Annual Meeting of American Society of Gene

Annual Meeting of American Academy of Neurology - April 26 2014

Agomelatine Increases Muscle Strength And Reduces The Expression Of Inflammatory Cytokines In mdx Dystrophic Mice

Alzira AS Carvalho,1Vinicius A. Tondato,1Larissa M. Iamnhuk,2Fernanda PR Gomiero,2Giuliana Petri,1Pamela Delgado,2Beatriz Alves,2Fernando Luiz Affonso Fonseca,1David Feder1
1Santo Andre, Brazil, 2Sao Paulo, Brazil

OBJECTIVE: Based on the antioxidant and anti-inflammatory properties of melatonin in DMD patients we decided to study agomelatine action in mdx mouse.
BACKGROUND: The new antidepressant agomelatine is an agonist of melatonergic MT₁/ MT₂ receptors as well as an antagonist of serotonergic 5-HT2C receptors. Among the pathogenic mechanisms proposed for DMD, oxidative stress and inflammation are directly involved in the dystrophic process.
DESIGN/METHODS: The study was conducted in male mdx with life 69.5 days (average of 31-114 days). The animals were maintained with food and water ad libitum. They were divided into two groups: Control (n = 22), agomelatine (n = 22). The first group received saline by gavage and the second one received agomelatine 30 mg /kg / day by gavage. The animals underwent treadmill exercise motorized 5 times / week, 18 cm / s for 10 min. Muscle strength was measured weekly. After five weeks, the diaphragm muscle was removed. Gene expression of TGF beta 1, TNF, and osteopontin were performed in muscle by real time PCR.
RESULTS: Agomelatine's group had a significant increase in muscle strength after 5 weeks treatment. The values of the measured cytokines were as follows:TGF beta1: Control (n=16)/Agomelatine (n=15):1,42 ±0,12/ 1,32 ± 0,17, p=0,05; TNF alpha: Control (n=16)/ Agomelatine(n=15): 1,46 ± 0,11; 1,36 ± 0,16,p=0,04; Osteopontine: Control (n=16)/ Agomelatine (n=15): 1,36 ± 0,11/1,26 ± 0,12, p=0.02.
CONCLUSIONS: The anti-inflammatory action of melatonin is accompanied by a reduction of proinflammatory cytokines such as IL-1b, IL-6, TNF-a and interferon (INF)-c in reducing oxidative stress and also increases IGF-I preventing muscle atrophy in mdx mice castrated. The study demonstrated that agomelatine reduce the expression of inflammatory cytokines and increase muscle strength in mdx mice, which could be an alternative option for the treatment of muscular dystrophy.

Results at 96 Weeks of a Phase IIb Extension Study of the Exon-Skipping Drug Eteplirsen in Patients with Duchenne Muscular Dystrophy (DMD)

Edward Kaye,1Jerry Mendell,2Louise Rodino-Klapac,2Zarife Sahenk,2Kandice Roush,2,2Loren Bird,2Linda Lowes,2,2Lindsay Alfano,2,2Ana Maria Gomez Ramirez,2Sarah Lewis,2,2Vinod Malik,2Kim Shontz,2,2Christopher Shilling,2,2Peter Sazani,1Jihad B. Saoud1
1Cambridge, MA, USA, 2Columbus, OH, USA

OBJECTIVE: The objective of this study is to establish long-term efficacy of eteplirsen treatment, targeted to skip exon 51.
BACKGROUND: DMD is a rare, degenerative, genetic disease that results in progressive muscle loss and premature death. Affecting 1 in 5000 male births, DMD is caused by the inability to produce the dystrophin protein. There are no approved drugs available to treat DMD. Eteplirsen is an investigational therapy designed to enable functional dystrophin production in boys who are amenable to exon 51-skipping therapy (~13%).
DESIGN/METHODS: Twelve boys aged 7-13 years with eligible genotypes were randomized 1:1:1 to 30 mg/kg, 50 mg/kg, or placebo. Upon completion of a 24-week double-blind, placebo-controlled study phase, all subjects were enrolled in an open-label extension and the placebo-treated subjects initiated eteplirsen treatment. The critical clinical endpoint was the change in 6-minute walk test (6MWT) distance from baseline compared to the placebo/delayed-treatment cohort.
RESULTS: After 96 weeks of treatment, a significant clinical benefit of 71 meters was observed (p≤0.001) on the 6MWT for ambulatory-evaluable subjects in the combined eteplirsen-treated cohorts (n=6) versus the placebo/delayed-treatment cohort (n=4). The eteplirsen-treated cohorts showed approximately 5% decline (19.3 meters) in walking ability from baseline. No clinically significant treatment-related adverse events were seen through 96 weeks.
CONCLUSIONS: Through 96 weeks of treatment, eteplirsen demonstrated a significant clinical benefit on the 6MWT with no clinically significant treatment-related adverse events. Data on these outcomes will be reported through 164 weeks of treatment.

Safety Profile and Pharmacokinetic Properties of Eteplirsen in the Treatment of Boys with Duchenne Muscular Dystrophy

Jihad B. Saoud,1,1Jerry Mendell,2Louise Rodino-Klapac,2Zarife Sahenk,2Kandice Roush,2,2Loren Bird,2Linda Lowes,2,2Lindsay Alfano,2Ana Maria Gomez Ramirez,2Sarah Lewis,2,2Vinod Malik,2Kim Shontz,2,2Christopher Shilling,2,2Peter Sazani,1Edward Kaye1
1Cambridge, MA, USA, 2Columbus, OH, USA

OBJECTIVE: The objective of this analysis is to examine the safety and pharmacokinetic profiles of eteplirsen, a PMO designed to skip exon 51 in patients with Duchenne Muscular Dystrophy (DMD).
BACKGROUND: Duchenne Muscular Dystrophy is a degenerative X-linked genetic neuromuscular disorder with an incidence of 1 in 5,000 boys caused by mutations to the gene coding for the protein dystrophin. Eteplirsen is an investigational therapy designed to enable functional dystrophin production in boys amenable to exon 51-skipping (~13%).
DESIGN/METHODS: Twelve boys, aged 7 to 13 were randomized 1:1:1 to 30mg/kg, 50mg/kg, or placebo/delayed treatment cohorts. Doses were administered weekly via IV infusion for 24 weeks, when all including placebo were rolled over to a long-term open-label extension study. Here we discuss the safety profile of eteplirsen through 96 weeks of treatment and touch upon its pharmacokinetic properties as determined through analysis of plasma and urine samples at weeks 12 and 24.
RESULTS: Patients experienced no clinically significant treatment related adverse events. Two cases of urine protein elevation resolved without intervention and one distal femur fracture was ruled as treatment unrelated. There were no hospitalizations or discontinuations and no clinically significant treatment related changes on any safety lab parameters including liver specific enzymes, kidney function, coagulation, or platelet counts.
Cmax and AUC increased proportionally with dose. The plasma half-life was approximately 3 hours long and therefore accumulation due to weekly dosing was not expected and was supported by the finding that the plasma concentration 5 minutes post-infusion at 12 and 24 weeks were similar. Approximately two thirds of the drug was cleared through the kidney.
CONCLUSIONS: Eteplirsen is generally well tolerated with no dose limiting toxicities observed at therapeutically relevant levels.

Baseline Performance on 6MWT Correlates with Other Functional Outcomes in a Phase IIb Study of Exon-Skipping Antisense Oligonucleotide Eteplirsen for Use in Duchenne Muscular Dystrophy (DMD)

Jihad B. Saoud,1Jerry Mendell,2Louise Rodino-Klapac,2Zarife Sahenk,2Kandice Roush,2Loren Bird,2Linda Lowes,2Lindsay Alfano,2Ana Maria Gomez Ramirez,2Sarah Lewis,2Vinod Malik,2Kim Shontz,2Christopher Shilling,2Peter Sazani,1Edward M. Kaye1
1Cambridge, MA, USA, 2Columbus, OH, USA

OBJECTIVE: The objective of this analysis is to evaluate the correlations at Baseline between patients' performance on the 6MWT and a variety of other functional measures.
BACKGROUND: Duchenne Muscular Dystrophy is a degenerative X-linked genetic neuromuscular disorder with an incidence of 1 in 5,000 boys caused by mutations to the gene coding for the protein dystrophin. Patients' qualifications for enrollment into clinical trials utilize several baseline functional measures before treatment initiation.
DESIGN/METHODS: Twelve boys, aged 7 to 13 were randomized 1:1:1 to weekly IV infusion with 30mg/kg, 50mg/kg, or placebo/delayed treatment cohorts. Pearson correlation while controlling for patients' age was used to evaluate the correlation between the maximum distance walked on the 6MWT administered at 2 consecutive days and the various functional outcomes (NSAA, rise time, 10-meter run time, timed 4-step test, FVC, FVC % predicted, MEP, and MIP) before treatment was initiated.
RESULTS: Maximum distance walked on the 6MWT was statistically significantly correlated with rise time (r=-0.69), 10-meter run time (r=-0.65), FVC (r=-0.76), and MEP (r=-0.71). Moderate correlations were observed with NSAA (r=0.57, p≤0.07) and MIP (r=-0.54, p≤0.09). Correlations with the remaining functional measures failed to reach statistical significance with the lowest correlation observed with FVC % predicted (r=-0.25) and the timed 4-step test (r=-0.47). When the same variables were evaluated by using the Spearman rho coefficient (ranked data), both the NSAA and timed 4-step test became statistically significantly correlated with maximum distance walked.
CONCLUSIONS: The high correlations evident among the various baseline measures suggest careful considerations to the patient selection criteria should be made at the time of designing study protocols. These findings suggest fewer criteria may be sufficient to qualify patients for clinical studies. Furthermore, and based on the mechanism of action of the experimental treatment, selecting a variety of uncorrelated measures for qualification may be warranted.

Human α7 Integrin Gene (ITGΑ7) Delivered By Adeno-Associated Virus Reverses The Phenotype Of The Double Knock Out (DKO) Mouse Devoid Of Dystrophin And Utrophin

Kristin N. Heller, Chrystal Montgomery, Kim Shontz, Paul Janssen, Reed Clark, Jerry Mendell, Louise Rodino-Klapac
Columbus, OH, USA

OBJECTIVE: Use human α7 integrin gene (ITGΑ7) delivered by adeno-associated virus (AAV) as an alternative strategy to treat Duchenne muscular dystrophy (DMD)
BACKGROUND: Dystrophin links extracellular matrix (ECM) proteins to the actin cytoskeleton. Limitations for DMD gene replacement therapy include the large size of the gene and immunogenic potential when mini-genes are expressed in deleted regions of patient's DMD gene (Mendell et al N Engl J Med 2010). α7 integrin is a laminin receptor in skeletal and cardiac muscle also linking the ECM to the actin skeleton. It is modestly upregulated in DMD and has been proposed to be an important modifier of dystrophy. rAAVrh.74.MCK.ITGΑ7 to the lower limb of mdx mice resulted in α7 overexpression at the sarcolemma and protected against loss of force following eccentric contraction-induced injury while specific force following tetanic contraction was unchanged (Heller et al. Mol Ther 2013).
DESIGN/METHODS:The present study further challenged rAAVrh.74.MCK.ITGA7 to improve the more severe DMD model: the mdx/utrn-/- dko. Vector was systemically delivered through the intraperitoneal injection compared to prior regional delivery studies in mdx. Outcome measures included functional studies of the diaphragm and EDL.
RESULTS:In the dko mouse, systemic delivery resulted in α7 expression in multiple muscle groups including the diaphragm, and upper and lower limb muscles. Most importantly ITGA7 significantly protected against loss of force following eccentric contraction-induced injury in the EDL and also improved muscle specific force post gene transfer. Preliminary studies further showed a reduction in kyphosis, improvement in body weight and an increase in myofiber diameter.
CONCLUSIONS:AAVrh.74.ITGA7 gene transfer improves eccentric contraction induced injury in the dko mouse. The data also indicates early treatment leads to increase in specific force, improvement in overall body weight and reduction in kyphosis. Taken together, AAVrh.74.ITGΑ7 gene transfer demonstrates promise as a viable treatment for DMD.

Plasmapheresis Enables Robust Micro-Dystrophin Expression Following Gene Transfer in Subjects with Pre-Existing Immunity

Louis G. Chicoine, Chrystal L. Montgomery, Thomas J. Preston, Danielle A. Griffin, William G. Bremer, K. Reed Clark, Zarife Sahenk, Christopher Walker, Jerry R. Mendell, Louise Rodino-Klapac
Columbus, OH, USA

OBJECTIVE: To test the hypothesis plasmapheresis attenuates the inhibitory effect of vector capsid binding antibodies on transgene expression in a non-human primate vascular delivery gene transfer model.
BACKGROUND: Gene therapy with adeno-associated viral (AAV) vectors for muscular dystrophies has taken several positive steps forward including ongoing development for muscle targeting with a vascular delivery approach. However, we and others have demonstrated the deleterious effects of pre-existing vector capsid neutralizing or binding antibodies on transgene expression.
DESIGN/METHODS: Prior to gene transfer, rhesus macaques were stratified based on the absence and presence of AAVrh.74 binding antibody titer (<1:50 were considered sero-negative, while ≥1:50 were considered sero-positive). Vascular delivery to the gastrocnemius muscle of an isolated limb (Rodino-Klapac et al. 2010) was performed on each animal with 2 x 1012 vg/kg of rAAVrh.74.MCK.micro-dys.FLAG. An additional group of sero-positive animals underwent 2 rounds of plasmapheresis on consecutive days prior to vector dosing. Three months after transfer, the macaques were euthanized, and the gastrocnemius muscle was harvested and evaluated by immunofluorescence with anti-FLAG antibody reported as percent positive fibers (mean % ± SEM).
RESULTS: Robust micro-dystrophin expression was visualized in AAVrh.74 sero-negative animals (mean 53.7 ± 7.6%) that were attenuated in sero-positive animals (10.1 ± 6.0%). Importantly, removal of AAV binding antibodies by plasmapheresis in AAV sero-positive animals resulted in high-level transduction (60.8 ± 18.0%).
CONCLUSIONS: The presence of pre-existing binding antibodies to vector capsid attenuates transgene expression, whereas, plasmapheresis mitigates this inhibitory effect. Plasmapheresis potentially increases the number of patients amenable to vascular gene therapy and provides a foundation for retreatment of patients whenever necessary.

Sociodemographic and Health Related Profile of Adults with Duchenne/Becker Muscular Dystrophy (DBMD): Data from the Muscular Dystrophy Surveillance, Tracking and Research Network (MD STARnet)

Shree Pandya,1Anil Kumar,2Kathy James,3Christine Westfield,2Emma Ciafaloni,1Christopher Cunniff,4Paul Romitti,5Richard Moxley,1MD STARnet
1Rochester, NY, USA, 2USA, 3Aurora, CO, USA, 4Tucson, AZ, USA, 5Iowa City, IA, USA

OBJECTIVE: To describe the sociodemographic profile and health related outcomes of adults with DBMD followed in MD STARnet
BACKGROUND: Improved respiratory and cardiac management along with glucocorticoid therapy have contributed to an increasing survival of patients with DBMD. This has led to a growing population of adults with DBMD.
DESIGN/METHODS: MD STARnet is a multi state population based surveillance system that collects data on individuals with DBMD born since 1/1/1982. In addition to epidemiologic data, MD STARnet collects extensive sociodemographic and clinical data related to natural history milestones and treatments and services received by individuals with DBMD. The cohort for this analysis was all individuals born from 1/1/1982 - 12/31/1992 and followed through 8/31/2011.
RESULTS: Overall 384 individuals were identified of which 32% have died [mean age 17.6 yrs (8.7-25.5)] and 20% were lost to follow up [mean age 15.3 (0.9-25.8)]. The remainder continue to be followed [mean age 21.5(17-28.6)]. Most individuals were white, non-Hispanic (59%) and live with parents/family (98%), have post high school/college training (24%). The number of individuals and the average age (range) at which they crossed clinical milestones or required specific treatments were as follows: Loss of ambulation[N=300,11.7yrs (6.9-24.5)], Scoliosis surgery[N=128,14.7yrs (10.3-20.2)], Non invasive ventilation[N=158, 16.9yrs (9.4-26.2)], Development of cardiomyopathy(SF<28%, EF<55%)[N=188, 16.5yrs (9.0-25.5)]Insertion of PEG[N=58,19.2yrs (11.8-26.0)]. Treatment with glucocorticoids [N=188, 8.3yrs (3.9-25.0)].
CONCLUSIONS: Our population based data describe an emerging population of adults with DBMD. This sociodemographic and health related profile provides information for providers, payors and policy makers to help design the appropriate care models to meet the needs of adults with DBMD and their families.

Electrical Impedance Myography Discriminates Congenital Muscular Dystrophy from Controls

Daniel Schwartz,1Anam Salman,2Janannaz Dastgir,1Barbara Lear,1Carsten Bonnemann,1Tanya Lehky1
1Bethesda, MD, USA, 2Valhalla, NY, USA

OBJECTIVE: To evaluate the discriminative value of electrical impedance myography (EIM) in distinguishing congenital muscular dystrophy (CMD) from controls.
BACKGROUND: EIM is an emerging non-invasive, highly reproducible electrophysiological technique that objectively characterizes muscle structure and composition by measuring bioimpedance. In EIM, a high frequency, low intensity electrical current is applied to a limb, and the resulting voltages are measured over a discrete region of muscle. In degenerative neuromuscular disorders, muscle fibers are lost and are replaced by fibrous and fatty tissue, altering the bioimpedance of muscle tissue. Here we evaluate the ability of EIM to discriminate CMD from controls.
DESIGN/METHODS: A total of 37 children, aged 4 to 19 years, mean age 10.8 years with genetically or biopsy confirmed CMD and a total of 9 healthy children, aged 7 to 16 years, mean age 11.6 years, were evaluated with 50-kHz EIM. The CMD group consisted of 19 children with collagen 6 related dystrophy, aged 7 to 19 years, mean age 11.6 years, and 18 children with LAMA2 related dystrophy, aged 4 to 18 years, mean age 9.9 years. EIM derived measures included: (1) reactance, a measure of muscle cell membrane integrity, (2) resistance, a measure of extra and intracellular fluid content, and (3) phase angle, which is derived from both reactance and resistance.
RESULTS: Mean 8-muscle EIM phase was significantly decreased (p<0.001) and resistance was significantly increased (p<0.001) in the CMD subjects versus healthy volunteers, but there was no significant difference in reactance between these two populations. Of note, mean 8-muscle EIM resistance was significantly increased (p=0.016) in LAMA2 related muscular dystrophy compared to collagen 6 related dystrophy, but there was no significant difference in phase or reactance between these two groups.
CONCLUSIONS: Our preliminary findings suggest that EIM may be useful in discriminating CMD from controls and may serve as a useful biomarker to follow disease progression in clinical trials.

To Report Clinical Heterogeneity of Facio-scapulo-humeral Muscular Dystrophy in Which Two Patients Presented with Recurrent Facial Palsy

Uzma Usman, Bilal Hameed, Johnny Salameh
Worcester, MA, USA

Objective: To report clinical heterogeneity of Facio-scapulo-humeral muscular dystrophy in which two patients presented with recurrent facial palsy
Background: FSHD is caused by a contraction mutation of D4Z4 macrosatellite repeats at the 4q35 locus in 95% of patients. Previous studies have shown that this deletion is found in patients with other phenotypes in addition to those with the classic Landouzy-Dejerine FSHD phenotype. It is not unusual for FSHD to present as isolated facial weakness that invariably progress to scapulo-humeral weakness10. However, recurrent facial palsy with limb-girdle involvement has never been reported to our best of knowledge.
Design/Method: We report a case of 62 year-old man who presented with five episodes of recurrent facial palsy with subsequent worsening of limb-girdle weakness at the age of 54 years. Interestingly, there was no family history of neuromuscular disease or muscular dystrophy. Second patient is an 82-year old woman who presented with 3 episodes of recurrent right facial palsy; last one was persistent, followed by limb-girdle atrophy.
Results: Both of these patients were found to have genetically proven FSHD with 4q35 deletion. Case 1 had restriction fragment of FSHD allele 1, size of 29 kb (Normal>40kb) and allele 2 was normal (>48 kb). Genetic testing of case 2 showed deletion in chromosome 4 with a restriction fragment of allele 2 (deletion of 28-kb with normal>40kb) and allele 1 was normal(>40kb).
Conclusion: Recurrent facial palsy in FSHD has not been reported yet. Recurrent facial palsy in our patients in the setting of FSHD genotype highlights the expanding clinical heterogeneity of this disease. Given the case histories of our patients, we propose that FSHD can be considered as one of the likely causes of recurrent facial palsy and genetic work up can be done for these patients to confirm the diagnosis of FSHD

Muscular Dystrophy Association U.S. Neuromuscular Disease Registry - Preliminary Findings

Jodi M. Wolff,1Joline C. Dalton,2Valerie Cwik,1Annie Kennedy,1Michelle Morgan,1Jane Larkindale,1Alan Beggs,3Joshua Benditt,4James Berry,3Thomas Crawford,5Emily Munson,1Rachel Richesson,6Jeffrey Rosenfeld,7Carly Siskind,8Kevin Flanigan9
1Tucson, AZ, USA, 2Minneapolis, MN, USA, 3Boston, MA, USA, 4Seattle, WA, USA, 5Baltimore, MD, USA, 6Durham, NC, USA, 7Fresno, CA, USA, 8Stanford, CA, USA, 9Columbus, OH, USA

OBJECTIVE:
To track, compare and improve patient care while gaining insight into natural history of disease states through a comprehensive neuromuscular disease registry across MDA clinics in the U.S.
BACKGROUND:
MDA began piloting its web-based clinical registry in January 2013 with 18 MDA-sponsored clinics entering demographic, genetic and clinical data for patients with ALS, Duchenne and Becker muscular dystrophy (DBMD), and SMA. The MDA Registry Advisory Board (RAB), comprising nine clinical and scientific professionals, maintains long-term oversight of the registry. The registry will be expanded to include all clinics and all diseases supported by MDA.
DESIGN/METHODS:
All patients with ALS, DBMD and SMA who are seen in the 18 pilot clinics are invited to participate. Demographic, diagnostic and longitudinal clinical information is collected on a prospective basis. Aggregate, de-identified data will be analyzed: descriptive statistics will be used to analyze and display demographic data and inferential and quantitative statistics will be used to analyze trends in patient care and compliance with care standards.
RESULTS:
Following informed consent, 442 patients have been entered into the registry. Of those with ALS (n=121), 56% of patients are male, 44% are female; average age of symptom onset is 56.9; average age at diagnosis is 57.6 years. 78.3% are Caucasian, 94.8% of patients have no known family history of ALS. Riluzole is taken by 43%, 74% do not use any nutritional therapies, and average BMI is 26.5. Mental status exam was performed for 27.4% of patients; ALS FRS was performed for 85.7%. Data for individuals with DBMD and SMA will also be presented. CONCLUSIONS:
Demographics of patients in the registry are currently skewed to those newly diagnosed; data illustrate opportunities to obtain longitudinal clinical information and inform standards of care for neuromuscular diseases. All data will be updated prior to presentation and compared to other surveillance efforts.

Quantitative Muscle Ultrasound: Comparison of Backscatter vs. Grayscale Methods in Duchenne Muscular Dystrophy Patients and Healthy Controls

Irina Shklyar,1,2Tom R. Geisbush,1Aleksandar S. Mijailovic,1Amy Pasternak,1Nicole Visyak,1Heather Szelag,1Jim S. Wu,1Basil Darras,1Seward Rutkove,1Craig Zaidman3
1Boston, MA, USA, 2New Haven, CT, USA, 3Saint Louis, MO, USA

OBJECTIVE: Compare two quantitative ultrasound measures of muscle pathology, quantitative backscatter analysis (QBA) and grayscale luminosity (GSL).
BACKGROUND: Ultrasound of dystrophic muscle shows abnormally bright echointensity that increases with age and pathology. Echointensity can be quantified by measuring the acoustic signal received by the transducer, known as backscatter (QBA), or the grayscale luminosity (GSL) of the post-processed image. QBA may yield more precise measures of echointensity because the data are collected before compression for display as grayscale images.
DESIGN/METHODS: We performed ultrasound on six upper and lower extremity muscles in 25 healthy boys and 25 with Duchenne muscular dystrophy (DMD) of similar (p>0.84) age (2-14 (mean 8) years). One rater measured the median GSL and QBA from each muscle image; two raters repeated 36 measurements from a representative sample. Age served as a surrogate for disease severity.
RESULTS: Average QBA correlated highly with average GSL (rho =0.89, p<0.0001). Both QBA and GSL showed excellent intra- and inter-rater reliability (intraclass correlation > 0.87). QBA and GSL from all muscles were higher in DMD subjects than controls (p<0.001). In DMD, age consistently showed stronger correlations with QBA than GSL; the strength of the correlations varied between individual muscles (rho=0.02-0.7). For example, age increased more strongly with QBA than GSL and in the biceps brachii (rho=0.54, p=0.006 vs. rho=0.38, p=0.061) and on average (rho=0.64, p=0.001 vs. rho=0.58, p=0.003) but did not vary with either QBA or GSL in the quadriceps or medial gastrocnemius (p>0.6).
CONCLUSIONS: Both QBA and GSL are reliably measured and are increased in dystrophic muscle. In DMD, QBA showed consistently higher correlations than GSL with age and may better quantify degree of pathology. Correlations between age and either QBA or GSL differed between muscles. Additional study using ultrasound will determine how QBA and GSL of individual muscles vary with function and over time.

Secondary Health Conditions Among Males With Duchenne Or Becker Muscular Dystrophy (DBMD)

Rebecca Napoliello,1Natalie Street,1Kristin Caspers,2Emma Ciafaloni,3Christopher Cunniff,4Jennifer Donnelly,5Deborah Fox,6Kathy James,7Joyce Oleszek,7Christina Westfield8
1Atlanta, GA, USA, 2Iowa City, IA, USA, 3Rochester, NY, USA, 4Tucson, AZ, USA, 5Denver, CO, USA, 6Albany, NY, USA, 7Aurora, CO, USA, 8Buffalo, NY, USA

Objective: To describe the frequency of secondary health conditions among males with Duchenne or Becker muscular dystrophy (DBMD).
Background: The full spectrum of secondary health conditions in males with DBMD is unknown. A few studies suggest a higher than expected prevalence of a sub-set of conditions such as autism spectrum disorder, obsessive-compulsive disorder, constipation, and gastroesophageal reflux.
Design/Methods: Caregivers of males with DBMD were identified through MD STARnet, a population-based surveillance system for DBMD in five states. Participants (n=209) were asked whether their child was ever diagnosed with any of twenty-nine conditions. Responses were categorized by affected male's age, ambulatory status, and steroid-use. The responses were compared to those of the National Survey of Children's Health (NSCH) when the questions were equivalent. Chi-square or Fisher's exact test was used to analyze associations between categorical variables. The differences in means of continuous variables were evaluated with T-tests.
Results: Males with DBMD aged >17 years were more likely to have depression (p<0.001), gastroesophageal reflux/heartburn (p= 0.029), and kidney stones (p<0.001) than males aged ≤17 years. Depression (p= 0.001), anxiety (p= 0.006), ADD/ADHD (0.022), gastroesophageal reflux/heartburn (p= 0.006), kidney stones (p= 0.007), trouble urinating (p= 0.010), and high blood pressure (p= 0.021) were more common among non-ambulatory males than ambulatory males. Cataracts were more common in long-term steroid users (≥6months) than short-term (<6 months) or non-users (p<0.001). In comparison to NSCH participants, MD STARnet males had a higher percentage of behavioral and/or conduct problems, developmental delay, and intellectual disability (p<0.001).
Conclusion: Males with DBMD have a higher frequency of behavioral and/or cognitive issues than NSCH participants. Certain conditions occur more frequently in males >17 years and those who are non-ambulatory. Identification of these secondary conditions can ultimately improve clinical care and direct future research in this population.

Future Clinical And Biomarker Development For SMT C1100, The First Utrophin Modulator To Enter Clinical Trials For Duchenne Muscular Dystrophy (DMD)

Jon Tinsley,1Neil Robinson,1Francis Wilson,1Graeme Horne,1Kay Davies2
1Abingdon, United Kingdom, 2Oxford, United Kingdom

Objective: To progress SMT C1100, Summit's utrophin upregulator molecule into DMD clinical trials. This is the first disease modifying approach potentially benefiting all DMD patients regardless of dystrophin mutation.
Background: DMD is the most common form of muscular dystrophy due to an inability to make dystrophin, resulting in loss of skeletal muscle. During foetal muscle development utrophin takes the functional role of dystrophin. Continual muscle expression of utrophin can functionally replace dystrophin potentially overcoming the dystrophin deficit in DMD. In animal studies, daily SMT C1100 treatment significantly reduced muscle pathology leading to better muscle function. A Phase 1, double-blind, placebo-controlled study has been completed in healthy male subjects. The data were supportive of SMT C1100 moving into DMD patient trials.
Design/Methods: Plans for the first patient trials of SMT C1100 have been developed consisting of two components; a safety and dose finding study in DMD boys in late 2013 followed by a proof of concept study in 2014. In order for proof of concept to be demonstrated in patients, a multicomponent biomarker strategy has been implemented that comprises of two modules. We aim to quantify utrophin RNA, total utrophin protein and utrophin fibre localisation derived from pre- and post-dose biopsies. To determine a reduction in the rate of degeneration, i.e. increase in mature fibre survival, changes in the percentage of newly regenerating fibres will be calculated from the biopsies. Using serum and urine samples we will quantify the levels of specific miRNAs associated with fibre leakage and protein markers of fibrosis and inflammation.
Results: Preliminary data from the Phase 1b safety and PK study will be discussed. The rationale for the choice of exploratory biomarkers proposed for the Phase 2 proof of concept trial will be described.
Conclusions: This is the first example of a utrophin upregulation therapy to enter the clinic with the potential to treat all DMD patients.

Bone Mineral Content As Measure Of Bone Health In Ambulatory Boys With Duchenne Muscular Dystrophy

Cuixia Tian,1Meilan Rutter,1Lindsey Hornung,1Jane Khoury,1Lauren Miller,1Jean Bange,2Brenda Wong1
1Cincinnati, OH, USA, 2CIncinnati, OH, USA

OBJECTIVE: To assess changes in Bone Mineral Density (BMD) and Bone Mineral Content (BMC) by functional status in ambulatory GC-treated DMD boys, to test the hypothesis that BMC is a more reliable measure for bone health in DMD.
BACKGROUND: Osteoporosis is a major problem in Duchenne Muscular Dystrophy (DMD). Dual-energy xray absorptiometry (DXA) assessment of bone health in DMD boys is challenging, as interpretation is affected by height, delayed bone maturation, puberty, and vertebral fractures. Accurate detection is important for improving clinical care in DMD.
DESIGN/METHODS: Retrospective study of fracture frequency, total body (TB) and lumbar spine (LS) BMD and BMC by DXA in ambulatory GC-treated DMD boys, assessed from 7/2006 to 7/2012. Age-adjusted z-scores (Z) and height-adjusted z-scores (HAZ) were derived using normal values. Generalized linear modeling was used to analyze changes.
RESULTS: 292 ambulant DMD boys were grouped by functional mobility score (FMS1, 2 or 3, by worsening status). GC durations were 2.5±1.5, 2.9±2.0 and 4.6±2.4 yrs; Limb fracture frequency were 16.4, 16.6 and 27.4%; spine fractures were 9%, 8.6% and 20.2% for FMS1, 2 and 3, respectively. TB bone indices all decreased with worsening FMS (p<0.05 for FMS 3 vs. FMS 1 or FMS 2). TB BMC was consistently lower than BMD for each group. LS BMD-Z was not different between FMS groups, but LS BMC-Z was lower for FMS3 than FMS1 or FMS2 (p<0.05). LS BMD-HAZ was higher for FMS3. LS BMC-HAZ was not different between groups.
CONCLUSIONS: TB bone indices worsened with declining mobility and increased fractures. BMC was significantly lower than BMD. LS height-adjusted BMD Z score paradoxically increased with increased fractures. Our study suggests that BMC may be a more reliable indicator of bone health in DMD, and should be considered as part of clinical care.

Physical Activity Profile and Barriers to Physical Activity in Individuals with Myotonic Dystrophy

Katy Eichinger,1Jeanne Dekdebrun,2Nuran Dilek,1Shree Pandya,1Ding-Geng Chen1
1Rochester, NY, USA, 2

OBJECTIVE:The purpose of this study was to document the physical activity profile and barriers to physical activity in persons with DM.
BACKGROUND:Individuals with myotonic dystrophy (DM) type 1 are less active than healthy individuals, which may result in secondary complications and decreased functional abilities.
DESIGN/METHODS:Individuals with DM were recruited during the Empower 2011 patient and family conference sponsored by the Myotonic Dystrophy Foundation. Participants were asked to complete a survey that included a Demographic/Clinical profile, the Barriers to Physical Activity and Disability Survey and the International Physical Activity Questionnaire. Physical activity in terms of number of days and time (in 10 minute increments) over the past 7 days was reported. A multiple regression model including age, number of current clinical problems, and the number of barriers reported was used to predict the amount of sedentary time (sitting time).
RESULTS:107 (53% male) participants with DM (65% DM1) reported finger and hand weakness, fatigue, day time sleepiness, gastrointestinal issues, and leg weakness as the most common clinical problems. Participation in at least 1 day of activity was reported by 15% of the participants for gardening, 69% for household activities, 42% for leisure time walking, and 22% for moderate intensity level leisure time activities. Lack of energy and motivation were the most common barriers to physical activity reported. Number of barriers was a significant predictor of the amount of time spent sitting.
CONCLUSIONS:Persons with DM have decreased physical activity and spend a significant time sedentary and are limited by a lack of energy and motivation. Administration of this survey at the conference may have influenced the report of physical activity as it may have been modified due to travel and conference participation. Further investigation is needed to better understand additional factors that contribute to inactivity.

Brain Gray Matter And White Matter Alterations In Patients With Myotonic Dystrophy 1

Stojan Peric,1Federica Agosta,2Francesca Caso,2Vidosava Rakocevic-Stojanovic,1Giancarlo Comi,2Vladimir Kostic,1Massimo Filippi2
1Belgrade, Serbia, 2Milan, Italy

OBJECTIVE: To investigate grey matter (GM) and white matter (WM) abnormalities in Myotonic dystrophy type 1 (DM1) using advanced Magnetic Resonance Imaging (MRI) techniques and assess their clinical correlates, and to evaluate separately childhood-juvenile-onset (jDM1) and classic adult-onset (cDM1) DM1 cases in order to explore the effect of age and disease durationin this condition
BACKGROUND: DM1 is a multisystem disease that affects also the brain. A few neuroimaging studies reported distributed patterns of GM and WM atrophy in DM1.
DESIGN/METHODS: Fifty-one DM1 patients (14 jDM1, 37 cDM1) underwent clinical and neuropsychological evaluations, and brain structural and diffusion tensor (DT)-MRI. WM hyperintensity (WMH) load was assessed. Voxel based morphometry (VBM) and tract-based spatial statistics (TBSS) were used to evaluate GM atrophy and WM microstructural damage. Voxel-wise analyses were adjusted for WMH load. A regression analysis was performed in order to evaluate clinico-anatomical correlations.
RESULTS: DM1 patients showed cognitive impairment involving especially visuospatial abilities, language, and orientation/attention. Visuospatial abilities/memory were more frequently compromised in cDM1 compared with jDM1 patients. WMH load was higher in both patient groups relative to controls. DM1 patients showed a widespread pattern of GM atrophy, which was more severe and distributed in cDM1 cases. DT-MRI showed significant damage of the main WM tracts similarly in cDM1 and jDM1 groups. Deficits in attentive-executive and visuospatial abilities were independently associated with WMH load and increased mean diffusivity of the left corona radiate, internal capsule, inferior fronto-occipital fasciculus and superior longitudinal fasciculus.
CONCLUSIONS: DM1 is clinically characterized by impaired cognitive performance and anatomically by widespread and severe GM and WM damage. Cognitive deficits of DM1 patients correlate with structural WM damage including both WMHs and WM microstructural damage. Despite being comparable clinically and cognitively, cDM1 patients experienced GM and WM damage, while jDM1 had a prominent WM damage.

A Population-Based Epidemiologic Study of Adult Neuromuscular Disease in the Republic of Ireland

Stela Lefter,1,2Orla Hardiman,2Aisling Ryan1
1Cork, Ireland, 2Dublin, Ireland

OBJECTIVE:
The objectives of this study are to provide data on the prevalence of adult neuromuscular diseases (NMD) in Ireland and to develop registries for these conditions.
BACKGROUND:
There have been no previous population-based studies of adult NMD in Ireland. In the era of potential therapeutic agents for neuromuscular disease, accurate disease-specific registries are essential to define phenotypes and allow participation in future therapeutic trials.
DESIGN/METHODS:
Study inclusion criteria are adults with neuropathies (acquired inflammatory demyelinating neuropathies, inherited neuropathies, spinal muscular atrophy type III and IV, Kennedy's disease, neuromyotonia), neuromuscular transmission disorders (congenital myasthenic syndrome, myasthenia gravis) and muscle diseases (muscular dystrophies, congenital myopathies, muscle channelopathies, inclusion body myositis, metabolic myopathies). Case ascertainment commenced in January 2012 and includes multiple sources: identification via neuromuscular clinics, referrals from adult / paediatric neurologists, neurophysiologists, neuropathologists, interrogation of searchable databases in public / private neurology departments in the country, interrogation of Hospital In Patient Enquiry data / chart review from 1990 - present in adult hospitals, liaison with the voluntary organization Muscular Dystrophy Ireland.
RESULTS:
To date 2564 cases have been identified: 1189 neuropathies, 882 myopathies and 493 neuromuscular junction disorders.
The preliminary prevalence for all NMD in Ireland is 75 x 105 adult population which includes Charcot-Marie-Tooth - 10.5 x105, limb-girdle-muscular-dystrophy - 2.6 x 105, facio-scapulohumeral muscular dystrophy - 2.4 x 105 and myotonic dystrophy type 1 - 6.6 x 105. Comparative data from published international studies suggests that we are approaching reported prevalence figures for many of these conditions. Case ascertainment continues until January 2014.
CONCLUSION:
Our study will generate for the first time prevalence data for adult NMD in Ireland. Defining the burden of NMD in Irish population will raise awareness of these conditions, facilitate earlier intervention where appropriate and provide a stronger argument for service provision for these patients

Genotype-Phenotype Evaluation In 476 Turkish Dystrophinopathy Patients

Hacer Durmus,1Nihan Erginel-Unaltuna,1Poda Mehves,1Arman Çakar,1Ayca Altinkaya,1Filiz Guclu-Geyik,1Feza Deymeer,2Yesim Parman,1Asli Tolun,1Piraye Serdaroglu1
1Istanbul, Turkey, 2Capa, Turkey

OBJECTIVE: In this study, we aimed to evaluate genotype-phenotype correlation in a large Turkish cohort.
BACKGROUND: Duchenne muscular dystrophy (DMD) and its less severe allelic form, Becker muscular dystrophy (BMD), are common X-linked recessive dystrophies caused by mutations in the dystrophin gene. Studies on the genotype-phenotype correlations on DMD/BMD in Turkey are limited. Identification of the characteristics of genotype and phenotype correlation may facilitate prognosis regarding clinical severity. Furthermore, several ongoing and planned clinical trials which aim at targeting specific DMD mutations highlight the importance of recognition of the genetic features in dystrophinopathy patients.
DESIGN/METHODS: Clinical and genetic findings of 476 patients diagnosed with dystrophinopathy at the Department of Neurology, Istanbul Faculty of Medicine between 1993 and 2013 were retrospectively evaluated. Multiplex DNA amplifications of the dystrophin gene were carried out according to Chamberlain et al and Beggs et al.
RESULTS: Two hundred eighty-nine patients were diagnosed with DMD and 187 with BMD. Deletions in the dystrophin gene represented 66.5 % of the mutations in DMD/BMD patients. Intron 44 was the most common starting breakpoint in Turkish DMD/BMD patients. In 76 % of the cases the deletions were detected in the region from exon 45 to exon 52 located in the central part of the gene. The most frequent deletions in DMD were deletions of exons 45-50 (8.6%), exon 45 (7%) and exon 44 (6.4%). Deletions in BMD were less heterogeneous, and the most frequent deletions were exons 45-47 (29.6%), exons 45-48 (21.3%) and exons 45-52 (7.4%). 76% of patients with deletions in dystrophin gene was consistent with the reading-frame hypothesis. The major exception was the deletions starting at exon 47.
CONCLUSIONS: The majority of the deletions in our population are clustered in the two known hot-spot regions, and in 76% of cases deletions were detected in the region from exons 45 to 52. Our findings are comparable to those reported in other populations.

Sarcoglycan Gene Transfer Leads to Functional Improvement in a Model of LGMD2E

Eric Pozsgai,1Danielle Griffin,2Kristin Heller,1Jerry Mendell,1Louise Rodino-Klapac1
1Columbus, OH, USA, 2

Objective: The goal of this study is to demonstrate efficacy of AAV-mediated β-sarcoglycan gene transfer in β-SG knock-out mice to provide proof-of-principle for gene replacement therapy for limb-girdle muscular dystrophy type 2E
Background: Limb-girdle muscular dystrophy (LGMD) type 2E results from mutations in the gene encoding β-sarcoglycan causing loss of functional protein. The sarcoglycans (α, β, γ, and δ-SG) are structural proteins localized at the cell membrane of muscle fibers that together with dystrophin and other proteins make up the dystrophin-associated protein complex (DAPC). Loss of functional protein in this complex results in an unstable sarcolemma leading to myofiber death and eventual muscle weakness.
Methods: We generated a self-complementary AAVrh74 vector containing a codon optimized human β-Sarcoglycan gene (hSGCB) driven by the muscle specific tMCK promoter. To demonstrate efficacy of vector delivery we injected two doses (3x1010 and 1x1011 vg) by direct injection to the tibialis anterior muscle of β-SG KO mice. We have also treated β-SG KO mice using a clinically relevant vascular delivery model to deliver AAV.tMCK.hSGCB to the lower limb muscles.
Results: Following direct intramuscular gene transfer in β-SG KO mice, we had clear demonstration of hSGCB expression and improvement in dystrophic pathology. We also showed hSGCB expression in lower limb muscles following vascular delivery. Functional outcome measures performed in the extensor digitorum longus muscle following vascular delivery showed an improvement in specific force generation in treated muscles.
Conclusions: Delivery of a normal copy of the β-SG gene to diseased muscle will allow for the production of functional wild-type protein, resulting in restoration of the DAPC and an improvement in muscle function. This pre-clinical study is pivotal for establishing proof-of-principle for translation of AAV.βSG gene transfer in LGMD2E patients.