Abstracts that will be presented on 57th Annual Meeting of American Academy of Neurology - Miami, April, 9-16,2005

Duchenne Muscular Dystrophy

Characterization of PTC124 Activity, Specificity, and Mechanism of Action for Nonsense Mutation Suppression

E. M. Welch, J. Zhuo, Y. Tomizawa, W. Friesen, A. Branstrom, S. Hwang, J. Babiak, L. L. Miller, S. W. Peltz, South Plainfield, NJ

OBJECTIVE: To characterize the suppression of nonsense mutations by PTC124. BACKGROUND: 15% of patients with Duchenne muscular dystrophy have disease due to a nonsense mutation. Aminoglycosides can suppress the effects of a nonsense mutation but are limited by toxicity and need for IV administration. PTC124 is a new drug that may offer a safer, nonantibiotic approach for promoting ribosomal readthrough of nonsense mutations. DESIGN/METHODS: PTC124-induced readthrough was assessed in HEK293 cells transfected with a luciferase gene harboring a premature stop codon at threonine 190, replacing ACA with a UAA, UAG, or UGA (reporter system). mRNA translation was assessed in a cell-free translation assay using the reporter luciferase RNA with a HeLa cytoplasmic extract. mRNA turnover was assessed in a real-time, quantitative, RT-PCR assay of reporter system cells. Coomassie-Blue-stained 2-D gels of reporter system cell extracts evaluated nonspecific ribosomal readthrough activity. Antibacterial assays determined if PTC124 had antibiotic activity. HeLa cell ribosomes were treated with PTC124-like compounds and dimethyl sulfate or kethoxal before primer extension analysis in RNA footprinting experiments. RESULTS: Time-course/washout experiments showed onset of PTC124 nonsense suppression within 2 hr, maximal activity by 20 hr, and, after drug removal, loss of activity by 6 hr. PTC124 activity was stop-codon-type- and concentration-dependent. At 20 hr, maximal readthrough activity over control was 12-fold (UGA), 4-fold (UAG), and 2-fold (UAA). With each codon type, readthrough was discernable at 0.1 μM and maximal at 1.0 μM. Gentamicin was less potent, requiring concentrations >100 μM for activity. PTC124 efficacy was unaltered by a 5-50% range of FBS concentrations, suggesting that drug-protein binding is not activity-limiting. In a cell-free translation system, 1 μM of PTC124 for 4 hr achieved maximal chemoluminescence over control of 18-fold (UGA), 5-fold (UAG), and4-fold (UAA). In cells treated with 30 μM for 48 hr, RT-PCR revealed luciferase mRNA was not increased over control, confirming the translation-specific action of PTC124. Activity was confined to nonsense mutations and not normal stop codons; 2-D gel analysis after 5 μM of PTC124 for 48 hr revealed a 62-kDa, pI-5.58 band consistent with full-length luciferase but no protein elongation due to ribosomal readthrough of normal stop codons. Nonsense suppression activity was separable from antimicrobial activity; PTC124 at 880 μM showed no antimicrobial activity against 6 strains of logarithmically growing gram+ and - bacteria. PTC compounds induced changes in the chemical footprinting pattern on conserved sites of the large rRNA that modulate nonsense suppression activity in prokaryotic systems; these are distinct from the small rRNA sites bound by aminoglycosides. CONCLUSIONS: These data document the potency, specificity, and mechanism of action of PTC124 for nonsense mutation suppression and suggest target plasma concentrations to be achieved in clinical studies.
 

PTC124 Nonsense Mutation Suppression Therapy of Duchenne Muscular Dystrophy (DMD)

E. Barton, M. Zadel, E. Welch, C. Trotta, S. Paushkin, M. Patel, J. Zhuo, Y. Tomizawa, M. Weetall, V. Northcutt, J. Babiak, L. Miller, South Plainfield, NJ, L. Sweeney, Philadelphia, PA

OBJECTIVE: To evaluate PTC124 efficacy in the mdx model of DMD (mice have a nonsense mutation in the dystrophin gene) and document PTC124 specificity. BACKGROUND: Nonsense mutations in the dystrophin gene cause 15% of cases of DMD. PTC124 is a small molecule that induces ribosomes to read through nonsense-mutation-containing mRNA and may offer a new oral therapy for such patients. DESIGN/METHODS: PTC124 was mixed with food for 8 weeks in efficacy studies. Groups (5-10 mice/group) were untreated mdx mice, treated mdx mice (PTC124 0.9 mg/mL and 1.8 mg/mL), and untreated wild-type C57 mice. Endpoints were PTC124 plasma concentrations, dystrophin immunofluorescence in tibialis anterior muscle, specific force in extensor digitorum longus (EDL), decrease in EDL force after 5 eccentric contractions, and serum creatine kinase. Unpaired t-tests compared treated with untreated mdx mice. Western blotting of high-abundance proteins in tissues (heart, kidney, liver, lung, small intestine, salivary glands, thymus, white cells) from rats and dogs given oral PTC124 at ≥1500 mg/kg for 14 days evaluated whether PTC124 induced protein elongation due to nonspecific readthrough of normal stop codons. RESULTS: PTC124 plasma concentrations at 8 weeks (mean [±SE] mcg/mL) were similar at both PTC124 dose levels: 0.9 mg/mL (1.85 [0.54]) and 1.8 mg/mL (1.03 [0.25]){p=.16}. Dystrophin was absent in untreated mdx mice, was restored similarly by PTC124 at both dose levels, and was present in wild-type mice. PTC124 partially improved EDL specific force (mean [±SE] N/cm²): untreated mdx (17.75 [0.65]), PTC124 0.9 mg/mL mdx (19.99 [0.63]){p=0.03}, PTC124 1.8 mg/mL mdx (19.18 [0.68]){p=0.16}although not to wild-type levels (22.44 [1.03]). PTC124 partially prevented EDL eccentric contraction injury (mean [±SE] % decrease in force): untreated mdx (42 [2]), PTC124 0.9 mg/mL mdx (19 [3]){p<0.001}, PTC124 1.8 mg/mL mdx (25 [3]){p<0.001}, and wild-type (11 [2]). PTC124 reduced serum creatine kinase (mean [±SE] IU/mL): untreated mdx (1654 [360]), PTC124 0.9 mg/mL mdx (858 [169]) {p=0.07}, PTC124 1.8 mg/mL mdx (848 [83]){p=0.04}, and wild-type (169 [86]). Western blots of proteins from rats (vimentin, a-actin, U1 snRNP A, and cofilin) and dogs (GAPDH, b-actin, and cofilin) evaluating each stop codon type (UGA, UAA, and UAG) showed no protein elongation in any tissue that would suggest that PTC124 induced changes in ribosomal readthrough of normal stop codons. CONCLUSIONS: Collectively, these preclinical efficacy and specificity data support the clinical development of PTC124 for nonsense-mutation-mediated DMD. Supported by: Study supported in part by PTC Therapeutics, Inc.
 

Phase 1 Multiple-Dose Safety and PK Study of PTC124 for Nonsense Mutation Suppression Therapy of Duchenne Musuclar Dystrophy (DMD)

S. Hirawat, V. J. Northcutt, E. M. Welch, G. L. Elfring, S. Hwang, N. G. Almstead, W. Ju, L. L. Miller, South Plainfield, NJ

OBJECTIVE: To assess the safety, PK, palatability, and nonspecific stop codon readthrough effects of multiple sequential doses of orally administered PTC124. BACKGROUND: PTC124 is a novel, orally bioavailable, nonantibiotic, small molecule that promotes ribosomal readthrough of mRNA containing a nonsense mutation (premature stop codon) and has the potential to restore dystrophin in patients with nonsense-mutation-mediated DMD. PTC124 induces production of full-length dystrophin and decreases muscle injury in mdx mice harboring a nonsense mutation in the dystrophin gene. Toxicology studies demonstrate that the drug is well tolerated in rats and dogs at doses ≥1500 mg/kg, shows no evidence of genotoxicity, does not induce QT-interval prolongation, and does not undergo extensive metabolism. In a Phase 1 single-dose study in healthy young adult volunteers, PTC124 safely achieved plasma concentrations that have been shown to be pharmacologically active in mdx myocyte cultures and in mdx mice. DESIGN/METHODS: This Phase 1 escalating multiple-dose study in healthy young adult volunteers is a prelude to studies in patients with DMD. The study will be conducted in 2 stages: in Stage 1, escalating doses of PTC124 will be administered over 7 days; in Stage 2, a PTC124 dose selected from Stage 1 will be administered over 14 days.
In Stage 1, 24 subjects (3 males, 3 females per cohort) are to receive PTC124 administered at 4 progressively higher dose levels in a dose-escalation design. The starting dosing regimen of 10 mg/kg BID with meals is based on the Phase 1 single-dose study and 28-day toxicology data. Subsequent dose levels will be based on safety and PK data obtained at each prior dose level. During each 7-day dosing, subjects will be followed with clinical observations, evaluations of safety and palatability, plasma sampling for PK analysis, and white blood cell collections for Western blotting to evaluate the hypothetical potential for protein elongation due to nonspecific normal stop codon readthrough. In Stage 2, 6 additional subjects (3 males, 3 females) are to receive PTC124 BID for 14 days at the highest safe dose derived from Stage 1. These subjects will be followed with clinical observations, safety laboratory testing, and plasma sampling to determine the safety and PK of PTC124 when given continuously for 2 weeks. RESULTS: Preliminary data from the first dose level of 10 mg/kg given BID for 7 days suggest that the drug is well tolerated and maintains target plasma concentrations of ≥10 μg/mL for >6 hr per day. Further study accrual and dose escalation is ongoing. CONCLUSIONS: Prior to the AAN meeting, the study will have completed all subject accrual and follow-up. Complete data on subject characteristics, dose ranging, safety, PK, palatability, and Western blotting analyses will be available for presentation. Results from this study will provide important safety information and will refine the PK model that will be used to project an appropriate dosing regimen for subsequent Phase 2 studies in patients with DMD.
 

Deflazacort Treatment in DMD Symptomatic Carriers

Alberto L. Dubrovsky, Laura N. Pirra, Andrea Lautre, Ana Lia Taratuto, Lilia Mesa, Jose Corderi, Buenos Aires, Argentina

OBJECTIVE: Some females carrying a mutation in the dystrophin gene can develop significant progressive motor disability with muscle weakness (Duchenne Muscular Dystrophy symptomatic carriers, DMDSC).
There is enough evidence in the literature to conclude that steroids, Prednisone or Deflazacort (Df), are effective in slowing the progressive muscle weakness that characterizes DMD.
However the effects of this medication in DMDSC has not been reported. BACKGROUND: Report the short term results of Df treatment in three cases of DMDSC. DESIGN/METHODS: Three DMDSC 56, 34 and 16 years old in which diagnosis was confirmed by muscle biopsy and/or DNA analysis were treated with Df in doses between 0.5 and 0.7 mg/Kg. All three cases presented with progressive severe muscle weakness and disability.
Clinical evaluation included manual muscle testing (MMT), timed and functional tests and an auto evaluation scale (0 worst, 1 same, 2 better, 3 much better).
Assessments were performed between 2 and 6 months after treatment was initiated.
Steroids unwanted effects were monitored in each patient. RESULTS: All three patients showed marked improvement albeit of a different degree in each case.
Timed Gowers sign and time to walk 10 meters showed the most significant improvement. One of the patients recovered the ability to rise from the floor. No change was detected in the MMT. Functional score (modified Scott score) also showed improvement. Auto evaluation scale was qualified as 2 (two cases) and 3 (one case)
No significant unwanted effects derived from medication developed in any of the cases. CONCLUSIONS: These cases suggest that steroid treatment might be beneficial in DMDSC.
In selected cases of DMDSC, Deflazacort treatment should be considered.
A larger trial is needed in order to confirm these findings and to define the appropriate dose as well as to establish the criteria for starting treatment.
 

Improved DEXA Methodology for Bone Mass Assessment in Boys with Duchenne Muscular Dystrophy

Wendy M. King, John T. Kissel, John D. Landoll, Howard S. Barden, Madison, WI, Velimir Matkovic, Columbus, OH

OBJECTIVE: To determine the most accurate method of assessing overall bone status in boys with Duchenne muscular dystrophy (DMD) for optimal assessment of fracture risk. BACKGROUND: Although dual-energy x-ray absorptiometry (DEXA) has become the gold standard for the assessment of bone mass in children, the two-dimensional (areal) measurement of bone mineral density (BD, g/cm²) currently used in DEXA is heavily influenced by bone size. We utilized new software that improves the assessment of bone status in children by including consideration of height for age, bone mineral content (BMC) for area, and bone area for height. We examined the value of including these body size ratios in BD determinations in boys with DMD. We also examined the role of the skull in total body BD in growing children. DESIGN/METHODS: We assessed bone status using DEXA (Lunar Prodigy) with experimental software in 22 DMD patients age 6-17 years (mean 11.5 years). Boys were evaluated for total body BD as a function of age (routine DEXA). This value was then compared to total body BMC as a function of bone area, height as a function of age, and total body bone area as function of height using Z-scores developed from 267 normal boys. Z-score equations were also developed for total body regional sites (head, arms, legs, trunk). All Z-scores, with the exception of height as a function of age, were calculated using male children from the Lunar pediatric reference population (mean 11.6 years). Height for age Z-scores were calculated using normal data from the USA Center for Disease Control. Both boys on steroids and steroid naive boys were included in the analysis. RESULTS: Although boys with DMD had a mean deficit in total body BD by standard DEXA compared to controls (Z=-1.2), this could be attributed to their moderately short stature (height for age Z=-1.2) and profoundly small, narrow bones (bone area for height Z=-2.8). Taking these size ratios into account, the boys had normal total body bone mineralization (BMC for bone area Z=0.04) [p<0.05]. Regionally, only the trunk showed a deficit in BMC for area (Z=-2.0). Subtracting the head from total body calculations produced further significant reductions in Z scores (less dense bones) for BD for age and BMC for area (p<0.001). Z scores for BD for age, height for age, and BMC as a function of area decreased in older subjects. CONCLUSIONS: Overall bone status in boys with DMD can most accurately be assessed by using DEXA technology that accounts for skeletal size rather than chronological age alone and excludes the assessment of the skull. This is particularly important for boys on corticosteroids. Such methodologies should improve our abilities to diagnose and treat reduced bone mass in boys with DMD.

Full Length-Utrophin-Expressing Gutted Adenovirus as an Ideal Therapeutic Agent for Duchenne Muscular Dystrophy

Jatinderpal R. Deol, Renald Gilbert, Joon-Shik Moon, Seoul, Korea, Mylene Bourget, Josephine Nalbantoglu, George Karpati, Montreal, QC, Canada

OBJECTIVE: To construct a fully deleted (“gutted”) adenovirus (AdV) capable of high expression levels of full-length utrophin (utr) in dystrophin (dys)- deficient muscle fibers. BACKGROUND: Duchenne muscular dystrophy (DMD) is characterized by a mutation in the gene encoding a sarcolemmal protein, dys and the deficiency of dys leads to progressive loss of muscle fibers. Gene replacement therapy using fully deleted AdV vectors shows great potential for the treatment of DMD. These vectors are less immunogenic than their predecessors and have the capacity of carrying large DNA inserts such as the full-length dys cDNA (13 kb). The use of fully deleted AdV vectors encoding full-length dys leads to significant improvement of the dystrophic phenotype of the mdx mouse, an animal model of DMD. However, dys behaves as a neoantigen in genetic dys deficiency states such as the mdx mouse and it causes immune responses in the treated muscles. An alternative approach would be to use utr, a functional homologue of dys, which is normally present only at the sarcolemma of the neuromuscular and myotendinous junctions. Therefore, utr is not expected to behave as a neoantigen in dys-deficient states. In fact, transgenic mdx mice expressing extrasynaptic utr in muscle fibers have shown a near total cure of their dystrophy. Thus, in an attempt to minimize the vector immunogenicity and to harness the therapeutic potential of utr, we proceeded to create a fully deleted AdV encoding full-length utrophin. DESIGN/METHODS: The expression cassette contains the full-length murine utr cDNA regulated by a hybrid promoter consisting of the chicken β-actin promoter and CMV enhancer. Plasmid, containing this cassette, was used to transfect and subsequently amplify the AdV of interest in a complementing cell line. Due to concerns regarding AdV vector size and their proper packaging, three different AdV vectors were constructed. The purified recombinant adenoviruses were tested in vitro by western blot and subsequently injected into neonate and adult mdx mice for in vivo evaluation. The mice will be euthanized at 10, 30, 90, 180, and 360 days post-injection and their injected muscles will be examined for utrophin expression, the restoration of the dystrophin-associated protein complex, and the reversal of the physiological dystrophic phenotype. RESULTS: Western blot analysis has shown adequate utrophin expression levels from in vitro studies involving early passages of two of the recombinant adenoviruses. In addition, the presence of viral DNA, isolated by HIRT extraction, has been detected by southern blots. Large scale production of all three utr-expressing AdV vectors is underway to continue the in vivo characterization of the therapeutic affects of utrophin expression vectors in mdx muscles. CONCLUSIONS: Helper-dependent, fully deleted (gutted) AdV expressing full length utrophin promises to be the ideal agent for gene replacement therapy in dys-deficiency states. Supported by: Canadian Institutes of Health Research.

Temporal and Spatial Expression Patterns of TGF-B1, 2, 3 and TGFBRI, II, III in Skeletal Muscles of Mdx Mice

Lan Zhou, John D. Porter, Betheda, MD, Georgiana Cheng, Bendi Gong, Denis Hatala, Anita Merriam, Xiaohua Zhou, Jill Rafael-Fortney, Columbus, OH, Henry Kaminski, Cleveland, OH

OBJECTIVE: To determine the temporal and spatial mRNA expression patterns of TGF-B1, 2, and 3 and their receptors in the quadriceps and diaphragm muscles of mdx mice. BACKGROUND: TGF-B1 is a multifunctional cytokine, regulating inflammation and fibrosis, which may play a role in the secondary pathogenesis of Duchenne muscular dystrophy (DMD). Expression of TGF-B1 is upregulated in skeletal muscles from DMD patients and DMD animal models. However, the temporal and spatial expression patterns of TGF-B1 and other TGF-B subfamily members, TGF-B2 and TGF-B3, as well as their receptors, and the correlation of their expression patterns with muscle inflammation and fibrosis, have not been well studied in mdx mice, a DMD mouse model. DESIGN/METHODS: We studied temporal mRNA expression of TGF-B1, 2, 3 and TGFBRI, II, III by quantitative RT-PCR, and the spatial expression patterns of TGF-B1 and TGFBRII by in situ hybridization in quadriceps and diaphragm of mdx and normal mice at age 3 weeks, 8 weeks, and 8 months. We assessed the muscle inflammation by immunostaining using an F4/80 antibody to identify macrophages and assessed fibrosis by immunostaining using a collagen III antibody. RESULTS: At age 3 weeks, inflammation was evident in mdx diaphragm and quadriceps, but more prominent by 8 weeks. By 8 months, only scattered macrophage infiltration was seen, but significant fibrosis had developed in mdx diaphragm but not in quadriceps. At age 3 weeks, the mRNA levels of TGF-B1, 2, 3 and TGFBRI, II, III were no different in mdx quadriceps and diaphragm as compared to controls, but by 8 weeks, coincident with inflammation becoming more prominent in both muscles, TGF-B1 mRNA expression was markedly increased in the mdx quadriceps (8.4-fold) and to a lesser degree in diaphragm (2.5-fold). Elevations in mRNA levels of TGF-B2 were modest (<2-fold) in both muscles. TGF-B3 was increased 2.3-fold only in mdx diaphragm. Gene transcripts of TGF-B receptors were slightly increased in both mdx muscles. At age 8 months, TGF-B1 mRNA was increased 4-fold in mdx quadriceps but not significantly increased in diaphragm. TGF-B2, TGF-B3, and TGF-B receptors were not significantly different at this stage. In situ hybridization confirmed higher levels of TGF-B1 mRNA in mdx muscles, being most prominent at age 8 weeks, and localized to the regenerating fibers and endomysial inflammatory cells. TGF-BRII mRNA showed a similar pattern of expression. CONCLUSIONS: The results strongly support a role for TGF-B1 in the pathogenesis of inflammation in mdx muscles. TGF-B1 is known to be a key pro-fibrotic cytokine; however, higher levels of TGF-B1 mRNA were identified in quadriceps. Therefore, mdx leg muscles may have properties that protect fibrosis, or alternatively, diaphragm may be particularly susceptible to development of fibrosis. Such variations could contribute an explanation to the differential involvement of muscle groups by muscular dystrophies. Supported by: Supported by R24 EY014837, R01 EY015306, and Alyce J. and Ann J. Metka Foundation.
 

Somatic Mosaicism in a DMD Carrier Detected by Use of Direct Sequence (SCAIP) Analysis

Karin M. Dent, Diane M. Dunn, Andrew C. von Niederhausern, Alex Aoyagi, Brett Duvall, Lynn Kerr, Therese Tuohy, T. H. Bui, Stockholm, Sweden, Robert B. Weiss, Kevin M. Flanigan, Salt Lake City, UT

OBJECTIVE: To describe the use of Single Condition Amplification/Internal Primer (SCAIP) methodology—a robust, rapid, and economical method of direct sequence analysis—in detecting a somatic mosaic carrier state for DMD. BACKGROUND: Duchenne muscular dystrophy (DMD) is an inherited disease of muscle caused by mutations in the DMD gene (MIM 300377). Approximately 60% of DMD patients have deletions of one or more exons, about 30% have point mutations, and less than 10% have duplications. SCAIP readily detects point mutations in carriers, but somatic mosaic detection by this method has not been reported. DESIGN/METHODS: Blood samples were obtained from a DMD patient and his mother. DMD in the patient was confirmed by clinical course and a muscle biopsy demonstrating a lack of dystrophin by immunohistochemical staining. There was no family history of DMD. The mother had no muscle symptoms, and had undergone a muscle biopsy that was normal including dystrophin immunohistochemistry studies. Genomic DNA was extracted from blood by standard techniques, and SCAIP analysis was performed as described (Flanigan et al., AJHG 2002). RESULTS: No DMD deletion was identified by multiplex PCR. SCAIP analysis identified a premature stop mutation in exon 12 in the boy's DNA sample (c.1465C>T, resulting in a Gln489X premature stop codon). However, direct sequencing of exon 12 in the mother identified a strong C peak and a minor T peak in both forward and reverse sequence reads. To determine the frequency of the C and T alleles, the exon 12 PCR product from the mother's amplification was cloned into vector PCR 2.1 using a TA cloning system (Invitrogen) and transformed into XL-1 cells. One hundred clones were sequenced; eighty-five (85%) were found to be the C allele, while 15% were the T allele, consistent with somatic mosaicism for this mutation. CONCLUSIONS: Discussion: The availability of SCAIP sequence analysis of DMD permits the economical and rapid identification of mutations that were previously difficult to detect, such as premature stop codon and small frameshifting mutations. The high sensitivity of heterozygote detection can be combined with TA cloning techniques as a semi-quantitative measure of somatic mosaicism. Our method facilitates determination of carrier status and somatic mosaicism, enabling more accurate genetic counseling regarding recurrence risk in somatic mosaic DMD carriers. Supported by: NIH/NINDS
 

“Becker-Type” Dysferlinopathy

Michael Sinnreich, Christian Therrien, George Karpati, Montreal, QC, Canada

OBJECTIVE: To describe clinical, biochemical and molecular findings in a family affected by a newly identified in-frame exon skipping mutation in the dysferlin gene giving rise to a shortened dysferlin molecule with milder disease phenotype. 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. Reports of genotype/phenotype correlations in patients with dysferlinopathies are lacking. Shortened dysferlin molecules which arise due to in-frame exon skipping mutations and which have residual biological activity to convey a milder disease phenotype, analogous to “Becker-type” dystrophinopathies, have not so far been described in patients with dysferlinopathies. DESIGN/METHODS: A woman presented in her fourth decade with a mild limb girdle muscular weakness and remained ambulatory at her current age of 75. Two of her daughters, currently in their fourth decade were severely affected by a disabling limb girdle muscular weakness with onset in their teens and were wheelchair dependent since their early thirties. Muscle biopsies of these three patients were processed for histological, biochemical and molecular analysis. Western blots were probed for dysferlin. Mutational analysis of the dysferlin gene was performed on reverse transcribed RNA isolated from skeletal muscle. RESULTS: On clinical examination the ambulatory mother had only moderate proximal weakness in upper and lower extremities. Both wheelchair dependent daughters had severe generalized weakness. Their father was clinically unaffected. Skeletal muscle histology showed a dystrophic pattern in all three patients. Mutational analysis revealed homozygozity for a null allele in both daughters due to a complex mutation [(4872delG) and (4876G→C)]; both parents were heterozygous for this allele. The mother harbored an additional in-frame splice mutation with skipping of exon 32, likely due to an identified A→G substitution of the putative branch point nucleotide at position 3443-32 in intron 31. This mutation partially deletes dysferlin's C2D domain. While Western blot showed absence of any dysferlin immunoreactivity in skeletal muscle of both daughters, the mother's skeletal muscle harbored reduced levels of a shortened dysferlin protein, possibly retaining sufficient biological activity to account for her significantly milder phenotype. CONCLUSIONS: We report on a mildly affected LGMD2B patient with one dysferlin null allele and one in-frame exon skipping allele. The in-frame skipping of dysferlin's exon 32 is likely due to a mutation of the branch point nucleotide in intron 31 that is implicated in RNA lariat formation. Despite partial deletion of dysferlin's C2D domain, the shortened dysferlin protein appears to retain sufficient biological activity to account for a significantly milder disease phenotype in the compound heterozygous mother, as compared to her two severely affected homozygous, dysferlin null, daughters. In vitro experiments with recombinant mini-genes are currently ongoing to demonstrate the implication of the putative intronic branch point nucleotide mutation in skipping of exon 32.
 

Regulation of the Utrophin Gene through the Use of Artificial Zinc Finger Proteins

Yifan Lu, George Karpati, Josephine Nalbantoglu, Montreal, QC, Canada

OBJECTIVE: To pursue transcriptional regulation of the utrophin (utr) gene through the targeting of artificial transcription factors to activate the utrophin A promoter in a constitutive manner (i.e. without need for the sustained presence of neural factors). BACKGROUND: Utr is a structural and functional homologue of dystrophin. In normal mature muscle fibers, utr is only present at the surface membrane of the neuromuscular and myotendinous junctions. Utr is anchored to the sarcolemma by the same molecular elements as dystrophin. From the high degree of homology between dys and utr, it is anticipated that if a sufficient amount of utr would be accumulated and maintained in the extrasynaptic sarcolemma, dystrophin deficiency would not have deleterious effects on muscle fibers. To upregulate the utr promoter we have chosen to use articficial zinc finger proteins (ZFPs) as transcription factors. DESIGN/METHODS: The Utr A promoter is a TATA-less promoter with a GC-rich 5' upstream area, being located within a CpG island. DNAse I mapping of the proximal 5' sequences of the utrophin A promoter revealed several DNAse I accessible sites, suggesting that these regions might provide good targets for ZFPs. Four GC-rich sequences were chosen and the appropriate ZFPs were designed based on the recognition modules described by Liu et al.(J Biol Chem, 2002, 277:3850). The modules were linked by the five residue linkers (TGEKP) that are conserved in many naturally occurring multi-finger domain proteins. These DNA binding domains were linked to an activation domain provided by the VP16 protein of herpes simplex virus, tagged with the Flag epitope for convenience of monitoring ZFP expression and a nuclear localization signal (NLS) to ensure nuclear trafficking. In subsequent experiments these ZFPs were compared to the previously described ZFP that activated utrophin (labeled Jazz). The ability of each ZFP to activate a reporter gene (luciferase) driven by the utrophin A promoter was assessed by transient transfection assays in 4 different cell lines (cos-1, NIH 3T3 fibroblasts, human embryonic kidney 293 cells, and mouse C2C12 myoblasts). The specificity of DNA binding was determined by electrophoretic mobility shift assay. The ability of the ZFP to activate the endogenous utr promoter in C2C12 cells was determined by stable transfection followed by quantitation of utr mRNA by Real-Time RT-PCR and Western blotting for utr protein. RESULTS: In reporter assays, one of the designed ZFPs (ZFP51) performed better than all the other ones, including the published one (Jazz). ZFP51 bound its recognition sequence with good specificity as determined by electrophoretic mobility shift assay. Furthermore, C2C12 cells stably transfected with ZFP51 expressed higher levels of utrophin mRNA as well as protein by Western blot analysis (on average ~3.5-fold increase as normalized to actin). CONCLUSIONS: Artificial ZFPs may provide a convenient way of upregulating endogenous utr promoter and could be used therapeutically in dystrophin deficiency. Supported by: Canadian Institutes of Health Research and Muscular Dystrophy Association (USA)
 

Myotonic Dystrophy

Excessive Daytime Sleepiness (EDS) and the Hypocretin Neurotransmission System in Myotonic Dystrophy

Emma Ciafaloni, Emmanuel Mignot, Stanford, CA, Valeria Sansone, Milano, Italy, James E. Hilbert, Michael L. Perlis, Xiaoyan Lin, Lynn C. Liu, Michael P. McDermott, Charles A. Thornton, Rochester, NY

OBJECTIVE: 1) Determine if a disrupted hypocretin (Hcrt) neurotransmission system is responsible for EDS in myotonic dystrophy type 1(DM1).
2) Compare the occurrence and severity of EDS in DM1 and DM2.
3) Analyze the Epworth Sleepiness Scale (ESS) in a large cohort of DM1 and DM2 patients in relation to age, number of CTG repeats, and other varables. BACKGROUND: EDS is a common and disabling aspect of the complex DM1 phenotype.
Abnormal REM pressure may occur in DM1, pointing to a central dysregulation of sleep similar to narcolepsy. It is unknown if EDS is also present in DM2. The underlying mechanism of EDS and other CNS manifestations in DM is unknown.
Hcrt levels in human narcolepsy are very low or undetectable and mutations in the Hcrt receptor gene are responsible for canine narcolepsy.
Symptoms of DM1 may results from abnormal regulation of alternative splicing in muscle and brain. DESIGN/METHODS: The ESS from 373 DM1 and DM2 patients from the National Registry of Myotonic Dystrophy and Facioscapulohumeral Muscular Dystrophy was analyzed.
Polysomnograms (PSG) and Multiple Sleep Latency Tests (MSLT) were performed in 8 DM1 patients.
Hcrt-1 levels were measured using a direct radioimmunoassay in genetically confirmed DM1 patients.
Because DM1 is associated with abnormal regulation of alternative splicing, we used RT-PCR to examine splicing of mRNA for Hcrt receptors 1 and 2. RNA samples were isolated from postmortem cerebral cortex. RESULTS: Hcrt1 CSF levels were in normal range in 7 DM1 patients (mean 243.67 pg/mL).
PSG in 8 patients showed significant abnormality of sleep continuity and architecture with sleep efficiency of 75.6% and high percentage of REM sleep (mean 26.3% +/-9.3) with 4 patients having >30% REM. Sleep latency on MSLT was< 10' in 5 patients.
Splicing of Hcrt Receptor 1 and 2 in cerebral cortex in DM1 was similar to controls without neurological disease.
Mean ESS was 10.7 in DM1 (n=313), 7.3 in DM2 (n=60) and 7.2 in control patients with FSH muscular dystrophy (n=85). ESS was significantly greater in DM1 than FSHMD (p= 0.0001) but not in DM2 compared with FSHMD (p=0.9). ESS >10 was found in 55% of DM1 (mean 14.4), 30% of DM2 (mean 13) and 25% of FSH patients (mean 12).
Although a statistically significant increase of 0.3 was seen at 1 year follow up in the ESS score of 228 DM1 patients, scores did not change in 167 of them. Further longitudinal analysis is undergoing to establish if EDS is progressive or static.
No significant correlation was found between ESS and age, BMI, duration of disease or number of CTG repeats (n=149). CONCLUSIONS: EDS is prevalent in DM1 but not in DM2.
There is no correlation between EDS and number of CTG repeats.
Despite sleep disturbance similarity with narcolepsy, EDS in DM1 does not result from Hcrt deficiency or defective splicing of Hcrt receptors. Supported by: This study was supported by an MDA grant.
The NIH sponsored National Registry of Myotonic Dystrophy and Facioscapulohumeral Muscular Dystrophy Patients and Family Members was used for this study.

Modafinil Does Not Reduce Daytime Somnolence in Patients with Myotonic Dystrophy

David Orlikowski, Pascal Laforet, Paris, France, Maria-Antonia Quera-Salva, Frederic Lofaso, Annie Verschueren, Jean Pouget, Marseille, France, Metropolitan, Bruno Eymard, Djillali Annane, Garches, France, Metropolitan, Sylvie Chevret, Paris, France, Metropolitan

OBJECTIVE: To determine efficacy and tolerance of Modafinil on reduction of daytime somnolence in patients with myotonic dystrophy (MD). DESIGN/METHODS: Prospective randomized, placebo-controlled study. Patients with genetically proven myotonic dystrophy were assigned to Modafinil (300 mg per day) or placebo for 4 weeks. Patients were included if they had an Epworth Scale Sleepiness (ESS) higher than 10 and a mean latency on the Multiple Sleep Latency Test (MSLT) lower than 8 minutes. Each patient was evaluated at study day 0, 7 and 28. The primary outcome measure was Maintenance of Wakefulness Test (MWT), secondary outcome were ESS, MSLT, nocturnal polysomnography and SF36 scale. Tolerance was assessed on clinical, electrocardiographic and biologic exams. Efficacy and tolerance were assessed at day 7 and 28 both by patients and neurologist by Visual Analogical Scale (VAS). Effect on mood was assessed by the Hamilton Depression scale. RESULTS: Twenty height patients were randomized and analyzed (Modafinil in 13 and placebo in 15). The 2 groups were well balanced for baseline characteristics except for ESS which was significantly higher in the placebo group. (p=0.01)
At 28-day, mean MWT was 15.8±3.8 in the placebo group and 16.4±3.3 in the Modafinil group (p=0.93). There was no significant difference between the 2 groups for any of the secondary outcomes. CONCLUSIONS: There was no argue for any benefit from Modafinil in MD patients with hypersomnia. Supported by: Association Francaise contre les myopathies (AFM)

Cerebrospinal Fluid Tau Protein Abnormalities and Executive Dysfunction in Myotonic Dystrophy Type 1 (DM1)

Valeria Sansone, Sandra Gandossini, Maria Cotelli, Marco Calabria, Antonella Alberici, Simona Signorini, Biagio Cotticelli, Roberta Ghidoni, Giuliano Binetti, Brescia, Italy, Giovanni Meola, San Donato Milanese, Milan, Italy

OBJECTIVE: To determine whether cerebrospinal fluid tau protein levels may account for executive dysfunction in myotonic dystrophy type 1 (DM1) and whether tau levels may function as appropriate biomarkers of cognitive impairment in this disorder. BACKGROUND: Neurofibrillary tangles (NFT) and specific tau aggregates are pathological hallmarks of many neurodegenerative diseases including frontotemporal dementia (FTD). NFT and specific tau variants have been found in the brain hippocampal, enthorinal and temporal cortex of DM1 patients, possibly correlating with (CTG)n repeats. How these neuropathology data correlate with neuropsychological and neuroimaging findings is still unclear in DM1. DESIGN/METHODS: 28 patients with DM1 (mean age 45.6 ± 13.3, 600-800 CTGn repeats) were subjected to neuromuscular, cognitive and neuropsychological assessments (Wechsler Adult Intelligence Scale-Revised, WAIS-R; Mini-Mental State Examination; Raven Progressive Matrices; Token Test; Verbal Fluency with phonetic and semantic cues; Story Recall; Digit Span; Corsi block tapping span; Rey's complex figure - Copy and Recall; Tower of London; Divided Attention; Trial Making test; Alertness). Results were compared to 20 age-, sex- and education-matched controls. Cerebrospinal fluid (CSF) determinations of total tau protein were determined in 19 DM1 patients and in 14 patients with frontotemporal dementia (FTD, mean age 60±6.6) by using ELISA (Innogenetics, Belgium). Brain 1.5 Tesla MRI (or 5 mm CT scans) to quantify general (GCA), focal cerebral atrophy (FCA) and white matter hyperintense lesions (WMHL) using an arbitrary scale of increasing severity from 0 to 3 as previously described ⁽¹⁾ were also performed.
⁽¹⁾Reduced cerebral blood flow and impaired visual-spatial function in proximal myotonic myopathy. Meola G et al. Neurology 1999 ;53 :1042-1050. RESULTS: Significant frontal function test differences (Divided Attention – number of omissions, p<0.05) were found between DM1 and controls (4.9 ± 3.7 vs 1.3 ± 0.7). Mean IQ of all DM1 patients was 90 ±12.6. In DM1 patients, CSF levels of total tau proteins were normal in 2/3 of patients (222.5 pg/ml ± 101.3) compared to normative values. In 6 of 19 patients with DM1, the levels of CSF tau were increased (625 pg/ml ± 297.6) and comparable to FTD patients. IQ levels in these 6 DM1 patients were lower than the remaining DM1 patients (85 ± 2.9 vs 92 ± 14.3). CONCLUSIONS: The WAIS-R profile and the specific impairment in tests of frontal lobe function emphasize that cognitive impairment is characteristic of adult DM1.The determinations of CSF tau proteins in DM1 patients, suggest that CSF tau may be a marker of executive dysfunction in DM1 like in FTD. Supported by: Support of the University of Milan, Italy (MIUR 60% and ex 40%) given to G. Meola
 

Progressive Frontal Lobe Impairment in Myotonic Dystrophy Type 1 (DM1) and Type 2 (DM2): A Longitudinal Study

Giovanni Meola, Valeria Sansone, Sandra Gandossini, Eleonora Cattaneo, Antonella Alberici, Biagio Cotticelli, San Donato Milanese, Milan, Italy, Orazio Zanetti, Marco Calabria, Maria Cotelli, Giuliano Binetti, Brescia, Italy

OBJECTIVE: To determine whether previously reported visual-spatial impairment and executive dysfunction, brain atrophy and white matter hyperintense lesions progress over time in myotonic dystrophy type 1 (DM1) and type 2 (DM2) and whether patients develop a dementia syndrome BACKGROUND: Neuropsychological tests, neuroimaging, presenile Alzheimer's neurofibrillary tangles without senile plaques in the brain of DM1 patients and more recent findings that toxic mRNA repeat expansions may alter tau expression, emphasize that myotonic dystrophies should also be considered brain disorders. The extent by which these abnormalities lead to a cognitive decline similar to a dementia syndrome over time is yet to be determined DESIGN/METHODS: 47 patients with DM1 and 30 patients with DM2, were subjected to: (i) brain 1.5 Tesla MRI (or 5 mm CT scans), to quantify general (GCA), focal cerebral atrophy (FCA) and white matter hyperintense lesions (WMHL) using an arbitrary scale of increasing severity from 0 to 3 as previously described ⁽¹⁾ and (ii) neuropsychological tests (MMSE, Raven progressive matrices, token, weigl, controlled association letters and categories, story recall, digit span, corsi and corsi supraspan, rey copy and recall). 30 of these DM1 (mean age 44.8 ± 10) and 15 of these DM2 (mean age 45.9 ± 16.1), repeated neuromuscular assessment; neuroimaging and neuropsychological tests over time (mean follow-up 5.5 years ± 3.4, range 2-12).
⁽¹⁾Reduced cerebral blood flow and impaired visual-spatial function in proximal myotonic myopathy. Meola G et al. Neurology 1999 ;53 :1042-1050. RESULTS: Results are expressed by comparing initial and final assessments. Muscle strength progressed significantly only in DM1 (p = 0.002) compared to DM2 (MegaMRC score: 133.4 ±9.0 vs 122.6 ±14.0 in DM1 and 116.7 ± 53.1 vs 116.5 ± 51.1 in DM2). In both DM1 and DM2, GCA (0.7±1.0 vs 1.1±1.0 in DM1, p<0.05 and 0.4±0.7 vs 1.3±0.5 in DM2, p<0.05), and WMHL (1.6±0.9 vs 1.8±0.8 in DM1, p<0.05 and 0.5±0.6 vs 1.4±0.5 in DM2, p<0.05) also progressed significantly. Among the frontal tests, Trial Making test B (p<0.0001), Alertness with (p<0.05) and without (p<0.001) warning signal and Divided Attention (p<0.05) worsened significantly in both DM1 and DM2. No additional areas of cognition were involved. No relation to muscle strength, paternal or maternal transmission, age at onset, CTG and CCTG expansions was found. CONCLUSIONS: Our longitudinal data suggest that, in DM1 more than in DM2 there is a progressive and significant impairment in muscle strength and in frontal performances (attentional), without further involvement of additional areas of cognition over time. These data indicate that adult DM1 and DM2 are characterized by a relatively specific progressive frontal cognitive impairment, but not fulfilling accepted criteria for dementia. This may have important prognostic and therapeutic implications. Supported by: Support of the University of Milan (MIUR 60% and ex 40%) given to G. Meola
 

Mexiletine: Effective Antimyotonia Treatment in Myotonic Dystrophy Type 1 (DM1)

William B. Martens, Richard T. Moxley III, Eric L. Logigian, Michael McDermott, Charles A. Thornton, Allen W. Wiegner, Boston, MA, Richard T. Moxley IV, Cheryl A. Barbieri, Christine L. Blood, Nuran Dilek, Rochester, NY

OBJECTIVE: To determine if mexiletine is safe and effective in reducing myotonia in DM1. BACKGROUND: Myotonia is an early, prominent, symptom in DM1 and contributes to decreased dexterity, gait instability, difficulty with speech/swallowing, and muscle pain. A few nonrandomized trials have suggested that the antiarrhythmic, mexiletine, is useful treatment for both nondystrophic myotonic disorders and DM1. DESIGN/METHODS: We performed two 7 week, double-blind, randomized, crossover trials, each having 20 ambulatory DM1 patients. All patients had delayed relaxation of grip and thenar percussion myotonia. The initial trial [mean age=45.8 yrs; range of CTG repeat size 169-885] compared placebo to 150 mg of mexiletine three times daily. The second, subsequent, higher-dose, trial [mean age=42.6 yrs; range of CTG repeat size 169-1731] compared placebo to 200 mg three times daily. The primary end point for quantitating myotonia was a computerized measurement of the time for the grip force to relax after a 3 second maximum isometric grip contraction from 90%-5% & 50%-5% of maximum. Other measures of myotonia included: electrically evoked myotonia of the first dorsal interosseous muscle; time to make 10 repeated isotonic grip contractions at 75% of maximum; and, the time required to swallow 100 ml of water. RESULTS: There was a significant (p<0.05) reduction in isometric grip relaxation time with both 150 and 200 mg doses of mexiletine. The greatest delay in relaxation and the greatest response to treatment occurred in the late phase of relaxation [50%-5% of maximum grip force]. Time to perform 10 repeated isotonic contractions, eg myotonia, decreased with 200 mg but not with 150 mg doses of mexiletine. No significant reduction in the time to swallow 100 ml of water occurred with 150 or 200 mg doses, nor was there a change in whole body strength using manual muscle testing or quantitative isometric myometry. Side effects were minimal for 150 and 200 mg doses of mexiletine [1 dropped out of the 150 mg and 1 from the 200 mg study from intolerable side effects]. No electrocardiographic changes occurred in serial recordings made during each of these studies. No worsening of first-degree heart block or bundle branch block occurred in patients having these alterations. CONCLUSIONS: Mexiletine at doses of 150 and 200 mg three times daily is well tolerated, safe, and effective as an antimyotonia treatment in patients with DM1. Longer term trials are necessary: a) to determine if mexiletine reduces the frequency and severity of muscle pain; and, b) if mexiletine exerts a beneficial effect in maintaining muscle strength and function.

Aberrant Splicing of Ryanodine Receptor and SERCA in Myotonic Dystrophy Type1

Masayuki Nakamori, Takashi Kimura, Canberra, ACT, Australia, Masanori P. Takahashi, Futoshi Aoike, Saburo Sakoda, Suita, Osaka, Japan

OBJECTIVE: To investigate splicing abnormalities of ryanodine receptor (RyR) and sarco/endplasmic reticulum Ca²⁺-ATPase (SERCA) in muscle from myotonic dystrophy type1 (DM1). BACKGROUND: In DM1, it is proposed that impaired function and localization of alternative splicing regulators contribute to its pathogenesis, and misregulated alternative splicing has been demonstrated for several mRNAs including cardiac troponin T, insulin receptor, and muscle chloride channel. In all cases, the normal developmental splicing switch is disrupted, resulting in expression of fetal or non-muscle isoforms that are inappropriate for adult muscle. Furthermore, the disturbance of intracellular calcium (Ca²⁺) homeostasis has also been suggested in DM1 muscle. The mRNA splicing of two major sarcoplasmic reticulum (SR) proteins, RyR1 and SERCA1, which play central role for intracellular Ca²⁺ homeostasis in skeletal muscle, have been demonstrated to be under developmental regulation. DESIGN/METHODS: We used 26 human skeletal muscle specimens (10 from DM1, 5 from ALS, 5 from polymyositis, and 6 from normal control). Using RT-PCR, we quantified the total amount of mRNA for RyR1 and RyR3, a predominant form in adult skeletal muscle and transiently expressed form in immature muscle, respectively. Total amount of SERCA1, SERCA2a, and SERCA2b, expressed in fast-twitch muscle, slow-twitch and cardiac muscle, and ubiquitous tissue respectively, were also quantified. The splicing manner of RyR1, SERCA1, SERCA2a and SERCA2b, regulated developmentally and/or tissue specifically, were compared between DM and non-DM muscle. RESULTS: In DM1, we found a significant existense of alternative spliced isoforms of RyR1 and SERCA1 which are normally expressed in immature skeletal muscle, but not in non-DM muscle. Although SERCA2a splice pattern was not changed, a novel SERCA2b splicing variant, which retained intron 19, was significantly decreased in DM1. The amount of mRNA for RyR1, RyR3, SERCA1, SERCA2a and total SERCA2 did not differ significantly. CONCLUSIONS: Alternative splicing of the RyR1 and SERCA1 pre-mRNA is aberrantly regulated in DM1 skeletal muscle. Especially, the increase of immature isoforms in DM1 indicates dysregulation of developmental splicing switch. Since RyR release Ca²⁺ from SR and SERCA reuptake Ca²⁺ into SR, the aberrant isoforms may be responsible for disruption of the intracellular Ca²⁺ homeostasis and resultantly for muscle degeneration in DM1. Supported by: Grants-in Aid from the Japan Society for the Promotion of Science (MPT) and Research Grant (14B-4) from the Ministry of Health, Labour and Welfare, Japan (SS)
 

Facioscapulohumeral Muscular Dystrophy

Myoblasts from Affected and Unaffected Muscles of Facioscapulohumeral Muscular Dystrophy (FSHD) Patients Display Differences in Morphology, Proliferation and In Vitro Differentiation Ability

Sabrina Sacconi, Jean Thomas Vilquin, Jean Pierre Marolleau, Jerome Larghero, Paris, France, Claude Desnuelle, Nice, France

OBJECTIVE: To assess the biological properties of myogenic cells prepared from unaffected muscles of FSHD patients and compared them with that of FSHD affected muscles and matched control myoblasts in the perspective of an autologous myoblasts transfer clinical trial. BACKGROUND: Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant disease linked to a deletion within tandem array of repeats termed D4Z4 located on chromosome 4q. It is characterized by a typical regional distribution, featuring composed pattern of affected and unaffected muscles. No treatment is currently available for this disease which physiopathological mechanism is still unknown. Autologous myoblast transfer from unaffected to affected territories could be considered in view of increasing the regenerating ability of affected muscles. DESIGN/METHODS: We produced 1) myoblasts from unaffected vastus lateralis muscle of 5 FSHD patients and myoblasts from the same muscles of 10 healthy controls; 2) myoblasts from affected shoulder girdle muscles of 4 FSHD patients and myoblasts from the same muscles of 4 healthy controls.
We evaluated in each FSHD cell culture at different time points: morphology, proliferation ability (proliferation rate, doubling time), purity of the cell culture (percentage of CD56 and desmin-positive cells), cell viability, telomere length and in vitro differentiation (myoblasts fusion index, expression of myosin heavy chain isoformes). RESULTS: Myoblasts prepared from unaffected muscles of FSHD patients presented no differences in morphology, proliferation ability and in vitro differentiation when compared to matched controls while myoblasts from affected muscles displayed several alterations. In particular, we could observe in these cells the appearance of “vacuolar-necrotic phenotype”, reduced proliferation ability with a significant increase on cell doubling time and an impaired differentiation with an important reduction in myoblast fusion index.
Interestingly, both affected and unaffected FSHD myoblasts showed normal telomere length at different passages. CONCLUSIONS: In contrast with myoblasts from affected FSHD muscle, myoblasts from unaffected territories presented no alteration in morphology, proliferation and differentiation properties when compared to controls. These findings could open the possibility of a future clinical trial on autologous myoblast transfer for FSHD patients. Moreover, the use of pure myoblasts culture model of affected and unaffected FSHD muscles will offer several advantages in claryfing the physiopathological mechanisms of this disease. Supported by: French Association Against Myopathies (AFM)
 

Oculopharyngeal Muscular Dystrophy

Electrophysiologic Findings in Patients with Oculopharyngeal Muscular Dystrophy

Lyell K. Jones, Jr., C. Michel Harper, Rochester, MN

OBJECTIVE: To characterize the electrophysiologic (EMG) findings in patients with oculopharyngeal muscular dystrophy (OPMD). BACKGROUND: OPMD is an inherited myopathy clinically characterized by onset in adulthood of ptosis and dysphagia, often accompanied by extraocular and limb muscle weakness. The phenotype may resemble presentations of different forms of neuromuscular disease, particularly disorders of neuromuscular transmission and other myopathies. Although the clinical, and more recently the genetic, features of OPMD have been well described, there remain questions regarding a possible coexistent neuropathy with this disease. There has not been a dedicated review of the electrophysiologic features of OPMD. DESIGN/METHODS: Following Institutional Review Board approval, the Mayo Clinic patient database was reviewed for patients with a diagnosis of OPMD. All patients who had 1) either a) genetically confirmed OPMD or b) the accepted, hallmark clinical features of OPMD and typical findings on muscle biopsy, 2) been evaluated by a Mayo Clinic neurologist, and 3) had an EMG performed at the Mayo Clinic, were included for analysis. RESULTS: Nine patients met the above criteria. The mean age of onset of symptoms was 51.0 years (range, 36-60 years). The mean age at the time of the EMG was 62.7 years (51-78 years). Clinical findings included ptosis in 9 (100%), dysphagia in 9 (100%), limb weakness in 9 (100%), ophthalmoparesis in 6 (66.7%), and distal paresthesias in 1 (11.1%). All patients had abnormal needle EMG findings. Six out 6 patients had small, rapidly recruited motor unit potentials in bulbar musculature. Nine out of 9 patients (100%) had small, complex, rapidly-recruited motor unit potentials in proximal limb muscles, and 8 of 9 (88.9%) had similar findings in distal muscles. Two of 5 patients had fibrillation potentials in bulbar muscles, and 9 of 9 patients had fibrillation potentials in at least one limb muscle, most commonly in the tibialis anterior (6 of 9, 67.7%). Seven of the 9 patients had normal repetitive stimulation studies. Six of the 9 patients, including the patient with the complaint of distal paresthesias, had distal lower extremity sensory studies; these were all normal (average sural SNAP amplitude of 13.2 μV, range 7 to 20 μV). CONCLUSIONS: All patients with OPMD in this series had an abnormal EMG. The number of patients with limb weakness was higher than in other series of OPMD patients; this may reflect a referral bias. The needle EMG findings were typical of other myopathies in that all had evidence of small, complex, rapidly-recruited motor unit potentials in most muscles examined. Fibrillation potentials were present in at least one muscle in every patient. These findings reinforce the utility of EMG in distinguishing OPMD from defects of neuromuscular transmission, and the pattern of findings may help direct more specific testing for myopathic patients in whom the diagnosis is unclear. None of these patients had findings suggestive of a length-dependent peripheral neuropathy, arguing against a coexisting neuropathic process in patients with OPMD.
 

Ullrich's Congenital Muscular Dystrophy

Ullrich's Congenital Muscular Dystrophy: An Analysis of 29 Families

Goknur Haliloglu, Ercan Demir, Trabzon, Turkey, Beril Talim, Nesrin Senbil, Melda Caglar, Betti Giusti, Guglielmina Pepe, Florence, Italy, Pascale Guicheney, Paris, France, Haluk Topaloglu, Ankara, Turkey

OBJECTIVE: The aim of our study is to evaluate the clinical spectrum, morphological, genetic and collagen VI involvement in our patients with an UCMD phenotype. BACKGROUND: Ullrich's congenital muscular dystrophy (UCMD) is a severe form of autosomal recessive congenital muscular dystrophy characterized by proximal contractures and distal laxity. Neonatal hypotonia, congenital arthrogryposis, torticollis and hip dislocation, a round myopathic face with high-arched palate and low set prominent ears, protruded calcaneus, increased subcutaneous fat tissue on the soles of the feet, skin features such as protruded bulbs of hair follicles and extensive cheloid formation and normal intelligence are other additional key features. Recessive and dominant mutations in the COL6A1, COL6A2 (21q22.3) and COL6A3 (2q37) genes encoding α1, α2 and α3 chains of collagen VI genes have been identified in patients with UCMD. DESIGN/METHODS: We analyzed 32 patients (15 girls, 17 boys) from 29 families with an UCMD phenotype. Collagen VI involvement is evaluated by immunohistochemical analysis in the muscle tissue and/or skin fibroblast cultures and/or genetic analysis. RESULTS: Mean age at the time of diagnosis was 7.7 y (1-13 y). Consanguinity was known in 22 of the families. Symptoms began at birth or infancy (n= 21), including neonatal hypotonia, congenital hip dislocation (n= 21), torticollis (n= 7) and arthrogryposis (n= 5). There is generalized muscle weakness with varying degrees of contractures in all patients. Additional spinal deformities are kyphoscoliosis (n=7) and rigid spine (n=13). Course is slowly progressive. Collagen VI abnormalities as total or partial reduction of collagen VI was found in 23 of the 29 families (79%). The haplotype analysis suggested linkage to COL6A1/COL6A2 locus in 7 of the families and COL6A3 locus in 3 of the families. One of the patients linked to COL6A3 locus had a homozygous nonsense mutation. In one of our patients linked to COL6A1/COL6A2 locus, although there was a positive immunostaining for collagen VI in the muscle tissue, fibroblast culture revealed absence of collagen VI expression. Age and presentation at onset, severity of weakness and contractures, frequency of ambulant and nonambulant patients were similar in patients with and without collagen VI involvement. Prenatal diagnosis was provided to two families with chorion-villus sampling and haplotype analysis. CONCLUSIONS: The diagnosis of UCMD depends on clinical grounds. 79% of the UCMD patients in this series had collagen VI involvement indicating genetic heterogeneity of the disease. The clinical features of the patients with collagen VI involvement are very similar to those ones without collagen VI involvement. Absence of collagen VI expression in fibroblast culture in a patient with normal expression in muscle may indicate that collagen VI localization can be normal although there may be loss of function. There may be other responsible proteins in this disorder. Supported by: Association Française contre les Myopathies (AFM), France and Telethon (Italy)
 

Limb Girdle Muscular Dystrophy

Mutational and Clinical Features of French Patients with Limb Girdle Muscular Dystrophy Type 2I (LGMD2I)

Helene Bourteel, Tanya Stojkovic, Jean-Marie Cuisset, Claude-Alain Maurage, Pascale Richard, Pascal Laforet, Paris, France, Patrick Vermersch, Lille, France

OBJECTIVE: To evaluate clinical, biological, and genetic characteristics of LGMD2I patients. BACKGROUND: Mutations of the fukutin-related protein (FKRP) gene cause LGMD2I.This pathology is associated with wide range of clinical severity correlated with a molecular heterogeneity. DESIGN/METHODS: We recruited 8 patients in 6 families from north of France, affected by LGMD2I. We reported for all patients : the sex ratio, the age at onset, the neurological examination, the evaluation of the cardiac and respiratory function, serum creatine kinase (CK) levels, the muscle biopsy with α-dystroglycan immunolabeling. Genetic analysis by direct sequencing of the FKRP gene was performed. RESULTS: We examinated four females and four males. The mean age was 31.7 +/- 17.9 years; the mean age at onset was 11.7 +/-8.1 years. Six patients had calves hypertrophy, five suffered from scoliosis or lumbar lordosis. Four presented symmetrical prominent scapular winging. There was no cranial nerve impairment. Except for a four years old patient, all showed progressive limb girdle weakness leading for four of them at the age of 25.7 +/-6.39 years to wheelchair dependence. Four females versus one male were affected by restrictive respiratory failure with difficulty of swallowing in one case. Three of them required nocturnal ventilation, one tracheotomy. Two males presented dilated myocardiopathy with left ventricular dysfunction . CK were always elevated (range: 1000-8865 IU/L). The histochemical analysis of muscle biopsy was characteristic of muscular dystrophy. Immunolabeling for α-dystroglycan was always reduced but never absent. We reported a homozygous L276I mutation in three cases. This homozygous mutation was found to be associated to three novel mutations in five other cases. One brother and his sister, both carriers of a L489R heterozygous mutation in addition to the L276I homozygous mutation, had different phenotype characterized by cardiac dysfunction for the brother and respiratory insufficiency and whellchair dependence for the sister. This identical phenotypic distribution, respectively for a brother and sister of another family was observed, and corresponded to a novel L322V heterozygous mutation associated with the L276I heterozygous mutation. Finally, a R275G heterozygous mutation with the L276I heterozygous mutation was discovered for the child, who presents only muscle pain. CONCLUSIONS: We observed various phenotypic expressions of LGMD2I from moderate to severe forms and intrafamilial phenotypic variability. These could be influenced by the gender. Indeed, we saw more frequently cardiac impairment in males and respiratory failure in females, especially into members of family carrying the same mutation. As expected, genetic analysis of the FKRP gene revealed the preponderant frequency of the L276I mutation, found in all patients. Three novels mutations not yet described were identified (L322V, R489L, R275G) in addition to the L276I homozygous or heterozygous mutation without any particular clinical pattern.
 

A New Diagnostic Approach to Molecular Diagnosis of LGMD 2A/Calpainopathy

Corrado Angelini, Marina Fanin, Annachiara Nascimbeni, Luigi Fulizio, Marco Spinazzi, Padova, Italy

OBJECTIVE: To improve detection rate of calpainopathy using a combination of protein (quantitative and functional assays) and molecular tests. BACKGROUND: LGMD2A is an autosomal recessive muscular dystrophy, which shows a wide phenotypic variability: the Erb scapulohumeral phenotype presents with onset in upper girdle, the classical LGMD phenotype has early (before age 12) or late (after age 30) pelvifemoral onset. While numerous gene mutations were described, the diagnosis has shifted from molecular genetic analysis towards protein biochemical assay by immunoblot. This latter approach is still a challenge, since using only protein studies may yield both false positive (secondary defects) and false negative results (normal protein with defective enzyme activity). On the other hand, if only mutation screening in CAPN3 gene is conducted in LGMD patients a low success rate is expected due to genetic heterogeneity. DESIGN/METHODS: By preliminary immunoblot in over 550 unclassified patients with various phenotypes (early and late-onset LGMD, Erb's dystrophy, hyperCKemia) we found 74 cases with calpain protein defect. In these patients mutation screening was done by SSCP, DHPLC, ARMS-PCR and sequencing. Patients showing normal amount of calpain-3 protein by immunoblot underwent a novel functional assay to test calpain-3 autocatalytic activity. The loss of this normal function suggested primary LGMD2A; in such cases the screening of gene mutations was conducted only by ARMS-PCR for known mutations and by SSCP to limit the cost and time effort. RESULTS: By preliminary immunoblot screening of calpain-3 protein defect, we identified 74 cases showing complete or partial protein defect. In patients with complete protein defect (early onset LGMD phenotype was common) we identified gene mutations in 86% of cases. In patients with partial protein defect we identified gene mutations in 50% of cases. In 140 patients showing normal protein amount, the assay for calpain-3 autocatalytic function allowed the identification of additional 11 cases, in whom mutations have been identified in 100% of cases. We demonstrate that when a defect of either calpain-3 protein amount or its autocatalytic activity is present, the overall probability of identifying gene mutations is 75%. We also show that patients with both normal calpain-3 protein amount and normal autocatalytic activity have a residual 5% probaility of yielding mutations in CAPN3 gene . CONCLUSIONS: This novel diagnostic approach consists of a combination of protein quantitative and functional assays and molecular screening methods. We show that this strategy allows molecular diagnosis in a large number of LGMD2A patients, sometimes in an early stage of the disease (asymtomatic hyperCKemia). The amount of calpain-3 protein appears to have a relative prognostic value: patients with early onset have absent protein, while almost all patients with normal protein levels have late or adult-onset muscular dystrophy. Supported by: Telethon Italy grant numbers GUP030516, GTF02009, EuroBioBank network QLRT2001-027769.
 

Phenotypic Marker Variability in Calpainopathy Patients of North America

Christopher J. Shilling, Daniel Darvish, Encino, CA, Steven A. Moore, Iowa City, IA, John T. Kissel, Wendy King, Xiomara Q. Rosales, Cheryl Wall, Jerry R. Mendell, Columbus, OH

OBJECTIVE: Establish phenotypic markers for calpainopathies (limb-girdle muscular dystrophy type 2A) in an outbred population of subjects. BACKGROUND: The diagnosis of specific LGMD subtypes requires a systematic approach starting with phenotypic features including the neuromuscular exam and muscle biopsy, followed by DNA confirmation of calpain-3 (CAPN3) gene mutations. In the inbred La Reunion population where LGMD2A was first described, stereotypic features included contractures at heel cords, hips, elbows, and fingers, proximal and distal weakness with scapular winging and sparing of the hip abductors, and a protuberant abdomen. Considering the broad differential diagnosis of LGMD and the necessity of western blotting for the detection of calpain-3 deficiency, we evaluated the clinical features of an outbred North American cohort of LGMD patients to identify strong indicators of LGMD2A. DESIGN/METHODS: Two hundred fifty unrelated LGMD patients were screened using clinical features, serum creatine kinase (CK), electrocardiogram, muscle biopsy analysis (immune staining, western blots) and CAPN3 mutational analysis. RESULTS: From this outbred population of unclassified LGMD patients sixteen (16) percent were found to have a calpain-3 deficiency by western blots. Of those deficient in calpain-3, DNA analysis showed potential mutations in up to 15 patients correlating to LGMD2A (78.9%). The phenotypic features in this group were found to be heterogeneous as follows: age of onset ranged from 7 to 42 years old (mean 17.2 +/- 9.6 years), CK from 152 to over 7000 (mean 1500 +/- 1700), average muscle strength on 10 point scale ranged from 8.1 to 4.0 (mean 6.6 +/- 1.3), three had scapular winging, one patient with ocular muscle weakness, and two with facial weakness. Distal lower limb weakness was common (approximately 50% with a score of 7 or below). Protuberant abdomen was infrequent. Contractures, a major feature of the La Reunion population, were seen in approximately 50% of the identified population. CONCLUSIONS: Our large outbred population of LGMD patients offers unique opportunities to test the hypothesis that particular LGMD subtypes present with stereotypic phenotypic features strongly indicative of the diagnosis. We examined the LGMD2A patients, one of the most common LGMD disorders in North America, for such features. The analysis failed to pinpoint specific surrogate markers for the disease, although elbow and ankle contractures had a frequency of approximately 50%. Thus, the diagnosis of LGMD2A remains arduous, by first excluding other disorders by employing immune stains of muscle sections. Negative biopsies, especially in patients with limb contractures, then require western blots to demonstrate calpain-3 deficiency. The final step remains confirmation of CAPN3 mutations. Supported by: The Muscular Dystrophy Association

General

Muscle Biopsy: Diagnostic Utility in Children Less Than Two Years

Deepa Arun, Alan Pestronk, Anne M. Connolly, St. Louis, MO

OBJECTIVE: To determine the diagnostic utility of muscle biopsy in children under two years of age. BACKGROUND: Timing of muscle biopsies in infants and young children is debated. Some centers biopsy children at the time of their presentation with weakness, while others follow them clinically for variable periods of time. The yield of an early biopsy in making a specific diagnosis remains uncertain. DESIGN/METHODS: We retrospectively reviewed 318 muscle biopsies (performed between 1980-2004) from children under two years (Mean 10m +/- 7m). These were divided into age-related groups 0-3 months (n=79), 3-12 months (n=113) and 12-24 months (n=126). We then determined biopsies to be diagnostic, non-diagnostic, or normal. RESULTS: In the 0-3 month group, 54% were diagnostic. These included congenital myopathies (19%), denervation (15%), and miscellaneous (20%) including congenital muscular dystrophy, mitochondrial myopathy, storage disorders, perifascicular myopathy and congenital fiber type disproportion. Non-diagnostic biopsies (29%) all showed abnormal fiber size variation (FSV). 17% were normal. In the 3-12 month group, 49% were diagnostic (14% denervation, 11% congenital myopathy, 4% congenital muscular dystrophy, 4% Duchenne dystrophy and 16% miscellaneous diagnoses). Non-diagnostic biopsies (41%) all showed FSV. 10% were normal. In the 12-24 month group, 44% were diagnostic (16% denervation, 13% muscular dystrophy, 5% congenital myopathy and 10% miscellaneous diagnoses). Non-diagnostic biopsies (46%) again showed FSV. 10% were normal. Children biopsied at less than 3 months of age were more likely to have a specific diagnosis when compared to children age 12-24 months (p=0.04). There were no differences in the ratios of specific diagnoses comparing 0-3 months to 3-12 months (p=0.18) or 3-12 months to 12-24 months (p=0.43). CONCLUSIONS: Overall, 47% of biopsies yielded specific diagnoses with prognostic, treatment, or genetic implications. Infants less than 3 months were more likely to have a specific diagnosis made compared to 12-24 month old children. FSV was the most common finding in all three age groups. Biopsies with FSV included those with small type 1, type 2 fibers or both. As the pathogenesis of FSV becomes clearer, the usefulness of this abnormality may increase. Our study supports the utility of biopsy at the time of presentation with weakness.

Dysferlin Deficiency

Altered Expression of Caveolin-3 in Muscles of Patients with Dysferlin Deficiency

Dubravka M. Dodig, Christian Therrien, Maria Y. Molnar, George Karpati, Michael Sinnreich, Montreal, QC, Canada

OBJECTIVE: To investigate the expression profile of caveolin-3 in muscles of dysferlin deficient patients. BACKGROUND: Caveolin-3 is a muscle specific surface membrane protein implicated in the formation of caveolae by acting as a scaffold to organize and concentrate lipid and protein building blocks at the sarcolemma. Mutations in caveolin-3 have been reported in limb girdle muscular dystrophy type 1C and in Rippling Muscle Disease. Dysferlin is a sarcolemmal protein involved in muscle surface membrane repair. It is deficient or defective in patients with limb girdle muscular dystrophy type 2B and Miyoshi Myopathy. Caveolin-3 was reported to interact with dysferlin in muscle homogenates in vitro, and isolated reports suggested a caveolin-3 reduction in patients with dysferlinopathies. However, 4 lines of dysferlin deficient mice failed to show reduction of caveolin-3 in skeletal muscle. Larger studies of caveolin-3 expression in human dysferlinopathies are lacking. DESIGN/METHODS: We screened 167 muscle biopsies from patients with a clinical phenotype of LGMD or distal myopathy for dysferlin deficiency by Western blotting. Skeletal muscle biopsies from identified dysferlin deficient patients were investigated for expression of caveolin-3 by immunohistochemistry. Mutational analysis was performed for dysferlin and caveolin-3. RESULTS: We identified 17 dysferlin deficient patients from 13 different families. Immunohistochemical analysis for caveolin-3 showed evenly reduced sarcolemmal staining in muscle fibers of 11 dysferlin deficient muscle biopsies when compared to normal control muscles and muscles of patients with dystrophies unrelated to dysferlin deficiency. Fibers with reduced caveolin-3 immunoreactivity were scattered throughout the muscle. Mutational analysis confirmed primary dysferlin deficiency in all cases. Caveolin mutational analysis is in progress.. CONCLUSIONS: In 11 out of 17 dysferlin deficient patients, immunohistochemical analysis of muscle revealed scattered fibers with reduced caveolin-3 immunoreactivity. These results in a relatively large series of dysferlin deficient patients support the isolated observations of caveolin-3 reduction in human dysferlinopathies and suggest that dysferlin and caveolin-3 may form molecular partnership in human skeletal muscle. This finding contrasts with observations in dysferlin deficient mice.
 

Phenotypic and Genetic Analysis in a Series of 55 Patients with Dysferlinopathy

Karine Nguyen, Guillaume Bassez, Bruno Eymard, Paris, France, Nicolas Levy, Jean Pouget, Marseille, France

OBJECTIVE: to study the phenotypic spectrum of dysferlinopathies and to identify mutations in the dysferlin gene (DYSF). BACKGROUND: Dysferlinopathies are autosomal recessive muscular dystrophies caused by mutations in DYSF and can present with two main phenotypes: a distal myopathy termed Miyoshi myopathy (MM), and a limb-girdle muscular dystrophy (LGMD2B). DESIGN/METHODS: Clinical, biological and pathological features in 55 patients with dysferlinopathy were analyzed, whereby the diagnosis was based on the absence or drastic decrease in dysferlin expression on muscle biopsy. Genetic analysis using SSCP (or dHPLC) screening of PCR products of each of 55 exons of the DYSF gene followed by sequencing was performed. RESULTS: Besides classical phenotypes of MM and LGMD2B, in 20 (37%), and 12 (22%) cases respectively, the authors identified unusual clinical findings including: 14 patients (26%) presenting with lower limb "proximodistal" (PD) weakness at onset, exercise intolerance (n=2), and asymptomatic hyperCKaemia at age 58 years (n=1). 15 patients (27%) had been misdiagnosed as polymyositis, and patients with histological features of inflammation tended to present with a PD phenotype and a more severe course of the disease. Gene analysis revealed mutations in the DYSF gene in 30 out of the 55 patients. CONCLUSIONS: The phenotypic spectrum of dysferlinopathy is wider than expected, ranging from asymptomatic hyperCKaemia in late adulthood to a rapidly disabling myopathy. A large proportion of patients have a "proximodistal" phenotype, which might correlate with muscle inflammation and increased severity.
 

Validation of a Time and Cost-Effective RNA Based Approach in Detecting Human Dysferlin Gene Mutations

Christian Therrien, Dubravka Dodig, George Karpati, Michael Sinnreich, Montreal, QC, Canada

OBJECTIVE: To validate an RNA-based gene analysis approach in detecting human dysferlin gene mutations. BACKGROUND: Dysferlin is the protein product of the gene that is defective in patients with limb girdle muscular dystrophy type 2 B and Miyoshi Myopathy. The gene is composed of 55 exons and the corresponding mature transcript encodes a multidomain molecule consisting of 2080 amino acids. Analysis of dysferlin gene mutations based on genomic DNA sequencing is cumbersome and expensive, and therefore not readily available. Additionally, exon-skipping mutations evade detection using a DNA based approach. We used an RNA based approach permitting improved and fast detection of dysferlin gene mutations in patients who had undergone a skeletal muscle biopsy. DESIGN/METHODS: Mutational analysis was performed on mRNA extracted from skeletal muscle biopsies from twelve unrelated patients with dysferlin deficiency. Dysferlin deficiency was established by Western blot analysis of skeletal muscle homogenates. Direct sequencing of the coding region of the dysferlin gene was performed using nine overlapping amplicons obtained by RT-PCR. Mutations were verified by sequencing of PCR products of corresponding genomic DNA fragments. Muscle sections were stored in a commercially available buffer for up to two weeks at room temperature and assessed for RNA integrity at different time intervals. RESULTS: Mutational screening by direct sequencing of mRNA derived amplicons revealed novel mutations throughout the dysferlin gene. In our 12 patients, the majority of mutations were in the C-terminal part of the protein. In three families, large deletions of cDNA fragments were observed, likely due exon skipping. RNA from muscle sections which were stored in a commercially available buffer for up to two weeks at room temperature allowed the generation of expected cDNA fragments, indicating that muscle tissue stored this way may be suitable for shipping. CONCLUSIONS: Determination of a particular mutation in any inherited disease is of value for a precise diagnosis and for pre-implantation or prenatal screening. This holds true also for dysferlinopathies. Moreover, new dysferlin gene mutations and their correlation with the clinical phenotype will enhance our understanding of the biology of dysferlin. RNA based mutational analysis is superior to a DNA based approach in detecting dysferlin gene mutations for two reasons: 1. It will identify any mutation in the coding region of the entire gene as well as potential exon skipping mutations which may go undetected on a DNA based approach. 2. It is time and cost efficient, as only nine amplicons need to be sequenced for a dysferlin gene analysis, in contrast to 55 exons, if a DNA-based approach with the same stringency was chosen. As most LGMD patients undergo a muscle biopsy, tissue is readily available and can be easily shipped for RNA-based gene analysis in a commercially available buffer without the need for cooling. This approach should facilitate gene analysis for large proteins encoded by multiple exons implicated in limb girdle muscular dystrophies.
 

Tamoxifen in ALS

Phase 2B Randomized Dose-Ranging Clinical Trial of Tamoxifen, a Non-Steroidal Tri-Phenyl-Ethylene Selective Estrogen Receptor Modulator [SERM], in Amyotrophic Lateral Sclerosis[ALS]: Increased Survival in 20-30-40mg Daily Compared with 10mg Weekly-10mg Daily Treatment Cohorts

Benjamin Rix Brooks, Mohammed Sanjak, Kathryn A. Roelke, Jennifer M. Parnell, Shirley M. Peper, Ann M. Houdek, David L. Lunney, Aaron P. Frye, H. Ian Robins, Andrew J. Waclawik, Madison, WI

OBJECTIVE: Determine if tamoxifen has a clinical survival benefit in ALS similar to its benefit in murine retrovirus-induced motor neuron disease. BACKGROUND: ALS patients were assigned to blocks [N=5] matched by age, sex, ALS onset site and baseline ALS Functional Rating Scale-total score[ALSFRSt]. Within each block, patients were randomized to 1 of 5 tamoxifen doses. Planned as a 12 month clinical trial, it was extended to 24 months when there was no statistically significant change in endpoints across the tamoxifen dose cohorts at 12 months. DESIGN/METHODS: ALS patients [N=60] were randomized within blocks. Evaluations were at 3 month intervals with assessment of adverse effects, serious adverse events, clinical measurements and survival over 24 months followup. Survival and event history data for achieving milestones in isometric muscle strength, vital capacity and ALSFRSt endpoints were analyzed by intention-to-treat allocation employing the Kaplan-Meier method with log-rank test. RESULTS: ALS patients entered [34 M; 26 F] had a mean age [51 yr ], ALSFRSt [ 24 ] with no significant difference in these parameters among the five dose groups at baseline. There was a statistically significant difference between survival of ALS patients in the10mg weekly vs the 40mg daily cohorts [p <0.05 one-sided safety predetermined p value]. Per protocol analysis of the difference in survival for the two lower dose cohorts together vs the three upper cohorts together was signficant [p<0.01 one-sided safety predetermined p value; p<0.03 two-sided safety predetermined p value]. Cox proportional hazard analyses supported a drug dose effect. Prolongation of 80% survival benefit was above 200 days in the 20mg, 30mg, 40mg daily tamoxifen treatment cohorts. Survival in the 10mg weekly-10mg daily cohorts was identical to that recorded for the ALS patients randomized to placebo in the Dutch ALS Creatine Study [Ann Neurol 2003;53(4):437-445]. No significant dose-related adverse events occurred throughout the clinical trial. CONCLUSIONS: Safety analysis in a single-site phase 2B dose-ranging clinical trial of tamoxifen in ALS patients indicates a survival benefit of tamoxifen treatment with 20-30-40mg daily compared with 10mg weekly-10mg daily. This survival benefit occurred in riluzole-treated ALS patients and was as large or larger than the improved survival in riluzole-treated compared with placebo-treated ALS patients. This observation needs to be replicated in an appropriately statistically powered clinical trial to establish whether tamoxifen should be considered as an adjuvant therapy with riluzole in the treatment of ALS. Supported by: Muscular Dystrophy Assocation - ALS Division and National Institute of General Medical Sciences General Clinical Research Center. Tamoxifen was provided by Astra-Zeneca