Selected Abstracts that will be presented on the Annual Meeting of American Academy of Neurology - march 29 - april 5,2003 Hawaii

1)      Orthopedic Effects of Steroid-Treatment in Duchenne dystrophy
Wendy M. King, Ryan E. Ruttencutter, Chad J. Hoyle, Chris J. Shilling, Jerry R. Mendell, John T. Kissel, Columbus, OH 

OBJECTIVE: To document the effect of long term daily steroid treatment on several orthopedic parameters in boys with Duchenne muscular dystrophy (DMD), based on a large clinic experience.
BACKGROUND: Although numerous clinical trials have documented increased strength in DMD boys treated with steroids, there have been few long term studies looking at side effects. Because The Ohio State University has been involved in steroid trials and also serves a large geographic area as a tertiary neuromuscular clinic, we have had an extensive population of DMD boys.
DESIGN/METHODS: The charts of 100 consecutive boys with genetically-confirmed DMD seen and still followed in the Muscular dystrophy Clinic at OSU over the past 3 years were retrospectively reviewed. The cohort included 31 boys who participated in the early CIDD prednisone trials and in a subsequent deflazacort trial. Charts were reviewed with attention to type and duration of steroid therapy (if any), as well as specific orthopedic complications including: long bone fractures, vertebral compression fractures, and scoliosis.
RESULTS: The mean age of the cohort was 15.2 years with an median follow-up of 8.5 years (1 to 29 years). Of the 100 young men, 49% had been on daily steroids for at least one year, while 51% were never treated with steroids. On average, boys on steroids walked independently 3.31 years longer than those never treated. (p <0.001) Boys never on steroids had an average scoliotic curve of 34.1 degrees (0^o /97^o  SD 20.4), while boys on steroids had an average curve of 14.3 degrees (0^o /45^o SD 14.1). Thirteen boys (23.2%) never treated with steroids reported one or more long-bone fractures; 18 boys (40%) treated with steroids reported fractures (p=0.01). Of the cohort, 24.4% of boys on steroids had one or more compression fractures while no such fractures were reported in the untreated boys.
CONCLUSIONS: Young men with DMD treated with daily steroids walked independently on average 3.3 years longer than untreated boys, and had an average scoliotic curve of 20 less degrees. There were increased long bone and vertebral compression fractures in the treated group. Whether the increased fracture rate in the treated group results from preserved activity, osteoporosis, or both remains to be determined.

 2)      Compositional Analysis of Muscle in Boys with Duchenne Muscular dystrophy Using MR Imaging
Franklin A. Marden, Anne M. Connolly, Marilyn J. Siegel, David A. Rubin, St. Louis, MO 

OBJECTIVE: To evaluate the ability of magnetic resonance imaging (MRI) to delineate the distribution and composition of diseased muscles in boys with Duchenne Muscular dystrophy (DMD).
BACKGROUND: DMD is the most common, inherited, degenerative disease of skeletal muscle, caused by mutations in the X-linked dystrophin gene. It affects boys by the age of 5 years, causing progressive weakness. Left untreated, the disease progresses so that boys are typically unable to walk without assistance by the age of 10 and succumb to cardiopulmonary complications by the second or third decade. The pathogenesis is unclear, although postulated mechanisms have focused on the lack of dystrophin and other structural proteins, leaky membrane channels, or a cascade of inflammation. Few studies have studied MRI as a possible adjunct to physical exam or as a means for further exploring the pathophysiology.
DESIGN/METHODS: Eleven boys between the ages of 5 and 10 years with disabling weakness due to DMD enrolled. Each boy underwent clinical and MRI examinations within the same week. Quantitative strength and timed functional testing were performed. Using the thigh as a surrogate for the disease as a whole, representative proximal, mid, and distal levels were evaluated with T1-weighted Spin Echo and Short-Tau Inversion Recovery (STIR) MRI sequences. Primary outcome measures included intra-muscular fatty infiltration, inter-muscle fat deposition, edema, and muscle size. Clinical correlation was performed.
RESULTS: All but 1 boy was able to tolerate the MRI. Muscles demonstrated selective fatty infiltration which correlated with patient age (p=0.05). There was preferential involvement of the gluteus maximus (10/10) and adductor magnus (8/10), followed by the rectus femoris, biceps femoris, and semimembranosus. The gracilis and sartorius muscles were relatively spared (10/10). Atrophy was more likely to occur in muscles heavily infiltrated with fat, and hypertrophy in those spared. Fat infiltrated between muscles in all patients as well. Hyperintense signal abnormalities on STIR sequences consistent with either edema or inflammation occurred in a geographic distribution, commonly affecting muscles spared of fatty infiltration.
CONCLUSIONS: MR imaging was able to show selective involvement of muscles which cannot be individually tested on physical examination, and thus may be more sensitive to changes over time and provide useful information for treatment of the disease. Furthermore, while fibrofatty changes have historically been described as a pathologic hallmark of the disease, MR imaging demonstrated that edema or inflammation may be an important component. This may help explain the response to immunomodulating agents such as Prednisone or Deflazacort. Improved imaging techniques to characterize the extent and severity of fatty infiltration and edema may provide a basis for exploring mechanisms of action of medications and ultimately assist in the management of the patient.

 3)      Echocardiography Shows Abnormal Ventricular Function Following Exercise in Healthy Carriers of Dystrophin Mutation
Katherine D. Mathews, Christina Trout, Campbell P. Kevin, Richard E. Kerber, Robert M. Weiss, Iowa City, IA 

OBJECTIVE: Exercise stress echocardiography in healthy DMD/BMD carriers tested the hypothesis that cardiac dysfunction would be similar to that seen with ischemia.
BACKGROUND: Cardiac dysfunction occurs in 18- 90% of women with dystrophin mutations. Cardiomyopathy is progressive, and symptomatic in ~10-20%. Mosaic dystrophin staining pattern is seen in cardiac biopsy from DMD/BMD carriers, as predicted by Lyonization. The basis for the cardiomyopathy is unknown. In animal studies, dystrophin deficient cardiomyocytes show increased susceptibility to injury by mechanical stress (Danialou, 2001; Kamogawa, 2001). Dystrophin-deficient skeletal muscle has increased susceptibility to oxidative stress (Rando, 2001). MR spectroscopy in DMD/BMD carriers shows reduced PCr/ATP ratio, also seen in ischemia (Crilley, 2000). We propose that progressive cardiomyopathy in DMD/BMD carriers results fromf relative ischemia; impaired ability to buffer oxidative stress and increased susceptibility to injury.
DESIGN/METHODS: Twelve healthy adult DMD/BMD carriers and 9 controls underwent 2-D echocardiography before and after symptom-limited exercise (Bruce protocol). Subjects had DNA confirmation of dystrophin mutation, or were obligate heterozygotes by family structure. No subject had pre-existing cardiac disease, risk factors for atherosclerosis, or abnormal fasting cholesterol. Echocardiography was reviewed by 2 cardiologists blinded to clinical status.
RESULTS: DMD/BMD carriers were younger than controls (37+/-10 yrs vs. 43 +/- 6.4 yrs, p = NS). Systolic blood pressure at rest were equal. Three subjects had low ejection fractions (EF) at rest, and mean EF was lower in subjects than controls (0.54 vs. 0.64, p = .01). Subjects had lower exercise tolerance than controls (time to target HR or symptoms 417 sec vs. 493 sec). After exercise, only 2 of 12 DMD/BMD carriers had completely normal findings, while all of the controls did. Two subjects developed ST segment changes, compared to 0/9 controls. Six subjects developed regional ventricular akinesis or hypokinesis (0 controls). In 10/12 subjects, EF fell after exercise, while it consistently rose in controls (mean delta EF -0.0555 +/- 0.099 in subjects; +0.078 +/- 0.057 in controls, p = .002).
CONCLUSIONS: 1. Stress echocardiography is a non-invasive way to detect subtle abnormalities in cardiac function in healthy adult DMD/BMD carriers.
2. Of 12 asymptomatic subjects, 83% have abnormal ventricular wall function following exercise, similar to that seen in patients with ischemic cardiomyopathy.
3. Relative ischemia of myocardium sensitive to injury because of decreased dystrophin, may cause the myocardial dysfunction seen in some DMD/BMD carriers. Medical intervention before cardiomyopathy becomes symptomatic may improve longevity and perhaps quality of life for these women. Information in this population will likely transfer to affected males with DMD/BMD.

 4)      Prolonged Dystrophin Expression and Functional Correction of mdx Mouse Muscle Using Helper-Dependent (Gutted) Adenovirus Expressing Isogenic Full-Length Dystrophin
Renald Gilbert, Roy W. R. Dudley, Montreal, QC, Canada, An-Bang Liu, Hualien, Taiwan, Basil J. Petrof, Josephine Nalbantoglu, George Karpati, Montreal, QC, Canada 

OBJECTIVE: To determine the duration and benefits of dystrophin expression following gene transfer into mdx mouse muscle using helper-dependent adenovirus (HDAd) encoding full-length murine dystrophin cDNAs.
BACKGROUND: HDAd holds great promises as a gene transfer vector for the treatment of genetic diseases such as Duchenne muscular dystrophy (DMD). It does no encode any viral genes (hence the name gutted) and is, thus, only weakly immunogenic. We have constructed a HDAd encoding two full-length human dystrophin cDNAs regulated by the powerful hybrid cytomegalovirus enhancer/ -actin (CB) promoter (Mol. Ther. 6: 501-509, 2002). Efficient, but only transient dystrophin expression occurred after gene transfer with this vector in dystrophin-deficient (mdx) mouse muscle, most likely because the dystrophin used was of human origin. We have now tested the same vector but encoding this time an isogenic transgene (murine dystrophin).
DESIGN/METHODS: A HDAd encoding two murine full-length dystrophin cDNAs regulated by the CB promoter was constructed and called HDCBDysM. The tibialis anterior (TA) muscle of neonatal and adult mdx mice was injected with 10 l or 30 l respectively of HDCBDysM at a titer of 2.0 X 10¹² virus particles/ml. At 10, 30, 60, 90 and 180 days post-injection the muscle were analyzed for the following parameters: dystrophin expression by immunohistochemistry and western blots; protection of muscle necrosis as measured by the % of muscle fibers with centrally located nuclei; improvement of the force-generating capacity of the muscle.
RESULTS: At 10 days post-injection, the average number of dystrophin positive (dys+) fibers was 712 in neonatally-injected animals (42% of the TA) and the amount of dystrophin produced corresponded to 230% that of normal muscle. This high level dystrophin expression remained unabated for the duration of this study. In adult-age-injected animals, the maximal transduction level occurred at 30 days post-injection with an average of 418 dys+ fibers (25% of the TA). An average of 204 fibers was still dys+ at 180 days post-injection, but this reduction was statistically significant. The centronucleation index was improved by treatment with HDCBDysM for the duration of the study in both animal groups. In neonatally-injected muscles at 60 days post-injection with HDCBDysM, both the maximal force-generating capacity of the muscle and its resistance to contraction-induced injury following a series of eccentric contractions significantly improved.
CONCLUSIONS: Long-term dystrophin expression as well as a correction of the dystrophic phenotype can be achieved following gene transfer using isogenic HDCBDysM in mdx muscle. HDCBDysM is, thus, a very promising vector for the treatment of DMD by gene therapy.

 5)      Long Term Expression of Transgene Delivered by Plasmid and Electrotransfer into Muscle Fibers of Immune Deficient Mice
George Karpati, Renald Gilbert, Yifan Lu, Montreal, Canada, Maria J. Molnar, Budapest, Hungary, Basil J. Petrof, Josephine Nalbantoglu, Montreal, Canada 

OBJECTIVE: To determine the duration and level of transgene expression in muscle fibers of immune deficient (SCID) mice after plasmid-mediated electrotransfer.
BACKGROUND: Plasmid-mediated gene transfer into muscle fibers shows poor efficiency unless it is accompanied by electrotransfer and hyaluronidase injection into the target muscles (Gene Ther. 8: 1264-1270; 2001). One of the unclear aspects of plasmid-mediated electrotransfer into muscle fibers is the duration of and level of transgene expression. Since the employed transgene protein ( -galactosidase, -gal) is immunogenic, we employed immune deficient mice
DESIGN/METHODS: Thirty micrograms (1 g/ l) of endotoxin-free plasmid containing an expression cassette of a strong promoter (a hybrid of chicken -actin promoter/cytomegalovirus enhancer) and bacterial Lac-Z gene encoding -gal plus hyaluronidase (Hy 0.4 U/ l) was injected into anterior tibialis muscles (ATM) of adult SCID mice. This was followed by electrotransfer to the ATM (175 V/cm, 8 pulses each of 20 msec duration, interval 1 sec). Cryostat cross sections from the entire ATM at its midpoint were stained for -gal at post injection days 7 (group 1), 10 (group 2), 90 (group 3) and 180 (group 4). The total number of -gal positive muscle fibers were counted in each muscle and the arithmetic mean of each group was calculated. Additionally, -gal level was quantitatively determined by luminometry on homogenates of pooled muscles from groups 3 and 4.
RESULTS: The means SE for the fiber counts of Group 1 was 2107 336 (N=6); Group 2: 1905 203 (N=7); Group 3: 1968 196 (N=9); Group 4: 850 155 (N=8). The difference of the means between either Group 1 or 2 or 3 versus Group 4 was highly significant (p=0.003), indicating a significant decline of the transfection level between 90 and 180 days. The pooled luminometric results normalized to protein content were 5495 lu in Group 3 and 1583 lu in Group 4.The normalized values indicated that the total -gal level dropped by approximately 72% between 90 and 180 days.
CONCLUSIONS: Even in the absence of significant immune activity of the host, there is a significant late decline of the transfection level in muscle after plasmid-mediated electotransfer although it still remains relatively high. The decline may be due to degradation of the DNA of the expression cassette or promoter attenuation. Studies are in progress to determine whether this decline continues up to one year post injection and the mechanisms responsible for the decline. The results suggests that if plasmid-mediated electrotransfer is to be used for therapeutic gene transfer in genetic muscle disease, repeat administration may be necessary but only at infrequent intervals

 6)      Platelet-Derived Growth Factor and Its Receptors Are Related to the Progression of Human and Mouse Muscular dystrophy: An Immunohistochemical Study
Kazuhiro Haginoya, Yajuan Zhao, Kazuie Iinuma, Sendai, Miyagi, Japan 

OBJECTIVE: To clarify the role of platelet-derived growth factor (PDGF) and its receptors in the progression of muscular dystrophy.
BACKGROUND: The progression of muscular weakness in patients suffering from Duchenne muscular dystrophy (DMD) and congenital muscular dystrophy (CMD) is directly correlated with the progressive loss of myofibers, which is accompanied by connective tissue and adipose tissue replacement. The mechanism of histological progression remains unclear. Previous studies suggest that there is a progressive loss of regenerating capacity in muscle fibers. Interstitial fibrosis may be attributed to a loss of regenerating capacity in satellite cells, which is caused by reduced blood supply to individual muscle fibers. Alternatively, recent studies also suggest that growth factors such as transforming growth factor beta 1 (TGFbeta1) and basic fibroblast growth factor (bFGF) have important roles in disease progression. However, the role of PDGF has not studied so far.
DESIGN/METHODS: Biopsied frozen muscles from patients with Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), and congenital muscular dystrophy (CMD) were analyzed immunohistochemically using antibodies raised against PDGF-A, PDGF-B, PDGFR alpha and beta. The expression of PDGFR-beta in CMD muscles was evaluated using western blot. Muscles from two dystrophic mouse models (dy and mdx mice) were also immunostained with antibodies raised against PDGFR alpha and beta.
RESULTS: In normal human control muscle, neuromuscular junctions and vessels were positively stained with antibodies against PDGF-A, PDGF-B, PDGFR alpha and PDGFR beta. In human dystrophic muscles, PDGF-A, PDGF-B, PDGFR alpha and PDGFR beta were strongly immunolocalized in regenerating muscle fibers and infiltrating macrophages. PDGFR alpha was also immunolocalized in the muscle fiber sarcolemma and necrotic fibers. The most significant finding in this study was a remarkable over-expression of PDGFR beta and, to a lesser extent, PDGFR alpha in the endomysium of DMD and CMD muscles. PDGFR was also over-expressed in the interstitium of muscles from dystrophic mice, particularly dy mice. Double immunolabeling revealed that activated interstitial fibroblasts were clearly positive for PDGFR alpha and beta. However, CMD muscles with advanced fibrosis showed no reactivity against PDGF and PDGFR. This was confirmed by western blot analysis for PDGFR-beta
CONCLUSIONS: These findings indicate that PDGF and its receptors are deeply involved in the active stage of tissue destruction, and are associated with the initiation or promotion of muscle fibrosis. They also have roles in muscle fiber regeneration and signaling at neuromuscular junctions in both normal and diseased muscle.

 7)      The Relationship of Clinical Severity to Dystrophin Immunohistochemistry and Western Blotting in Duchenne and Becker Muscular Dystrophies
Sheila Kumar, Thomas Crawford, Ahmet Hoke, Baltimore, MD 

OBJECTIVE: To correlate clinical severity in Duchenne muscular dystrophy (DMD) and Becker's muscular dystrophy (BMD), and dystrophin abnormalities on immunohistochemistry and Western blotting.
BACKGROUND: Abnormalities of dystrophin have been documented in both DMD and BMD. However, clinical correlations with the protein abnormalities have not been studied in the past.
DESIGN/METHODS: We stratified 19 patients with an ambiguous dystrophin genotype-phenotype relationship into 5 groups by clinical severity during late childhood to early teen years. Dystrophin Western blotting was classified as either absent or reduced. The quality of dystrophin immunohistochemical staining for rod domain, N-terminus and C-terminus antibodies was sorted into those with normal to moderately reduced dystrophin staining and those with severely reduced or absent dystrophin staining. Clinical and dystrophin classifications were blinded.
RESULTS: In this cohort Western blotting has a positive predictive value (ppv) of 67% for BMD and 62% for DMD phenotype. Dystrophin immunohistochemistry has a ppv of 70% and 78% for BMD and DMD, respectively. The combination of Western and immunohistological data has substantially better ppv, with congruent Western-immunohistological classification having a 100% ppv for both BMD and DMD.
CONCLUSIONS: In patients with an ambiguous genotype-phenotype relationship the combination of Western blotting and immunohistochemical dystrophin testing has higher predictive value for prognosis than does either test in isolation.

 8)      Expression Profiling of 16-wk-Old mdx Mouse Skeletal Muscle Shows an Upregulated mRNA Creatine Biosynthetic Pathway - A Possible Salvage Pathway?
Brian S. Tseng, San Francisco, CA, Po Zhao, Washington, DC, Scott J. Pattison, Columbia, MO, Joseph A. Granchelli, Buffalo, NY, Richard W. Madsen, Frank W. Booth, Columbia, MO, Eric P. Hoffman, Washington, DC 

OBJECTIVE: The absence of a functional dystrophin protein leads to a severe phenotype in boys with Duchenne Muscular dystrophy (DMD), unlike mdx mice. Our objective is to better understand the underpinnings that enable this ameliorated phenotype in mdx mice in spite of the shared biochemical defect with DMD.
BACKGROUND: These mRNA expression data may provide insights of the pathogenesis of DMD, a human disease where the absence of dystrophin is associated with a progressive and severe skeletal muscle degeneration process that leads to death before 20 years of age. The mdx mouse model is one to analyze since it is relatively unimpaired yet lacks a dystrophin protein and does have elevated serum CKs.
DESIGN/METHODS: The current experiment employed a microarray scan of 12,488 mRNAs in 16-wk-old mouse mdx muscle at a time when the skeletal muscle is avoiding severe dystrophic pathophysiology in spite of the absence of a functional dystrophin protein (J Appl Physiol 93:537-545, 2002). These findings are compared with those of wild littermates with intact dystrophin protein. Preliminary cross-comparisons with a human DMD expression profile database will also be made.
RESULTS: Among this expression profiling comparison, one intriguing mRNA species found to be differentially expressed is the guanidinoacetate methyltransferase mRNA, whose protein catalyzes the last step in creatine biosynthesis, increased 3.1-fold in mdx muscle from wild, whereas a previous report by Chen et al. (J. Cell Biol. 151:1321-36, 2000) showed a 4-fold decrease in DMD muscle. From a metabolic standpoint, nearly all other clustered groups of mRNAs for oxidative and glycolytic enzymes in mdx are not downregulated to the extent as that seen in DMD.
CONCLUSIONS: These observations suggest a differential or compensatory mRNA expression pattern of creatine biosynthesis in whole skeletal muscle of the mdx mouse compared to wild littermates and may be essential for salvage or adaptation pathways that maintain skeletal muscle integrity in spite of an absent dystrophin protein.

 9)      Analysis of Calcium-Dependent Proteinase and Cytoskeletal Protein in Muscular dystrophy Muscles
Ken-ya Murata, Miwa Takamure, Satoshi Ueno, Kashihara, Nara, Japan 

OBJECTIVE: To examine whether the calpains and their substrate alpha-fodrin are expressed in the muscles of patients with Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) and to clarify their involvement in muscle fiber degeneration.
BACKGROUND: Dystrophin is recognized as the protein responsible for the pathogenesis of DMD and BMD. However, the mechanism underlying degeneration and necrosis of the muscle fibers is still unclear. Altered muscle membrane permeability caused by dystrophin abnormalities may induce calcium influx into the muscle fibers, resulting in calcium accumulation. Calpain is a calcium-dependent proteinase that is activated by the elevation of intracellular calcium. Alpha-fodrin (280 kDa) is a substrate of the calpains that digests them into 150-kDa fragments. The calpains are important candidate enzymes for the proteolysis that occurs in DMD and BMD; thus, their localization and relation to the substrate needs to be clarified.
DESIGN/METHODS: Muscle biopsy specimens were obtained from 3 patients with DMD, 3 patients with BMD, 5 patients with untreated polymyositis (PM), and 5 normal controls. Serial frozen sections were examined by single and double immunocytochemistry, with monoclonal antibodies against human alpha-fodrin, beta-spectrin, calpain I, calpain II, and NCAM, and observation under fluorescence and confocal laser scan microscopy. Muscle specimens were also examined by western blotting to identify the alpha-fodrin fragments.
RESULTS: Immunostaining for alpha-fodrin, calpain I, and calpain II was very weak in normal control muscles. Muscles of PM patients showed a strong reaction for fodrin at the surface and for calpain in the cytoplasm of some atrophic fibers, though other fibers showed very weak reactions. On the contrary, expression of both alpha-fodrin and calpain was highly upregulated in DMD and BMD muscles. Almost all DMD muscle fibers showed positive reactions despite the size of the fiber. The fodrin-expressing muscle fibers were also calpain-positive in all except the normal control specimens. Western blot analysis revealed normal alpha-fodrin fragments in all specimens and 150-kDa fragments in all but normal control muscles. These 150-kDa fragments were not detected by polyclonal antibody, which reacts only with fragments cleaved by caspase 3.
CONCLUSIONS: According to our observations, calpain cleaved alpha-fodrin into 150-kDa fragments in DMD and BMD muscles, and these fragments are expressed at the surface of the muscle fibers. Although these changes are not specific to DMD and BMD, they are highly increased in comparison to those characterizing inflammatory myopathies, indicating that calcium-dependent proteinase plays an important role in the muscle fiber degradation associated with DMD and BMD.

 10)  The Role of Inflammatory Cytokines and Calpain in the Upregulation of Extrasynaptic Utrophin in Inflammatory mdx Muscles
Ishrat Waheed, Renald Gilbert, Basil J. Petrof, Josephine Nalbantoglu, Karpati George, Montreal, QC, Canada 

OBJECTIVE: To dissect the pathogenic mechanism(s) in the extrasynaptic utrophin upregulation in inflammatory mdx muscles.
BACKGROUND: It has been shown that chronic low grade inflammation induced by transduction of mdx muscle with first generation adenovirus vector (FGAV) encoding eta galactosidase ( -gal) causes significant extrasynaptic utrophin upregulation (Neurology, Suppl 3, A458, 2000). This was found to be sufficient to mitigate the dystrophic manifestations of the mdx muscle fibers (centronucleation index, restoration of dystrophin or utrophin-associated proteins and force generation properties). The experiments reported here addressed some aspects of the pathogenic mechanism(s) operating in this process.
DESIGN/METHODS: Anterior tibialis muscles (AT) of adult mdx and appropriate control (C57/BL6) mice were injected with 30 micro liters of high titer (1X10/12 virus particles/ml) FGAV encoding -gal or with a saline solution (a total of four groups). Thirty days later the AT muscles were tested for the prevalence of various classes of inflammatory cells, extrasynaptic utrophin upregulation by immunocytochemistry and immunoblots, and the activity of Ca⁺⁺-sensitive calpain level and activity. Similar experiments were performed in mice in which the genes encoding either interleukin-6 (IL-6) or the tumor necrosis factor- (TNF- ) were ablated.
RESULTS: In the AT muscles of IL-6 knockout animals, the degree of inflammation and extrasynaptic utrophin upregulation was similar to saline controls suggesting a redundancy of IL-6 activity. By contrast, in the TNF- knockout mice, the endomysial inflammatory response and extrasynaptic utrophin upregulation was absent. The calpain activity was significantly reduced in AT muscle homogenates of inflammatory muscles of mdx and control animals although the immunoreactive protein was increased.
CONCLUSIONS: The results suggest that TNF- is required for extrasynaptic utrophin upregulation in mouse muscle while 1L-6 is not. Furthermore, the inhibition of Ca⁺⁺-dependent calpain in the inflammatory muscles seems to have a role in extrasynaptic utrophin upregulation. This has been confirmed by in vitro studies with mdx myotubes showing that utrophin is indeed a target of calpain and calpain inhibitors augment extrasynaptic utrophin upregulation. Thus, non-toxic inhibitor molecules of calpain are likely to be useful for extrasynaptic utrophin upregulation and for the treatment of dystrophin deficiency diseases.

 11)  Rapid Direct Sequence Analysis of the Dystrophin Gene: Genotype/Phenotype Correlations
Kevin M. Flanigan, Andrew von Neiderhausern, Diane M. Dunn, Alex Aoyagi, Robert Weiss, Salt Lake City, UT 

OBJECTIVE: To identify novel subexonic mutations in the dystrophin gene and determine cases with unusual phenotype/genotype significance.
BACKGROUND: Duchenne (DMD) and Becker (BMD) Muscular dystrophy are caused by mutations in the dystrophin gene, which is spread over approximately 2.2 million bases on the X chromosome. Commonly-available testing detects deletions of one or more exons, accounting for approximately 60% of patients. Subexonic deletions or insertions and premature stop codon mutations have only been readily detectable by a first using one of a variety of polymorphism screening techniques, followed by sequencing of variant regions. We have developed a novel method to directly, rapidly, and economically screen the entire dystrophin gene, which allows the ready identification of missense, nonsense, and frameshifting mutations.
DESIGN/METHODS: We used a novel method of direct sequence analysis of the dystrophin gene to identify mutations in 20 probands without deletions or duplications one or more exons in size. For each patient, high-quality double-stranded coverage of approximately 110 kilobases was obtained. Mutations were detected in 16/20 probands.
RESULTS: Of 16 mutations detected, 10 were premature stop codon mutations; two frameshifts resulting from a single base pair (bp) deletion (1 proband) and single bp insertion (1 proband); and two splice-site mutations. Two probands had novel missense mutations. One patient with DMD (diagnosed at age 5 years with loss of ambulation at age 11) had a Cys3313Phe mutation in the dystroglycan binding domain encoded in exon 68. One patient with BMD (diagnosed at age 6 years and still ambulant at age 16) had a Asp165Val mutation in an actin binding domain encoded in exon 6. Both of these amino acid residues are highly conserved throughout evolution (including in C. elegans). An additional mutation of significant interest found in a man with BMD who is still ambulant at age 58 years, and who had a stop codon mutation at the third amino acid residue (Trp3X). The nearest in-frame translation initiation signal is >120 codons away.
CONCLUSIONS: Missense mutations have been reported in BMD but are somewhat controversial in DMD. In methods that rely on a mutation screen rather than direct sequence analysis of the entire gene, it is possible that putative missense mutations are false and the disease can be explained by another coding region change not found by a screening method. Our method of direct sequence analysis allows us to unequivocally describe the entire coding sequence of the gene, and attribute disease in these cases to missense mutations in highly functionally conservered regions. Identification of individuals with unusual mutations, such as Trp3X, may shed light on alternate methods of translation intitiation, which might elucidate potential therapeutic pathways.

 12)  Historical Origins of Duchenne Muscular dystrophy
Kenneth L. Tyler 

OBJECTIVE: To review the historical origins of the disease today known as Duchenne muscular dystrophy (DMD) as revealed in the medical literature of the 19th Century.
BACKGROUND: In 1868 GBA Duchenne provided a definitive depiction of "pseudohypertrophic muscular paralysis, " a disease he had initially described in 1861. His contemporaries were aware of several earlier descriptions of this disease, now mostly forgotten. We trace the historical origins of "Duchenne muscular dystrophy " as revealed in the 19th Century medical literature.
RESULTS: In 1830, Charles Bell briefly described a boy with DMD. At age 8 the child developed the onset of progressive leg weakness associated with wasting of some muscles and remarkable prominence of others. In 1847, Partridge described two brothers with progressive proximal leg weakness. At autopsy he found fatty degeneration in involved muscles (the first autopsy in DMD). The first definitive depiction of the disease was by Meryon in 1852 (9 years before Duchenne 's initial report). Meryon described a family of ten children in which all 4 boys and none of six girls were afflicted. The boys developed progressive weakness starting in infancy in the absence of sensory symptoms or signs of brain or spinal cord disease. Meryon described calf enlargement and the tendency to contractures in late disease. He provided the first microscopic analysis of the muscles, noting that the "striped fibres were completely destroyed and in many areas replaced "oil globules". Initially suspecting spinal cord disease, his pathological studies convinced him the process was due to some intrinsic "deficiency of the elements " in the muscle itself. Duchenne first briefly described the disease in 1861, delaying a more detailed report until 1868, at which time he had pathological data from both autopsy and his 'histological harpoon'  to support his contention this was a new disease. He identified the cardinal clinical features of the disease including: (1) progressive decrease in strength beginning in the legs, (2) excessive development of volume in weakened muscles, (3) absence of fever, sensory, or sphincter disturbance. He did not initially emphasize the pattern of inheritance. Gowers saw his first cases of DMD as a medical apprentice in the early 1860's and later published both definitive clinical lectures (1879) and the outstanding early monograph on this disease. He recognized the importance of inheritance and the male predilection, and provided complete clinical details (including his description of the mode afflicted boys used to rise from the ground-Gowers' sign), and provided the best microscopic analysis of the associated muscle pathology. He reluctantly acquiesced to the association of Duchenne's name with the disease, but emphasized the importance of earlier contributions (not initially acknowledged by Duchenne).
CONCLUSIONS: Many investigators provided recognizable descriptions of DMD before Duchenne. Meryon's  contribution is particularly notable, and deserves to be rescued from the dustbin of history . Gowers depictions are at least equivalent to Duchenne s as masterpieces of clinical description.

3)   Acute Respiratory Failure as an Unusual Presentation of Myotonic dystrophy
Nizar Souayah, Peter Siao Tick Chong, Jeremy Schmahmann, Didier Cros, Boston, MA 

OBJECTIVE: To report a previously undiagnosed case of adult myotonic dystrophy whose presenting symptom was acute respiratory failure, not triggered by anesthesia or sedation. To our knowledge, this has not been previously reported in English literature.
BACKGROUND: Myotonic dystrophy is an inherited multisystem disease with variable severity and age of presentation. Although respiratory failure is a common cause of death, it is an unusual presenting symptom. We report an adult patient with acute respiratory failure who was later found to have myotonic dystrophy.
DESIGN/METHODS: Case report
RESULTS: A previously "healthy " 32-year-old woman developed progressive shortness of breath and generalized weakness. Over the next 48 hours, she was noted to be cyanotic and confused. She was brought to the emergency room. Examination revealed focal weakness and preserved deep tendon reflexes. She was intubated for acute respiratory failure and propofol was started for agitation. Initial work ups for cardiovascular, pulmonary, infectious, metabolic and central nervous system disorders were negative. Electromyography was requested to rule out Guillain-Barre syndrome. Nerve conduction studies was normal. Needle EMG showed widespread fibrillation potentials, positive sharp waves and brief runs of myotonic discharges. The patient 's mother, who was not known to have myotonic dystrophy, was at the bedside and she was noted to have facial features of myotonic dystrophy and percussion myotonia. The diagnosis of myotonic dystrophy was later confirmed by genetic test which showed abnormal expansion of CTG repeats on chromosome 19. A follow up needle EMG when the patient was off propofol showed long runs of myotonic discharges.
CONCLUSIONS: Myotonic dystrophy should be considered in the differential diagnosis of acute respiratory failure in young adults. The absence of any prior history of myotonic dystrophy in the patient or in family members does not exclude this possibility. Furthermore, our observation of an attenuation of myotonic discharges with propofol suggests that it may be the anesthetic of choice for patients with myotonic dystrophy.

 14)   Characterization of Pattern of Cognitive Impairment in DM1 Patients
Anna Modoni, Gabriella Silvestri, Fortunato Mangiola, Pietro Attilio Tonali, Camillo Marra, Rome, Italy 

OBJECTIVE: To analyze the neuropsychological performances in congenital and adult DM1 patients and correlate the results with the genotype (nCTG in blood) and the phenotype(degree of muscle impairment).
BACKGROUND: DM1 is characterized by a variable degree of Central Nervous System involvement, widening from mental retardation affecting congenital cases to mood and behavioural changes typical of adult forms. Brain CT and MRI can show frontal and temporal white matter lesions and/or mild atrophy. Neuropathology of DM1 brains document neurofibrillary tangles and an abnormal pattern of tau expression in various brain areas,especially hyppocampus. Neuropsychological studies documented an impairment of memory, general intelligence and executive functions. However, these studies show divergent results regarding correlations between degree of cognitive impairment, muscle phenotype and genotype (nCTG). Moreover only few studies analyse differences in the pattern of cognitive impairment between congenital and adult forms of DM1.
DESIGN/METHODS: We studied 30 adult and 8 congenital DM1 patients. The pathological CTG expansion was detected in all on blood DNA by long-PCR and subsequent Southern Blot. The severity of muscle involvement was established by MDRS scale. Neuropsychological examination included MMSE, memory, linguistics, praxis, attention, level and executive tasks. Statistical analysis was carried out by ANOVA for multiple test.
RESULTS: Patients did not differ in educational level; congenital patients were significant younger than other DM1 patients.All patients had performances below the normal scores in executive function and frontal tasks. Congenital patients performed worse than adults on most of cognitive domains with the exception of the memory functions that result similarly impaired in both groups.Interestingly, comparison among classical DM1 harboring different range of CTG expansion, matched for age and educational level, showed that patients with smaller CTG expansion were more impaired in memory tasks (p <.001) and measures of 'semantic abilities'  (i.e. naming nouns, semantic verbal fluency)(p <.01), while no differences were found in the other domains. There was no correlation between degree of muscular involvement and severity of cognitive impairment.
CONCLUSIONS: Congenital forms showed a global cognitive impairment indicative of mental retardation related to brain developmental delay. Adult DM1 patients showed an impairment of frontal and temporal cognitive functions. According to these data we speculate that the presence of RNAs containing very large CUG expansions in brain embryonic tissues would exert a toxic effect, probably sequestering RNA-binding proteins involved in the regulation of brain development, while the finding of a fixed pattern of intellectual impairment in adult forms, not correlated to nCTG expansion, suggest that in adult life only specifical subsets of neurons may be sensitive to the toxic effect of expanded CUG-RNAs.

 15)   Global and Regional Brain Atrophy in Myotonic dystrophy (DM1)
Giovanni Antonini, Andrea Romano, Vanessa Ceschin, Francesca Gragnani, Caterina Mainero, Francesca Caramia, Stefania Morino, Elisabetta Bucci, Marco Fiorelli, Luigi Bozzao, Rome, Italy, Italy 

OBJECTIVE: To quantify brain volumes and regional atrophy in DM1 patients using voxel based morphometry (VBM).
BACKGROUND: Neuroimaging studies have shown brain abnormalities (ventricular dilatation, brain atrophy and white matter lesions) in DM1 patients. VBM is a technique designed to evaluate both global and regional brain atrophy.
DESIGN/METHODS: Twenty-two consecutive DM1 patients (13 males and 9 females; median age 33, range 20-55 years) and 22 age- and sex-matched healthy controls were submitted to brain MRI (spin-echo axial T1-weighted, proton density- and T2-weighted images). Brain volumetric analysis was performed from the axial T1-weighted images using SPM99. Fractional brain volumes (grey matter fraction [GMF], white matter fraction [WMF] and brain parenchymal fraction [BPF]) were calculated from the segmented images after the elimination of non-brain voxels. Regional atrophy was calculated according to the optimized method for VBM, which consists in segmentation, normalization to a grey matter template, smoothing and modulation of T1-weighted images. Lesion load (LL) for white matter lesions was calculated on T2-weighted images using a semi-automated local thresholding contouring program (Dispunc). Two-sample t-test was used to compare global and regional atrophy between patients and controls; multivariate analysis was used to evaluate the independent effect of demographic, clinical, genetic data and LL on brain atrophy.
RESULTS: Fractional brain volumes were lower in DM1 (BPF: 0.788 0.0349; GMF: 0.504 0.0314; WMF: 0.283 0.0137) than in controls (BPF: 0.804 0.0171; GMF:0.515 0.0173, WMF: 0.288 0.0151), though this difference did not reach statistical significance. BPF and GMF inversely correlated with age both in patients (p <0.000) and in controls (p <0.003). Regional analysis revealed local areas of grey matter atrophy in the frontal lobes (right superior frontal gyrus and precentral gyrus, left middle frontal gyrus), in the parietal lobes (left postcentral gyrus, left inferior parietal gyrus and bilateral superior parietal lobules), in the temporal lobes (left superior temporal gyrus and bilateral middle temporal gyri), in the left superior occipital gyrus and in the left caudate (p <0.05).
CONCLUSIONS: This study shows that DM1 patients exhibit cortical atrophy that involves specific brain areas and progresses with aging more rapidly then in healthy subjects. The brain areas most affected are those involved in cognitive processes, which are known to be frequently impaired in DM1. Further studies will evaluate the correlations between regional brain atrophy and neuropsychological functions in DM1.

 16)   Type 2 Nuclear Clump Fibers: A Distinctive Early Histopathological Feature in Proximal Myotonic Myopathy (PROMM) - Myotonic dystrophy Type 2 (DM2)
Giovanni Meola, Valeria Sansone, San Donato Milanese, Milano, Italy, Anna Vihola, Vaasa, Finland, Enzo Mancinelli, Milano, Italy, Giuseppe Rotondo, San Donato Milanese, Milano, Italy, Ralf Krahe, Houston, TX, Bjarne Udd, Vaasa, Finland 

OBJECTIVE: To demonstrate that type 2 nuclear clump muscle fiber atrophy is a distinctive histopathological diagnostic feature in PROMM/DM2 and that this muscle biopsy finding may even identify asymptomatic patients with this disorder.
BACKGROUND: For many centers the diagnosis of PROMM/DM2 is still one of exclusion, based on the finding of autosomal dominant inherited muscle weakness, EMG myotonia, cataracts and the mandatory exclusion of (CTG)n expansion on chromosome 19q13.3 associated with the myotonic dystrophy type 1 locus (DM1). Although PROMM/DM2 has been shown to be caused by a (CCTG)n expansion mutation in the 3q21 locus, leucocyte DNA testing for PROMM/DM2 is too much of a research procedure. Muscle biopsy findings have been reported to be similar to those in DM1 and have been considered, so far, a non-specific supportive feature of PROMM/DM2.
DESIGN/METHODS: Besides routine histochemistry, myosin heavy chain (MCH) isoform immunohistochemistry for fiber type differentiation and subsequent morphometric analysis were performed on muscle biopsies from the biceps brachii of 15 Italian 3q21-PROMM/DM2 confirmed mutation patients and from the vastus lateralis of 15 Finnish 3q21-PROMM/DM2 confirmed mutation patients. Atrophy factor using MCH immunohistochemistry was correlated with biceps and vastus lateralis muscle strength MRC score for each patient.
RESULTS: Routine histochemistry did not reveal preferential type 2 atrophy in PROMM/DM2 whereas MCH immunohistochemistry identified a distinctive subpopulation of type 2 nuclear clumps of very small size ( 20 m), which expressed almost exclusively fast MCH (type 2) isoforms. Type 2 nuclear clump muscle fiber atrophy was prominent in both biceps brachii and in vastus lateralis. Atrophy factor detected by MCH immunohistochemistry was significantly elevated even in mildly weak (grade 4.5 MRC) or in normal strength biceps brachii. Muscles from patients with longer disease durations or with more severly affected biceps brachii strength (MRC 4) showed higher atrophy factors (800-1000).
CONCLUSIONS: Type 2 nuclear clump fiber atrophy detected by MCH immunohistochemistry is a distinctive histopathological feature in PROMM/DM2. The finding of a significantly high atrophy factor detected by MCH even in normal/mildly weak muscles suggests that MCH may be considered as a method for preliminary diagnosis in sporadic patients and as a possible preclinical test to detect at-risk asymptomatic individuals in families with PROMM/DM2 until DNA testing for PROMM/DM2 will be available for the biomolecular diagnosis on a routine basis.
Supported By: This study was supported by MIUR ex 40% (Cofin) to G.M.

 17)  Expression Profiling in Limb-Girdle Muscular dystrophy 2B
Corrado Angelini, Marina Fanin, Padova, Italy, Yukiko Hayashi, Tokyo, Japan, Stefano Campanaro, Chiara Romualdi, Gerolamo Lanfranchi, Padova, Italy 

OBJECTIVE: To delineate gene modification in dysferlin deficient patients using a novel dedicated microarray platform with 3'-end skeletal muscle cDNAs.
BACKGROUND: Limb -girdle muscular dystrophy type 2B (LGMD2B), and the distal muscular dystrophy of Miyoshi (MM), are caused by mutations in dysferlin gene, mapped to chromosome region 2p13. Dysferlin immunolocalizes to the sarcolemma similarly to dystrophin, but it is not associated with the dystrophin-glycoprotein complex. Its function and the mechanism by which its defect causes muscle fibres necrosis is still unknown. Molecular diagnosis of dysferlinopathy is now available, and a number of patients have been diagnosed and analysed. The disease onset occurs in the second decade, despite the fact that dystrophic muscle pathological changes and increase CK level are evident earlier. Modifier genes play a role in modulating clinical phenotype and expression profiling by microarray might be useful in order to advance our understanding of muscle pathogenesis in this disorder.
DESIGN/METHODS: In this study we compared 8 biopsies (7 vastus lateralis, 1 deltoid muscle) of dysferlinopathy patients defined by Western Blot and molecular analysis to control quadriceps or deltoid muscle. Altered expression pattern was analized both in pooled patient RNAs and by hierarchical clustering tree formed by individual patients. We analyzed in each case the disease clinical severity and pathological features comparing it with concurrent gene upregulation or under regulation observed by microarray technology.
RESULTS: MHC class I gene and genes involved in protein biosynthesis were upregulated. The expression of genes codifying the sarcomeric proteins titin, nebulin and telethonin were down regulated. There was a major upregulation of protein interacting with calcium, namely S100 calcium binding protein and sarcolipin, a sarcoplasmin calcium regulator.
CONCLUSIONS: The biological processes that are most affected in this type of muscular dystrophy appear to be an increased expression of inflammatory response and genes involved in protein biosynthesis, there is also a major upregulation of protein interacting with calcium, while there is under expression of genes that code for the giant structural proteins of the sarcomere (titin and nebulin) and the small Z line protein telethonin. We hypothize in LGMD2B Ca²⁺ alteration due to membrane damage leads to an altered regeneration pathway, this altered regeneration determines reduction of sarcomeric and Z line proteins resulting in a loss of muscle fiber functionality.

 18)  Limb-Girdle Type Myopathy Associated with Circulating Autoantibody Against Giantin in Golgi Apparatus
Ko Sahashi, Tohru Ibi, Naoki Nakao, Aichi, Japan, Kentaro Sahashi, Nagoya, Japan, Kinji Ohno, Rochester, MN, Hisao Kondo, Cambridge, United Kingdom 

OBJECTIVE: To characterize autoimmune origin of a limb-girdle type myopathy (LGMy).
BACKGROUND: (1) LGMy is a heterogeneous disorder. Much progress has been made in our understanding of genetic bases of limb-girdle muscular dystrophies (LGMD) in the last decade. Autoimmune origin of LG-My, however, has been rarely reported. (2) Giantin is a 376 kDa Golgi complex membrane protein that contributes to formation of a tethering bridge between a transport vesicle and the Golgi membrane before docking of a vesicle. (3) Anti-giantin autoantibody is reported in four patients with rheumatoid arthritis, scleroderma, and Sj gren syndrome, but none has overt myopathy (J Autoimmun 7: 67, 1994; Biochem Biophys Res Commun 205: 1399, 1994; Cell Struct Funct 22: 565, 1997). Anti-giantin autoantibody is also reported in 18 out of 164 HIV patients, but at low titer levels (J Autoimmun 7: 67, 1994).
DESIGN/METHODS: Muscle biopsy, immunostaining of cultured cells, immunoblot, and immunoprecipitation.
RESULTS: Patient: A 59-year-old non-consanguineous female homemaker developed difficulty in squatting and arising at age 44. Muscle biopsy at age 47 revealed chronic inflammatory myopathy. Muscle weakness slowly progressed even on oral corticosteroid therapy. The patient currently shows severe waddling gait and a positive Gowers sign. Facial, bulbar, and distal limb muscles are spared or minimally affected. CK was repeatedly normal. Anti-nuclear, anti-ENA, and anti-SS-A antibodies were positive. Genetic analysis of recessive types of LGMD revealed no mutation. Muscle pathology: Repeated muscle biopsy revealed degenerating small fibers with cytoplasmic body, rod formation, and vacuolar degeneration, as well as sparse inflammatory cells. Immunostaining demonstrated normal dystrophins, utrophin, sarcoglycan complexes, calpain, dysferlin, telethonin, emerin, and laminin subunits. Immunological studies: The patient s serum stained perinuclear membrane structures in Hep-2 human laryngeal carcinoma cells. Immunoblot of Hela cell extracts probed with the patient s serum showed a 300 kDa band. The mobility of this band on SDS-PAGE was similar to that of giantin. Denative immunoprecipitation revealed that anti-giantin antibody recognizes a protein immunoprecipitated by the patient s serum, providing direct evidence that the patient carries an autoantibody against giantin.
CONCLUSIONS: (1) We first report serum anti-giantin autoantibody in LGMy. (2) Fukuyama-type congenital muscular dystrophy, muscle-eye-brain disease, and LGMD2I are hereditary Golginopathies that impair glycosylation of -dystroglycan and cause deficiency of laminin- 2, neurexin, and agrin (Nature 418: 417, 2002). Positive staining for laminin- 2 in our patient indicates different pathomechanisms, which likely include impaired protein trafficking in muscle Golgi apparatus.

19)  Oculopharyngeal Muscular dystrophy: An Uruguayan Population with 11 (GCG) Repeats
Mario Medici, Mercedes Rodriguez, Claudia Camejo, Juan Jos Castagneto, Leda Roche, Montevideo, Uruguay, M. M.Rodríguez, J.P. BouchardQuebec, Canada, B. Brais>Montreal, 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 an lisis, 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 more than 90 individuals of 22 unrelated families, with ages ranging from 22 to 80 years ( mean age 52 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, 55% were symtomatic and 45% were asymtomatic. Eyelid ptosis was the most commom sign, 100%, while dysphagia was found in 87% 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 57% 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 10 repeats. In the asymtomatic patients 21% showed 11 (GCG) repeats (at risk group), the other 79% 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.

20)  Mutations in M-Line Titin Cause Tibial Muscular dystrophy and LGMD2J
Bjarne Udd, Anna Vihola, Vasa, Finland, Henna Haravuori, Helsinki, Finland, Isabelle Richard, Paris, France, Peter Hackman, Helsinki, Finland 

OBJECTIVE: To identify the gene for tibial muscular dystrophy (TMD), a dominant late-onset distal myopathy with slowly progressive foot drop and severe LGMD in rare homozygotes.
BACKGROUND: Previous studies have shown linkage on chromosome 2q31 with titin as the major candidate.Titin is the biggest known protein in the human body with an in vivo length of 2 microns spanning over one half of the sarcomere from the Z-disc to the M-line. Titin appears very early in myofibrillogenesis and is thought to rule the assembly of the contractile elements. Later titin serves a number of mechanical and regulatory functions. Some of the known interacting ligands are calpain3, a-actinin, myosin, myomesin, MYBP-C and telethonin. Recently mutations in I-band and A-band titin were shown to cause cardiomyopathy both in humans and in the zebrafish, and a severe muscular dystrophy (MDM) in a natural mouse mutant.
DESIGN/METHODS: Sequencing the giant titin gene and immunohistochemical titin assessment in muscle biopsies of patients.
RESULTS: A very unique 11 bp mutation changing four amino acid residues in the last Mex6 exon was discovered in the Finnish TMD patients. In a French TMD family, a Leu Pro mutation in Mex6 position 293357 and in a Belgian family an Ile Asn mutation in the same exon at position 293329 were found. Mex6 is adjacent to the known calpain3 binding site Mex5 of M-line titin. Immunohistochemical analysis using two doifferent M-line titin specific antibodies showed loss of C-terminal epitopes, further emphasizing the functional defect caused by the mutation in the Finnish patients and in agreement with the previous results of secondary calpain3 defect in TMD/LGMD2J.
CONCLUSIONS: These are the first mutations in titin shown to cause human skeletal myopathy.

 21)  Distal Muscular dystrophy Affecting the Calves with Onset after Age 30: Not Due to Dysferlinopathy
Jonathan Katz, Palo Alto, CA, Richard Barohn, Kansas City, KS, Thomas Rando, Palo Alto, CA, Carlayne Jackson, Matthew Wicklund, San Antonio, TX, David Saperstein, Kansas City, KS, Anthony Amato, Boston, MA 

OBJECTIVE: To identify patients presenting with calf myopathy with and without dysferlinopathy.
BACKGROUND: Miyoshi myopathy is an autosomal recessive disorder caused by mutations in the membrane protein dysferlin. The condition is defined clinically by its presentation, with weakness predominantly isolated to the calf muscles. This myopathy characteristically begins between the ages of 15 and 30 years and is associated with marked elevations of serum creatine kinase (CK). In contrast, little is known about the same clinical phenotype (calf myopathies) beginning after the age of 30. It is not known whether such patients commonly have dysferlinopathy or a distinct type of myopathy.
DESIGN/METHODS: Muscle was analyzed by immunoblot for both dysferlin and dystrophin in patients who met the following criteria: (1) weakness that began either unilaterally or bilaterally in the gastrocnemius muscles; (2) elevated CK levels; (3) myopathy suggested by electrodiagnostic testing and muscle biopsy. These included 5 patients presenting after the age of 30 and 13 patients presenting before age 30. We contrasted creatine kinase levels and clinical course between these two populations.
RESULTS: Four of the five patients presenting after age 30 had both normal dysferlin and dystrophin immunoblots. These patients all had elevated CK levels, but never exceeding 10 times the upper limit of normal. They showed little progression of weakness during follow up periods ranging from five to fourteen years. None of these patients had a family history of myopathy. Muscle histology showed myonecrosis without vacuoles. Magnetic resonance imaging of the lower limbs in two patients showed fatty change limited to the posterior compartment of the calves, sparing the thighs. The fifth patient had a truncated dystrophin by Western blot. Mutational analysis using polymerase chain reaction and Southern blot, however, did not detect a deletion in the dystrophin gene. In contrast, all 13 patients presenting before age 30 had dysferlinopathy by immunoblot. These patients had CK levels of at least 13 times the upper limit of normal often reaching much higher levels and showed progressive clinical courses typical of Miyoshi myopathy.
CONCLUSIONS: Our findings suggest that the calf myopathy phenotype presenting after the age of 30 is rarely due to dysferlinopathy. These patients usually have less elevated CK levels and may have a slower progression compared with classic Miyoshi myopathy presenting under age 30. Dystrophin abnormalities may also rarely lead to this late-onset phenotype.

 22)   Mutational and Clinical Features of Japanese Patients with Dysferlinopathy
Toshiaki Takahashi, Masashi Aoki, Maki Tateyama, Yoshiaki Onodera, Emi Kondo, Hitomi Sato, Mariko Ito, Masaru Yoshioka, Hidehiko Konno, Sendai, Japan, Robert H. Brown, Jr., Charlestown, MA, Hiroshi Saito, Yasuto Itoyama, Sendai, Japan 

OBJECTIVE: To analyze clinical features of Japanese patients with defined mutations in the dysferlin gene (dysferlinopathy).
BACKGROUND: Mutations in the dysferlin gene cause both Miyoshi myopathy (MM) and limb girdle muscular dystrophy 2B (LGMD2B).
DESIGN/METHODS: We examined 41 patients with dysferlinopathy in Japan, including 20 with MM and 21 with LGMD2B. Genomic DNA was extracted from the peripheral lymphocytes of the patients with informed consent. The PCR products of all 55 exons were screened by single strand conformation polymorphism (SSCP) or direct sequencing from the PCR fragments.
RESULTS: We identified 16 and 11different mutations respectively in MM and LGMD2B patients. Mutations in Japanese patients were distributed along the entire length of the gene. The mean age at onset of the patients with MM was 21.8 7.4 years (range 14 - 37 years) and that of the patients with LGMD2B was 26.2 9.2 years (range 14 - 41 years). On the average, the first use of a cane was at 35.5 years (16.0 years after the onset) for MM and 39.3 years (13.6 years after onset) for LGMD2B. Patients became wheelchair-bound at 42.8 years (22.8 years after onset) in MM and 45.1 years (21.4 years after onset) for LGMD2B. The mean maximum serum CK level at any age of the patients was 5,829 4,273 IU/l (range 1,289 - 12,566 IU/l) for MM and 3,787 2,493 IU/l (627 - 10,000 IU/l) for LGMD2B; in both disorders, the serum CK level fell in proportion to the duration of the illness.
CONCLUSIONS: We have identified four common four mutations (C1939G, G3370T, 3746delG, and 4870delT) in Japanese patients with MM, accounting for 60 percent of all MM mutations in this population. Three of the four mutations (C1939G, G3370T, and 4870delT) accounted for 58 percent of the mutations in LGMD2B patients, while the 3746delG mutation was not found in patients with LGMD2B. The G3370T mutation may be associated with a milder form of MM and LGMD2B. By contrast, the G3510A mutation appears to be associated with a severe form of MM and the C1939G mutation with a severe form of LGMD2B.

 23)  DNA Single-Strand Breaks Increase in OPMD (Oculo-Pharyngeal Muscular dystrophy)
Muneshige Tobita, Watari-gun, Japan, Maki Tateyama, Atsushi Takeda, Yasuto Itoyama, Sendai, Japan, Ikuya Nonaka, Tokyo, Japan, Yuzo Iwasaki, Watari-gun, Japan 

OBJECTIVE: To investigate DNA single-strand breaks (SSBs) increase in the frozen muscle biopsy specimens from the OPMD patients.
BACKGROUND: Recently, DNA damage has been reported to be involved in the process of cell death in some muscular disorders. However, little is known about the role of DNA damage in the pathogenesis of OPMD. To detect early DNA damage, we used in situ nick translation procedure and evaluated SSBs increase in the muscle biopsy specimens from the OPMD patients diagnosed by PABP-2 gene analysis.
DESIGN/METHODS: The in situ nick translation procedure was carried out for detection of SSBs in frozen muscle tissues from four OPMD patients, five ALS patients and four normal controls. ³H dCTP (specific activity: 68 Ci/mmol, Amersham, UK) was used for the purpose of autoradiographic detection. The reaction mixture containing 30 mM each of dATP, dGTP, dTTP, dCTP, 200 U/ml of Klenow fragment, 5mM MgCl₂ and 10 mM 2-mercaptoethanol in 50 mM Tris-HCl, was applied on each 10 m-thickness section. After incubation at room temperature for 60 min, the sections were rinsed, dehydrated and dipped in emulsion. They were then exposed for 4 weeks, developed and counterstained.
RESULTS: The SSBs were recognized as silver grains in this method. In controls or ALS, SSBs were not increased. The number of grains was 0-2/nucleus in 99% of myonuclei. In contrast, a marked increase in SSBs (number of grains: 10/ nucleus or more) was recognized in the myonuclei in OPMD. About 12 % of muscle fibers had such SSBs-positive myonuclei. SSBs-positive myonuclei were observed in apparently affected small angulated fibers and interestingly, in morphologically normal fibers in OPMD. These findings suggest that SSBs increase may reflect early DNA damage in the pathological process involving DNA damage.
CONCLUSIONS: Using the in situ nick translation procedure, we detected an increase in SSBs in the myonuclei in OPMD. This increase may reflect early DNA damage which leads to cell death or pathological processes. It is suggested that the increase in SSBs plays an important role in the pathogenesis of OPMD.

 24)Analysis of del521T LGMD2C Phenotype Related Factors on the Basis of a Genetic Epidemiological Study in Tunisia
Mounir Kefi, Rim Amouri-Malouki, Mongi Ben Hamida, Fayçal Hentati, Tunis, Tunisia 

OBJECTIVE: This study was based on a genetic epidemiological survey in order to collect accurate informations, from a large number of genetically homogeneous patients with LGMD2C. The aim of the study was to analyze the phenotypic features and environmental and genetic factors that could be related to the disease severity.
BACKGROUND: Limb-girdle muscular dystrophies (LGMDs) are a genetically heterogeneous group of muscular dystrophies (MDs). LGMD2C is associated with a mutation in the -sarcoglycan gene ( -SG). This -sarcoglycanopathy is prevalent in Tunisia where only one homozygous mutation, a 521-T deletion has been identified. Despite the genetic homogeneity of patients observed in Tunisia, an important clinical variability is encountered. The cause of this clinical variability remains unknown.
DESIGN/METHODS: From 28 selected index patients with LGMD2C sharing the 521-T mutation, we carried out a field survey during which all index cases were visited at home and examined with their family members. Correct pedigrees were established and 104 secondary cases were newly identified. Informations related to onset, wheelchair-bound age (WCB), life conditions (physical activity and distance between home and school), clinical stage and pattern of muscle wasting were recorded on a questionnaire. The patients were classified as severe, moderate or mild on clinical parameters. Immunostaining for the SG subunits were performed on 50 muscle biopsies. A genetic linkage study with markers spanning the -sarcoglycan gene was carried out.
RESULTS: The 132 LGMD2C patients showed a wide spectrum of clinical course with 46.9% severe, 24.2 % mild and 28.8% intermediate. In 21 families (75%) including 114 patients the phenotype was heterogeneous between siblings. The -SG was absent in all cases whereas the , and -SG showed a variable expression. The analysis of the different data could not give out any relationship between the disease severity and environmental factors (such as life conditions or physical activity) or clinical defined parameters (age of onset and WCB, duration of the disease). In addition, the pattern of SG subunits expression is not related to the clinical phenotype
CONCLUSIONS: LGMD2C patients with delT-521 mutation showed a phenotypic inter and intra-familial variability. The presence of such variability may be related to a modifier gene(s) which did not seem to interact directly with the sarcoglycan protein complex but possibly with the cascade of biochemical pathways leading to muscle fiber necrosis. The identification of this modifier gene(s) could help in the understanding of the pathogenic mechanisms involved in MD.