Resumos que serão apresentados no Encontro Anual da Academia Americana de Neurologia em abril de 2004 em São Francisco USA relacionados a distrofia muscular

[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.

[P05.131] A Peculiar Form of Limb Girdle Muscular Dystrophy with Combined Dysferlin and Calpain-3 Deficiency and Probable Autosomal Dominant Inheritance

Michael Sinnreich, George Karpati, Montreal, QC, Canada

OBJECTIVE: To report clinical, histopathological and biochemical findings in a mildly affected mother and two severely affected daughters presenting with a limb girdle muscular dystrophy with combined dysferlin and calpain-3 deficiency. BACKGROUND: Dysferlin is the protein product of the gene that is defective in patients with limb girdle muscular dystrophy type 2B (LGMD2B) and Miyoshi myopathy. Calpain 3 is the muscle-specific member of the calcium activated neutral protease family and primary mutations in the CAPN3 gene cause limb girdle muscular dystrophy type 2A. Both these disorders are known to be inherited only as an autosomal recessive trait. Secondary reduction of calpain-3 levels have been observed in primary dysferlin and titin deficiency, fascioscapulohumeral dystrophy and Duchenne muscular dystrophy. DESIGN/METHODS: A mother of two affected daughters presented with onset of a mild limb girdle muscular weakness in the fourth decade. Two of her daughters presented with a disabling limb girdle muscular weakness with onset in the second decade. Muscle biopsies of these three patients were processed for histological and biochemical analysis. Western blots were performed for dysferlin and calpain-3. RESULTS: Histological analysis of the mother’ s muscle biopsy showed certain fascicles which contained up to 50% of lobulated fibers. Other fascicles contained predominantly small fibers with numerous nemaline rods. The muscle biopsy of both daughters showed advanced dystrophic changes with large variability in fiber size, necrotic fibers undergoing phagocytosis, and replacement of muscle by adipose and fibrous tissue. There was complete absence of dysferlin immunostaining and near complete absence of calpain-3 immunostaining on Western blots of the biopsy samples from all three patients. CONCLUSIONS: We describe three unsual features of a limb girdle syndrome in this family: 1. The inheritance pattern appears to be autosomal dominant with anticipation. 2. All affected members show combined absence of two different molecules implicated in LGMD2A and LGMD2B respectively. 3. The two generations show marked discordance in the myopathology. Mutational analysis of the patients and full investigation of the father will clarify the genetic background in this unique family. Supported by: the Canadian Institutes of Health Research, the Muscular Dystrophy Association of Canada and USA, the Association Francaise contre les Myopathies and the Lichtensteinstiftung, Basel, Switzerland.

[S62.005] Clinical Heterogeneity in Limb Girdle Muscular Dystrophy Type 2I

Elena Pegoraro, Chiara Boito, Eleonora Mondelli, Paola Prandini, Alessia Bagattin, Marina Fanin, Corrado Angelini, Padova, Italy

OBJECTIVE: 1) To identify limb girdle muscular dystrophy type 2I (LGMD2I) patients in a large cohort of undiagnosed limb girdle muscular dystrophy patients and 2) to derive genotype/phenotype correlations. BACKGROUND: LGMD2I is due to mutations in the fukutin related protein gene (FKRP). FKRP is a putative glycosyltransferase involved in the alpha dystroglycan glycosylation. DESIGN/METHODS: 214 patients who showed muscle histopathology consistent with a muscular dystrophy or myopathy with normal dystrophin, alpha sarcoglycan, calpain and dysferlin were studied. The entire entire 1.5 kb FKRP coding sequence from patient DNA was analysed by single strand conformational polymorphism (SSCP) or denaturing high performance liquid chromatography (DHPLC) of overlapping PCR products followed by direct sequencing of heteroduplexes. RESULTS: Thirteen LGMD2I patients were identified by FKRP gene mutations analysis (6% of all patients tested). Missense mutations were detected in about 80% of the mutant chromosomes (21/26). Of the six missense mutations identified, three were novel. The C826A common mutation was present in 38% of our LGMD2I patients (4 patients were homozygous and 1 compound heterozygous). a-dystroglycan immunofluorescence in patients muscle biopsies showed a severe reduction in most of the cases. Clinical phenotype in our LGMD2I patients varied from asymptomatic hyperCKemia, to severe early-onset to mild late-onset muscular dystrophy. A dilated cardiomyopathy and respiratory insufficiency was a common feature. Among the C826A carrier great intrafamilial and interfamilial clinical variability was observed. CONCLUSIONS: Our data suggest that FKRP gene mutations are a relatively frequent cause of LGMD. Disease progression was complicated by respiratory insufficiency or cardiomyopathy.

[P05.136] Oculopharyngeal Muscular Dystrophy: An Uruguayan Population with 11 (GCG) Repeats

Medici Mario, Mercedes Rodríguez, Claudia Camejo, Leda Roche, Juan Jose Castagneto, Claudia Braida, María Mirta Rodríguez, Montevideo, Montevideo, Uruguay, J. Pierre Bouchard, Quebec, QC, Canada, Bernard Brais, Montreal, QC, Canada

OBJECTIVE: The aim of this study was better caracterise clinically and molecularly Uruguay OPMD cases: 1-Determine the number of affected individuals and develop molecular análisis, in order to confirm clinical diagnosis and detect at risk relatives.
2-Establish the genotype-phenotype correlation. BACKGROUND: Oculopharyngeal muscular dystrophy (OPMD) is a late onset autosomal dominant muscular dystrophy characterized by bilateral eyelid ptosis, progressive dysphagia and proximal limb weakness, beginning usually in the fifth or sixth decade of life. The OPMD mutations(14q11.2–q 13) have been identified as short (GCG) 8-13 expansions in the PABPN1 gene coding for polyalanine.
A group of 22 families coming from Canary Islands have been studied in Uruguay since 1971. DESIGN/METHODS: A prospective study including 120 individuals of 27 unrelated families, with ages ranging from 21 to 87 years ( mean age 50 years), took place during the past twenty four months. The protocol for consented patients included the collection of personal and family background data, and the pedigree charts. Muscle power was clinically assessed and dysphagia was quantified through the cold water test. All patients signed informed consent and agreed to blood withdrawal and determination of number of repeats by means of a non radioactive PCR and haplotypes studies. All the individuals over 20 years old, with a known family diagnosis, with at least one member with intranuclear filaments found by electron microscopy were included in the study. RESULTS: Among the patients studied, 54% were symtomatic and 46% were asymtomatic. Eyelid ptosis was the most commom sign, 100%, while dysphagia was found in 70% of patients. In the symptomatic group eyelid ptosis was the initial symptom in 52%, while dysphagia was present at onset in only 16%. Ophtalmoparesis was observed in 54% of cases while proximal weakness ocurred in 44%. Moleculal analysis confirmed clinical diagnosis in all symtomatic patients, all showed 11 (GCG) repeats and one only 9 repeats. In the asymtomatic patients 25% showed 11 (GCG) repeats (at risk group), the other 75% showed normal repeats. CONCLUSIONS: This work is the first to establish that Uruguay OPMD cases are mostly carriers of a (GCG)₁₁ mutation. This large cohort suggests that the onset of OPMD in these patients is slightly earlier than in French-Canadian (GCG)₉ carriers and that both, ophthalmoparesis and proximal weakness, may be more important. Lastly, the molecular data suggest that this OPMD cluster is due to a founder effect probably originated from the Canary Islands.

[P05.133] Titin M-Line Mutations: New Phenotypes besides Tibial Muscular Dystrophy and Severe LGMD2J

Bjarne A. Udd, Anna Vihola, Vasa, Finland, Jaakko Sarparanta, Henna Haravuori, Isabelle Richard, Paris, France, Peter Hackman, Helsinki, Finland

OBJECTIVE: Assessment of the expanding TMD phenotype. BACKGROUND: We have previously reported the first mutations in the titin gene to cause human skeletal muscle disease. Three different c-terminal mutations have been identified to be responsible for the autosomal dominant distal myopathy phenotype Tibial muscular dystrophy (TMD). Moreover, one of these mutations FINmaj, occurred in homozygous form in one large complex Finnish kindred and caused a completely different LGMD phenotype, termed LGMD2J, because this phenotype segregated as a recessive trait. DESIGN/METHODS: We have analyzed 386 Finnish patients presenting with distal myopathy or other peculiar undetermined phenotypes for the identification of the FINmaj titin mutation causing TMD/LGMD2J. The molecular genetic methods used were direct sequencing, SSCP, and DHPLC of DNA samples of the patients. RESULTS: We have identified 202 patients heterozygous for the c-terminal titin mutation FINmaj. Among these patients 92% had a mainstream phenotype compatible with the description of TMD in the previous clinical reports. However, 8% had unusual phenotypes including both proximal leg or posterior lower leg muscle weakness at onset. Three patients with LGMD2J were confirmed homozygotes. CONCLUSIONS: The phenotype caused by c-terminal titin mutations is expanding. The large variability of phenotypic expression of just one mutation, the Finnish titin mutation FINmaj, indicates that no phenotype of dominant myopathy/dystrophy can be excluded from being caused by mutated titin. Homozygous or compound heterozygous mutations without phenotype in the heterozygote carriers may also be responsible for undetermined recessive myopathies and LGMD.

[P05.130] Clinical and Genetic Features of a Novel Autosomal Dominant Limb-Girdle Muscular Dystrophy 1F (LGMD 1F)

Lluis Palenzuela, Antoni L. Andreu, Andrea Kattah, Josep Gamez, Israel Fernandez-Cadenas, Barcelona, Barcelona, Spain, Michio Hirano, New York, NY

OBJECTIVE: To characterize a novel autosomal dominant limb-girdle muscular dystrophy (LGMD) in a large Spanish kindred spanning five generations. BACKGROUND: LGMDs comprise a heterogeneous group of hereditary diseases characterized by progressive and predominantly proximal limb weakness with histologic signs of necrosis and regeneration in muscle. To date, 15 genetically distinct types of LGMD have been identified and include only 5 autosomal dominant forms. DESIGN/METHODS: 61 individuals were examined clinically. Twelve underwent nerve conduction and electromyography studies and 5 had muscle biopsy. Electrocardiograms were performed in 12 patients, echocardiography in 6 and brain MRI in 2. For the genetic analyses we studied 47 individuals (27 affected and 20 unaffected) from three generations of the family. After extracting genomic DNA, a genome-wide screen was performed with 400 polymorphic microsatellite markers spaced throughout the 22 autosomal chromosomes. After calculating 2-point LOD scores and fine-mapping the critical region, candidates genes were identified and sequenced. RESULTS: Of 61 examined individuals, 32 showed muscle weakness involving the pelvic and shoulder-girdle muscles predominantly. Patients could be divided into two groups based on the age at onset: juvenile, before age 15 and adult, >20 years old. In later generations, individuals had younger ages-at-onset, suggesting genetic anticipation.
Linkage of the disease to the 5 known AD-LGMD chromosomal loci was excluded.
Within the chromosomal segment between markers D7S487 and D7S640, 16 microsatellite markers revealed an identical haplotype in almost all affected relatives. Two individuals that harbored recombinations allowed us to narrow further the linked region to an interval between D7S182 and D7S2519, spanning 3.68 Mb.
Filamin C, an actin-binding protein highly expressed in muscle was considered the best candidate gene within the disease locus; however, DNA sequencing of the exons and flanking introns showed no mutations. A Northern blot analysis did not show alterations in the gene transcript and a Western blot analysis showed no abnormalities in the protein. CONCLUSIONS: The LGMD1F disease locus maps to 7q32.1-32.2. Among the candidate genes, filamin C was excluded. Within the critical region, there are 23 additional known and 24 predicted genes that are candidates. Supported by: NIH (R01AR47989) and the Spanish Ministry of Science and Technology SAF 2001-1998

[P05.129] Establishing the Prevalence of LGMD Genotypes in North America: An Ongoing Multi-Center Collaborative Study

Christopher J. Shilling, Columbus, OH, Matthew P. Wicklund, Steven A. Moore, Kevin P. Campbell, Wendy King, Cheryl Wall, Katherine D. Mathews, Alan Pestronk, Robert C. Griggs, Gerald M. Fenichel, Catherine A. Stolle, Charlotte A. Brown, Julaine M. Florence, Ami Mankodie, Jenny Robison, Nick Laubenthal, Shree Pandya, Ann Galgon, John T. Kissel, Jerry R. Mendell

OBJECTIVE: To establish the prevalence of limb-girdle muscular dystrophy (LGMD) genotypes in the USA. BACKGROUND: LGMD has been linked to at least 16 chromosomal loci (six autosomal dominant, LGMD1A-F; and ten autosomal recessive, LGMD2A-J). The majority of LGMD subtypes have been described in founder populations, mostly outside the USA. DESIGN/METHODS: Six collaborative American centers studied 310 unrelated patients with a suspected diagnosis of LGMD. Criteria included symmetrical, proximal greater than distal weakness and demonstrated normal dystrophin on muscle biopsy. Patients diagnosed with congenital myopathy, congenital muscular dystrophy, or mitochondrial disorder were excluded. A muscle biopsy was performed unless tissue was available from a previous biopsy and blood was obtained for DNA analysis. A battery immune studies on tissue sections and western blots were performed against dystrophin, α-,β-,γ-,δ-sarcoglycans (SG), α-,β-dystroglycan (DG), caveolin-3, dysferlin, telethonin (antibody contributed by G. Faulkner; Trieste, Italy), emerin, and calpain-3 (Calp3). Mutation screening was performed based on immune staining abnormalities. RESULTS: Out of 310 cases, 22 (7%) had been erroneously diagnosed with LGMD (6 Becker, 11 facioscapulohumeral, 2 X-linked Emery Dreifuss, 2 merosin, and 1 hereditary inclusion body myopathy). 286 were diagnosed with LGMD. 155 (54%) had a protein or molecular abnormality corresponding to an LGMD diagnosis. Of these, 47 (16%) had dysferlin abnormalities; 45 had SG mutations (15%); 33 had α-DG abnormalities (11%) which strongly favor mutations of FKRP; 11 demonstrated mutations in the LMNA gene (4%); 3 showed caveolin-3 deficiency (1%); 2 had myotilin mutations (<1%). Calp3 deficiency was found in 16% of available samples. Calp3 deficiencies were highly unlikely to be secondary to mutations of the titin gene based on family history (none of these subjects had a family history indicative of LGMD2J or tibial muscular dystrophy). No telethonin-deficiencies were encountered in this population. CONCLUSIONS: This is the largest, most comprehensive prospective study of LGMD ever done in an outbred population. Based on the described data we suggest screening for dysferlinopathies, calpainopathies and sarcoglycanopathies (each composing ~16% of the cases) as a priority in diagnosing LGMD followed by α-DG and Lamin A/C. With current technology it is possible to identify a protein abnormality or genetic mutation in half of the patients with LGMD. This is highly relevant to any future planned pathogenic or therapeutic studies to be performed in the USA.

[S62.003] Dysferlinopathy : Study of Differential Muscle Groups Involvement Using CT Scan and MRI

Corrado Angelini, Marina Fanin, Roberta Padoan, Padova, Italy

OBJECTIVE: To determine differential and sequential muscle involvement in upper/ lower limb-girdle and leg muscles in LGMD 2B patients. This is useful for monitoring disease progression and could be used both for natural history and clinical trials. BACKGROUND: Recently LGMD2B and Miyoshi myopathy were found to be allelic disorders arising from defects in a novel gene that encodes dysferlin. Dysferlin is localized to the sarcolemma. This protein has been recently demonstrated to be involved in repair of skeletal muscle plasmalemma. Western Blot analysis for dysferlin has been shown a reliable and rapid method for the diagnosis of this type of muscular dystrophy. The involvement of muscle groups and clinical course is variable; however the clinical rate and progression of the disease has not been so far evaluated by imaging studies in molecularly defined patients. DESIGN/METHODS: We investigated by muscle CT scan or MR imaging a group of 15 primary dysferlinopathy patients. Dysferlin gene mutations were identified by SSCP and sequencing. Western blot analysis revealed absent or markedly reduced dysferlin protein in all patients. Four patients presented at onset proximal limb girdle muscle involvement, ten distal Miyoshi myopathy, and one had myalgia with elevated CK level. All patients were examined using four functional tests and assigned to a clinical severity scale (CSS). Muscle strength was evaluated on nine muscle groups according to MRC scale. Upper limb , thigh and leg muscles were scanned by CT scan in all 15 cases, in two siblings MRI of lower extremities ( T1 weighted sequences ) was performed. Outcome measures included intramuscular fatty infiltration, intermuscle fat deposition, and muscle size.We scored skeletal muscle groups investigated radiologically as: stage 0 : normal; stage1:early moth-eaten appearance; stage 2 : late moth-eaten appearance; stage 3:washed-out appearance; stage 4 : end-stage apparance. A correlation between CSS and radiological features was statistically evaluated. RESULTS: Fatty infiltration occurred in a characteristic pattern with distal muscles more commonly involved and proximal muscle most frequently spared. There was a spread from distal to proximal muscles in posterior compartment of lower limbs: medial head of gastrocnemius and hamstring demonstrated extensive disease,whereas quadriceps femorus and vastii were spared. Patients in advanced stage of the disease had atrophic thigh muscles and prominent involvement of biceps brachii and deltoid on muscle CT scan.On MRI there was evidence of selective involvement of posterior compartment ,myoedema and inflammation in presymptomatic muscle groups. CONCLUSIONS: This study defines differential involvement of muscle groups in molecularly diagnosed dysferlinopathies and suggests widespread inflammation and selective involvement of posterior compartment muscles in a characteristic pattern. These changes may provide the basis for exploring mechanisms of action of medications and monitoring their effects.