednisolone improve strength and prolong survival of laminin α2-deficient mice. J Neuroimmunol 2002;127:80-87.
2. Connolly AM, Schierbecker J, Renna R, Florence J. High dose weekly oral prednisone improves strength in boys with Duchenne muscular dystrophy. Neuromuscul Disord 2002;12(10):917-925.
3. Connolly AM, Keeling RM, Mehta S, Pestronk A, Sanes JR. Three mouse models of muscular dystrophy: the natural history of strength and fatigue in dystrophin-, dystrophin/utrophin-, and laminin α2-deficient mice. Neuromusc Disord 2001;11:703-712. Supported by: Muscular Dystrophy Association

[P05.134] Testing Stabilizing Function of Dystrophin in Human Duchenne Muscle Dystrophy Cells (DMD) vs. Control Cells (CC) Due to Lowered Stress Tolerance: Promising In Vitro Testing Tool for Possible Functional Vital Transfection Efficiency Measurements

Denis Bron, Sima Dadelahi, Ashley Hayes, Denise Brenklé, Friedel Wenzel, Steck Andreas, Basel, Switzerland

OBJECTIVE: DMD is a X-linked myopathy and is caused by mutations in the dystrophin encoding gene. As generally known, the absence of dystrophin causes muscle fibre necrosis in DMD patients for example due to lowered stress tolerance by membrane fragility. BACKGROUND: Current results in mouse myotubes support suggested mechanical role of dystrophin in the context of the membrane-cytoskeletal complex. To support the results in mouse myotubes, we tested human DMD cell resistance due to osmotic stress. Further, we evaluated these results for possible functional consequences of in vitro transfection methods. DESIGN/METHODS: Human non transfected DMD- and control- muscle cells were stressed by hypoosmotic Dulbecco buffer (100mosm). Pictures were taken before buffer exposure and after 3 and 12 minutes. Transcellular cell diameter measurements were taken and analysed statistically. In addition, fluorescent enzymatic probes such as EthD-1 (intranuclear cell death marker) and Calcein AM (cytoplasmatic vitality marker) were used as an cell membrane and nuclear permeability marker. RESULTS: Statistically significant differences in cell swelling comparing each cell line (DMD p=0.044, CC p=0.43) were found after hypoosmotic exposure. The difference between each cell was significant (p<0.001) higher in the DMD cell line. Further, the DMD lysis rate differs statistically significant (p<0.0001) compared to CC. CONCLUSIONS: Our results confirmed the mechanical role of dystrophin in human DMD cells. Further, our in vitro study using the same cells demonstrates osmotic resistance as promising tool testing cell transfection functionally. Combining markers such as cell diameter measurements, lysis rate and fluorescent stains increase diagnostic relevance. Further work is underway studying additional parameters such as bleb manifestations and cytoplasmatic granulation.

[P01.118] Creatine Monohydrate Supplementation Appears Safe for Children with Neuromuscular Disorders

Jonathan B. Strober, San Francisco, CA

OBJECTIVE: To evaluate the safety and efficacy of creatine supplementation in a select cohort of children with neuromuscular disorders. BACKGROUND: Creatine, muscles main store of energy, is transported in the blood by a carrier protein and actively taken up by tissues with high energy demands with 95% of the total body creatine found in skeletal muscle Creatine supplementation has been shown to raise its plasma concentration, sustainable with repeated dosing, and can increase the total creatine content of muscles. Patients with neuromuscular disorders have been found to have reduced concentrations of phosphocreatine, a marker for total creatine. In two reports, renal dysfunction has been exacerbated in patients receiving creatine supplementation. However, in several studies of creatine supplementation in neuromuscular disorders no significant side effects were reported. Some of these studies found mild, but significant, benefit from supplementation, however, others found no benefit. Therefore, continued investigation is warranted in children with neuromuscular disorders. DESIGN/METHODS: Five children with neuromuscular disorders (2 with Duchenne muscular dystrophy (DMD), 2 with limb-girdle muscular dystrophy and 1 with CIDP) were enrolled in this cohort study, 2 males (DMD) and 3 females with an age range of 6 – 10 years. Patients took an initial loading dose of creatine monohydrate 25 mg/kg/dose four times a day (100mg/kg/day) for a total of 5 days, followed by 50 mg/kg/day in one daily dose for 30 days. Each child underwent baseline testing, repeat testing at the end of the 30 days of supplementation and one month off creatine. Strength was tested using myometry and functional assessment was made using Jebsen hand-grip test and the Gross Motor Function Measure. Muscle mass was determined by urine 3-methylhistidine levels. To monitor for toxicity a complete blood count, serum electrolytes, serum glucose and calcium, liver function tests, blood urea nitrogen and creatinine, routine urinalysis and 24 hour urine creatine levels were obtained. Toxicity was assessed using a modified common toxicity criteria from the Cancer Therapy Evaluation Program. Any toxicity graded 3 or higher was considered unacceptable. RESULTS: All five children completed the study without significant side effects. The only child with any significant improvement was the girl with CIDP, who showed a 200% increase in hip extension, sustained over the 2-month testing period. Three of the patients with muscular dystrophy showed a 25-50% decrease in hip flexion strength and one boy with DMD showed a 50% decrease in hip abduction over the 2 months. All other measurements were not significantly changed. CONCLUSIONS: While creatine monohydrate appears safe in children with neuromuscular disorders, preliminary data suggest no short-term benefits in muscle mass, gross motor function or strength. Supported by: This study was funded in part in the UCSF Pediatric Clnical Research Center with funds provided by the National Center for Research Resources M01RR01271 and the UCSF

[P05.135] Missense Mutations in the Actin Binding Domain (ABD) of Dystrophin Cause Muscular Dystrophy by Disrupting Protein Stability

Fiona L. Norwood, Southampton, Hampshire, United Kingdom, Andrew Sutherland-Smith, John Kendrick-Jones, Cambridge, United Kingdom

OBJECTIVE: To determine the effects of pathogenic mutations on dystrophin protein stability. BACKGROUND: Dystrophin is a large cytoskeletal protein whose absence or disruption is associated with Duchenne (DMD) or Becker muscular dystrophies (BMD) respectively. The N-terminal domain of dystrophin has been shown to bind to F-actin in vitro and in vivo and is termed the actin binding domain (ABD). Several pathogenic mutations have been detected within the human ABD of dystrophin. DESIGN/METHODS: Wildtype ABD GST-fusion protein constructs were expressed, purified using affinity chromatography, cleaved to remove their GST tags and their solubility and actin binding function assessed. Further constructs in which the two cysteine residues in the wildtype ABD were replaced by serine residues were made and the functional assays repeated. Mutant ABD GST-fusion protein constructs were made and the relative solubility of each expressed protein was assessed. RESULTS: Expression and characterisation of the human dystrophin ABD led to the observation that when the two cysteine residues normally present in this domain were replaced by serines it greatly enhanced the subsequent purification and stability of the ABD while preserving its ability to bind to F-actin. The introduction of pathogenic mutations, associated with specific clinical phenotypes in muscular dystrophy patients, into the ABD affected its ability to fold correctly and thus to be purified and its properties tested. Furthermore, incorporation of these pathogenic mutations into the cysteine exchanged wildtype ABD showed that these single residue substitutions still caused the expressed domain to be unstable and not to correctly fold. CONCLUSIONS: We predict that these pathogenic residues must be in crucial positions in the actin binding domain so that when mutated they destabilise its structure. Our structural analysis of this domain has confirmed this conclusion. Supported by: Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge, UK.

[P02.157] Combined Deficiency of Calpain and Dystrophin Mutually Reduce the Severity of Phenotypes?

Maria J. Molnar, Michael Sinnreich, Agnes Herczegfalvi, Eva Siska, Budapest, Hungary, George Karpati, Montreal, QC, Canada

OBJECTIVE: To determine the effect of an abnormal dystrophin and calpain expression profile on the skeletal muscle phenotype in patients with muscular dystrophy. BACKGROUND: Dystrophin deficiency is an X chromosomal inherited disease causing Duchenne/Becker muscular dystrophy. Calpainopathy is a recessive disorder usually resulting in limb girdle muscular dystrophy. Both gene defects usually result relatively severe phenotypes. DESIGN/METHODS: The 40 years old proband had mild quadriceps weakness and myalgia since age 32 years. EMG revealed myopathic changes, serum CK activity was x 15 times elevated. His sister is asymptomatic with serum CK 329U/l. The son of her sister was a clumsy child and at age 6, mild leg weakness was observed. By age 10, his signs did not progress. His serum CK activity is x 20 elevated. RESULTS: Histological examination of the proband's muscle showed moderate dystrophic changes. Immunocytochemical staining with Novocastra DYS1 and DYS3 antibodies revealed only a few revertant fibers while with DYS2 antibodies, all fibers were dystrophin positive. Western blot analysis of the muscle showed total absence of calpain and a reduced size and amount of dystrophin shown with the DYS2 antibody, confirming the diagnosis of Becker dystrophy and calpainopathy. Mutational analysis of the dystrophin and calpain genes is in progress. CONCLUSIONS: This is the first report of a patient with combined dystrophinopathy and calpain deficiency. The unusually mild phenotype suggests that the combined deficiency of calpain and dystrophin may mutually reduce the severity of phenotypes. This observation might have an important impact on the molecular therapy of the muscular dystrophies.

[EV.013] Factors Influencing the Transduction Efficiency and Duration of Transgene Introduced into Muscles by Plasmid-Mediated Electrotransfer (PMET)

Renald Gilbert, Nancy Larochelle, Yifan Lu, Maria J. Molnar, Budapest, Hungary, An-Bang Liu, Hualien, Taiwan, Basil J. Petrof, Kristian Orlopp, Hanns Lochmuller, Munich, Germany, Josephine Nalbantoglu, George Karpati, Montreal, QC, Canada

OBJECTIVE: To determine factors that influence the level and duration of transgene expression in muscle fibers in dystrophin-deficient (mdx) and of immune incompetent (SCID) mice after PMET. BACKGROUND: Plasmid-mediated gene transfer into muscle is a potentially safe and cost-effective procedure to treat genetic deficiencies such as Duchenne muscular dystrophy (DMD). The efficiency of plasmid-mediated gene transfer can dramatically be increased by the application of an electric current (PMET) to the target muscle. One of the unclear aspects of PMET into muscle fibers is the level and duration of transgene expression in the presence versus absence of immune competency of the host, as well as the role of isogenicity and size of the transgene. DESIGN/METHODS: The tibialis anterior muscle (TA) of adult SCID mice was pretreated with hyaluronidase followed by the electrotransfer of 30 μg of plasmid (pCBLacZ) coding for β-galactosidase (β-gal). The TA of adult mdx mice was treated in the same manner with pCBLacZ and with plasmids coding for the human microdystrophin (9-kb, pHDysMic) or the murine full-length dystrophin (19-kb, pMDysFl). The number of transduced fibers per muscle (β-gal+ or dys+) was analyzed at 10, 90, 180 and 360 days post-treatment. In the case of SCID mice, the amount of β-gal produced and the amount of plasmid DNA in the muscle were also measured by luminometry and real time PCR, respectively. RESULTS: At 10 days post-injection, SCID muscle was transduced much more efficiently (1981 β-gal+ fibers) than mdx muscle ( 312 β-gal+ fibers) after PMET of pCBLacZ. In mdx muscle, more muscle fibers were transduced using small plasmids such pCBLacZ and pHDysMic (505 dys+ fibers) compared with pMDysFl (148 dys+ fibers). Although no significant reduction in the number of β-gal+ fibers was observed in the SCID up to 360 days post-treatment, there was a reduction (2.8X) of the amount of β-gal produced at 360 days. A significant reduction (12X) in the amount of plasmid DNA in SCID muscle was demonstrated at 180 days post-treatment. In mdx muscles injected with pMDysFl, the number of dys+ fibers was also reduced at 180 ( 51) and 360 days (41) post-treatment. CONCLUSIONS: Transduction efficiency of PMET varies greatly with the mouse strain and the transgene size. Furthermore, even in the absence of a significant immune competency of the host (SCID), there is a slow but significant decline of the plasmid DNA and transgene protein level. This implies that repeated but relatively infrequent administration of plasmid DNA may be necessary for continued high-level production of therapeutic gene product in muscle.

[P02.156] Gene Expression Profiling in Duchenne Muscular Dystrophy Patients Using Specifically Dedicated Oligonucleotide Microarray

Armelle Magot, Martine Le Cunff, Nolwenn Le Meur, Jean Marie Mussini, Jean J. Leger, Yann Pereon, Nantes, France

OBJECTIVE: To examine the pathogenic pathways and identify new or modifying factors involved in Duchenne Muscular Dystrophy (DMD) using microarray technology with muscle specific oligonucleotide chips. BACKGROUND: The development of DNA microarrays for comprehensive RNA expression analysis raises the exciting opportunity to examine biological pathways and to compare the hypotheses deduced from the study of histological pathology with the findings of molecular pathology. In particular, although the histopathological pathology of dystrophic tissue is well documented, the underlying molecular pathways remain poorly understood. DESIGN/METHODS: Skeletal muscle mRNA from four DMD patients was compared with normal skeletal muscle mRNA. We developed a muscle specific microarray comprised of 3588 oligonucleotides. The latter represented genes involved in neuromuscular physiology and related diseases. They were selected from suppression subtractive hybridization libraries, pangenomic microarray hybridization data and literature databases. Two replicates of each experiment were done using different microarray slides. Two statistical analysis tools specifically dedicated to microarrays data (SAM, Significance Analysis of Microarray and Limma, LInear Models for MicroArray data) were used. Only genes commonly yielded by both methodologies were admitted in the final list of genes differentially expressed in DMD biopsies compared with unaffected biopsies. RESULTS: 1009 genes were differentially expressed in dystrophic muscles as in comparison with normal skeletal muscle samples. Part of them reflected changes in the histological pathology (e.g. extracellular matrix, muscle structure and regeneration). Among differentially expressed genes, a few ones were related to two specific molecular pathways that could contribute to the pathogenic process. Genes encoding matrix metalloproteinase 2, 3 and 14 and tissue inhibitor metalloproteinase 1 and 2 were overexpressed, suggesting a dysregulation of tissue remodeling pathways. Genes encoding caveolin structure (caveolin 1, 2 and flotillin 1) were also overexpressed, revealing that this signaling pathway might be substantially affected in DMD muscle fibers. CONCLUSIONS: The present study illustrates the potential interest of microarray technology in the comprehension of neuromuscular disorder pathophysiology. In particular, it suggests that metalloproteinase and caveolin signaling pathways might be affected in the DMD dystrophic process. It also highlights a large number of genes whose involvement in the muscular dystrophy pathogenesis was unknown. Their identification might be important in the understanding of the disease process.