Research that will be presented in 2012 Annual Meeting of American Academy of Neurology

 

1) [P04.088] A Phase 2, Randomized, Placebo-Controlled, Multiple Ascending-Dose Study of ACE-031, a Soluble Activin Receptor Type IIB, in Boys with Duchenne Muscular Dystrophy (DMD)

Craig Campbell, London, ON, Canada, Diana Escolar, Baltimore, MD, Jean Mah, Calgary, AB, Canada, Mark Tarnopolsky, Hamilton, ON, Canada, Kathy Selby, Vancouver, BC, Canada, Hugh McMillan, Ottawa, ON, Canada, Yijun Yang, Dawn Wilson, Rachel Barger, Matthew Sherman, Kenneth Attie, Cambridge, MA

OBJECTIVE: To evaluate the safety, tolerability, pharmacokinetics, and pharmacodynamic (PD) effects of ACE-031 in DMD. BACKGROUND: ACE-031 is a soluble activin type IIB receptor (ActRIIB-IgG1) that binds negative regulators of muscle, including myostatin and activin. In phase 1 studies in healthy adults, ACE-031 treatment resulted in dose-dependent increases in lean mass. DESIGN/METHODS: Ambulatory (30ft walk/run <12sec), steroid-treated DMD boys, age ≥4yr, were randomized to 12 weeks treatment with either escalating doses of ACE-031 or placebo (9:3 in each cohort), followed by 12-weeks follow-up. The primary objective was safety/tolerability. PD endpoints included lean/fat/bone mass (DXA), thigh muscle, fat volume (MRI), strength, 6-minute-walk-test-distance (6MWD), and other motor/pulmonary function tests. RESULTS: Two cohorts completed treatment (C1: 0.5mg/kg SC q4weeks, C2: 1mg/kg SC q2weeks). Mean age was 10.3 years (range 6-17). There were no AEs that were serious, severe, or resulted in discontinuation. Reversible telangiectasias and mild epistaxis were reported in C2. Mean total body lean mass by DXA increased 5.2% in C2 (p=0.015 vs baseline) vs 2.6% in placebo (p=0.20) after 12 weeks. Thigh MRI (C2) showed a trend for decreased muscle volume in placebo group which was attenuated in ACE-031 group. The least-squares mean
Δ6MWD was +12meters in combined ACE-031 groups vs -30meters in placebo group after 24 weeks (p=0.06, ANCOVA). The %change in 6MWD correlated with that for 2MWD (r=0.67, p<0.001) and 10m walk/run (r=-0.53, p=0.009) after 24 weeks. Dose-related trends for decreased gain in fat mass and increased lumbar spine BMD by DXA were seen after 12 and 24 weeks. CONCLUSIONS: In this phase 2 study of ACE-031 in steroid-treated DMD boys, reversible telangiectasias and mild epistaxis were reported at higher dose. Significant dose-dependent increases in lean mass were observed. There were trends for maintained 6MWD, decreased gain in fat mass, and increased lumbar spine BMD with ACE-031 treatment. Supported by: MDA, PPMD.

2) [P04.084] Motor and Cognitive Assessment of Infants and Young Boys with Duchenne Muscular Dystrophy; Results from the Muscular Dystrophy Association DMD Clinical Research Center Network

Anne Connolly, Julaine Florence, Mary M. Cradock, Elizabeth Malkus, Jeanine R. Schierbecker, Catherine Siener, Charlie O. Wulf, Pallavi Anand, Saint Louis, MO, Linda Lowes, Lindsay N. Alfano, Laurence Viollet-Callendret, Kevin Flanigan, Jerry Mendell, Columbus, OH, Craig McDonald, Erica Goude , Linda Johnson, Alina Nicorici, Sacramento, CA, Peter Karachunski, John Day, Joline Dalton, Minneapolis, MN, Janey M. Farber, Saint Paul, MN, Karen K. Buser, Minneapolis, MN, Basil Darras, Susan O. Riley, Elizabeth Schriber, Rebecca E. Parad, Boston, MA, Kate Bushby, Newcastle Upon Tyne, United Kingdom, Michelle Eagle, Newcastle Upon Tyne, United Kingdom, MDA DMD Clinical Research Network, Tucson, AZ

OBJECTIVE: To implement motor and cognitive assessments in infants and young boys with Duchenne Muscular Dystrophy(DMD). BACKGROUND: Therapeutic trials in DMD exclude young boys because traditional outcome measures rely on cooperation. The Bayley Scales of Infant and Toddler Development(BAYLEY-III) have been validated in both typically-developing(TD) children and those with developmental disorders but have not been studied in DMD. Initial work in the Hammersmith Functional Motor Scale Extended (HFMSE) and North Star Ambulatory Assessment (NSAA) suggest these may be useful in this young DMD population. DESIGN/METHODS: Clinical evaluators from six neuromuscular centers were trained in the use of the BAYLEY-III, NSAA, and HFMSE. Infants and and young boys with DMD(N=25) (mean=1.84 ± 0.7 years (range=0.37-2.99) were assessed with the above measures. RESULTS: The Bayley-III mean motor composite score was 83.5±12 with range 58-103 (TD 100±15). Gross motor function was more severely involved than fine motor with mean scaled scores of 6.2±1.5 and 7.8±1.9 respectively (TD 10±3). The mean cognitive comprehensive score (N=25) was 89.6±8.7 with range of 65-115 (TD 100±15). The mean receptive and expressive language scores (N=24) measured 7.1 and 7.9 respectively (TD 10±3). Age was negatively associated with gross motor scores (linear regression slope p=0.008) but not with cognitive and language scores. HFMSE was tested in 24 boys and showed a mean score of 31±13. NSAA was performed in18 boys with a mean age of 2.2±years with a mean score of 12±5. CONCLUSIONS: These outcome assessments of young infants and boys with DMD are possible in a multicenter trial with careful clinical evaluator training. Ongoing studies will document change over two years. Supported by: Muscular Dystrophy Association.

3) [P04.085] Outcomes Measure Reliability in Non Ambulatory Boys and Men with Duchenne Muscular Dystrophy (DMD): Results from the Muscular Dystrophy Association DMD Clinical Research Network

Julaine Florence, Anne Connolly, J. Philip Miller, Elizabeth C. Malkus, Jeanine R. Schierbecker, Catherine A. Siener, Charlie O. Wulf, Pallavi Anand, Saint Louis, MO, Craig McDonald, Erica Goude, Linda Johnson, Alina Nicorici, Sacramento, CA, John Day, Peter Karachunski, Joline Dalton, Minneapolis, MN , Jason M. Kelecic, Kelley Paulson, St. Paul, MN, Cameron E. Naughton, Minneapolis, MN, Linda Lowes, Lindsay N. Alfano, Laurence Viollet-Callendret, Kevin Flanigan, Jerry Mendell, Columbus, OH, Basil Darras, Janet Quigley, Amy E. Pasternak, Elizabeth Shriber, Rebecca E. Parad, Boston, MA, MDA DMD Clinical Research Network, Tucson, AZ

OBJECTIVE: To establish optimal and reliable assessments in non-ambulatory boys and men with DMD. BACKGROUND: Therapeutic trials in DMD most often exclude non-ambulatory individuals and consensus has not been reached regarding which measures are most reliable and feasible. DESIGN/METHODS: Clinical evaluators(CEs) from the five MDA-DMD CRN sites were trained in all assessments. Ninety-three non-ambulatory boys/men with DMD who were not on continuous ventilation (mean age 16.8±3.6 years) were assessed twice(AM/PM) on one day with a battery of assessments. Reliability using intra-class correlation coefficients(ICCs) was determined by comparing AM and PM measures. Feasibility was determined by the percentage that could perform the task. RESULTS: Forced Vital Capacity(FVC)(100% of subjects) showed mean FVC 47±23% predicted with an ICC of 0.97. Manual muscle testing was performed for shoulder abduction (88% of subjects; ICC=0.95), elbow flexion (96.8% of subjects; ICC=0.98), wrist flexion (70% of subjects; ICC=0.92) and wrist extension (96% of subjects; ICC=0.89). Hand Held myometrywas performed for supine elbow flexion (71% of subjects; ICC=0.93), supine elbow extension (70% of subjects; ICC=0.94), grip (97% of subject; 0.92), pinch grip(95% of subjects; ICC=0.84), and key grip (99% of subjects; ICC=0.93). Upper extremity active range of motion(ROM) ICCs ranged from 0.89-0.99 and passive ROM ranged from 0.71 to 0.97. Nine-hole peg was performed in 77% of subjects with ICC ranging from 0.96-0.97 for dominant and non-dominant hands. Brooke upper extremity scale was performed on 100% of subjects with an ICC=0.99. Egen Klassifikation(EK) scale was performed in 100% of subjects with ICCs on individual questions ranging from 0.93 to 0.99. Jebsen-Taylor Hand Function test was performed in 84% of subjects with ICCs of individual tasks ranging from 0.64 to 0.98. CONCLUSIONS: This study demonstrates excellent reliability across most measures with well-trained CEs from five centers. Ongoing studies will test sensitivity to change across time. Supported by: Muscular Dystrophy Association.

4) [P04.087] Guidelines and Outcomes; Standardizing Care for Duchenne Muscular Dystrophy

Michele Scully, Rochester, NY, Valerie Cwik, Tucson, AZ, Bruce Marshall, Bethesda, MD, Emma Ciafaloni, Rochester, NY, Thomas Getchius, Saint Paul, MN, Robert Griggs, Rochester, NY

OBJECTIVE: To review evidence-based and consensus guidelines for the treatment of Duchenne Muscular Dystrophy (DMD) and consider how outcome measures could standardize and improve patient care. BACKGROUND: There are no evidence-based quality care indicators to standardize outcomes across Muscular Dystrophy Association (MDA) clinics. We reviewed the Cystic Fibrosis (CF) model for standardizing CF clinic outcomes and consider potential outcomes to assess the quality of care in DMD clinics. For forty years, the CF foundation has maintained a nationwide patient registry and acquired patient data from each CF clinic. The data, analyzed yearly, is reviewable on the CF website. Comparing individual and national clinic outcomes challenges clinics to improve clinical care. This data also provides potential patient populations for future investigations, including clinical trials. DESIGN/METHODS: We reviewed: published guidelines and consensus statements on the care of DMD; current efforts to obtain outcomes from DMD clinics; standardized data obtained annually from CF clinics; and published approaches to improving outcomes by attention to the fidelity of health care delivery. RESULTS: There are multiple approaches within the DMD field for surveillance and collection of patient information, including MDSTARNet, Parent Project Muscular Dystrophy, and MDA DMD Clinical Research Network. Based on CF analyzed data, a similar patient registry and nationwide reporting of standard outcomes across MDA clinics appears feasible. Monitoring data including molecular diagnosis, age at loss of ambulation, FVC, and survival, could provide metrics for quality of care. CONCLUSIONS: There are logistic challenges to reporting nationwide outcomes; however the influence this model has had on outcomes for CF patients has changed survival. In 2009, the median life expectancy for a patient with CF was 35.9 years, compared to 27 years in 1985. Implementation of clinical data monitoring in MDA clinics will determine if similar benefit accrues in patients with Duchenne Muscular Dystrophy.

5) [S15.003] Natural History of Cardiomyopathy in Duchenne Muscular Dystrophy and the Effects of Angiotensin-Converting Enzyme Inhibitor with or without β-Blocker

Philip T. Thrush, Laurence Viollet, Kevin Flanigan, Jerry Mendell, Hugh Allen, Columbus, OH

OBJECTIVE: To prospectively examine the natural history of cardiomyopathy (CM) in Duchenne muscular dystrophy (DMD) patients & assess responses to treatment utilizing angiotensin-converting enzyme inhibitors (ACEI) with or without concomitant
β-blocker (BB). BACKGROUND: Cardiomyopathy is an invariable consequence of DMD. Suggested treatments include ACEI and/or BB, but few large series are reported. We present 65 DMD patients with CM treated with ACEI or ACEI+BB, including their natural history & therapeutic responses. DESIGN/METHODS: Serial echocardiograms were performed and reported for natural history analysis prior to initiation of therapy when available. Adequate ejection fractions (EF) were obtained in 45 patients at initiation of therapy (EF<55%). Twenty patients were excluded due to pre-existing ACEI therapy (n=11), inadequate follow-up (n=8), or inadequate echocardiogram (n=1). ACEI dosage adjustments were made if a continued decrease in EF was noted. BB therapy initiated when average heart rate on Holter >100 beats/min. Data analyzed using paired t-test & linear regression. RESULTS: Natural history data (n=24) demonstrated decreased EF over time (r2=0.22). At ACEI therapy initiation, the mean age was 14.7 ± 4.4 years & mean EF was 44.3 ± 8.3%. BB therapy was used in 25/45 patients. Mean age for ACEI+BB group was 16.1 ± 3.9 years. Both groups demonstrated significant improvement compared to natural history (p≤0.0001 for ACEI, p<0.001 for ACEI+BB). No significant difference noted between treatment groups. CONCLUSIONS: Patients with DMD demonstrated gradual decline in myocardial function. Treatment with ACEI or ACEI+BB resulted in significant improvement compared to the natural history. No significant difference was noted in EF improvement between treatment groups. Treatment with ACEI or ACEI+BB can delay progression of CM. Supported by: Data collection was made possible by the Muscular Dystrophy Association & Wellstone Muscular Dystrophy Cooperative Research Center (NIH sponsored) and led to creation of the MDA DMD Clinical Network. Mutational analysis for most patients performed as part of the United Dystrophinopathy Project-supported by NINDS (R01 NS043264)[K.M.F.].

6) [S15.002] Osteopontin in Duchenne Muscular Dystrophy

Elena Pegoraro, Luca Bello, Luisa Piva, Andrea Barp, Mario Ermani, Padova, Italy, Luisa Politano, Naples, Italy, Eugenio Mercuri, Roma, Italy, Stefano Previtali, Yvan Torrente, Milan, Italy, Claudio Bruno, Carlo Minetti, Genova, Italy, Angela Berardinelli, Pavia, Italy, Giacomo Comi, Milano, Italy, Adele D'Amico, Rome, Italy, Gianni Soraru', Padova, Italy , Sonia Messina, Messina, Italy, Tiziana Mongini, Torino, Italy, Enrico Bertini, Rome, Italy, Alessandra Ferlini, Ferrara, Italy, Francesca Gualandi, Genova, Italy, Roberta Battini, Pisa, Italy, Patrizia Boffi, Turin, Italy, Marika Pane, Roma, Italy, Giuseppe Vita, Messina, Italy, Eric Hoffman, Washington, DC, Corrado Angelini, Padova, Italy

OBJECTIVE: To test the effect of the SPP1 rs28357094 in a longitudinal cohort of ambulatory DMD patients and to dissect the molecular mechanisms of increased disease severity associated with the G allele at rs28357094. BACKGROUND: Osteopontin (OPN coded by the SPP1 gene) is a secreted glycoprotein expressed by multiple cell types including myoblasts. In mdx muscle OPN modulates inflammation and fibrosis via a reduction of transforming growth factor-b (TGFB). In Duchenne muscular dystrophy (DMD) a polymorphism in the SPP1 promoter region (rs28357094) is a determinant of disease progression. DESIGN/METHODS: 80 DMD patients were genotyped at rs28357094 and stratified in TT vs TG/GG according to a dominant model. Genotypes were compared to phenotypes using the North Star Ambulatory Assessment (NSAA) and the 6 Minute Walk Test (6MWT) score at T0 and T12. Muscle biopsies from selected patients were studied for quantification of TGFB and transforming growth factor-b receptor-2 (TGFBR2) mRNA and SPP1 mRNA and protein, muscle histology and infiltrating inflammatory cells. A polymorphism in the TGFBR2 gene (rs4522809 ) was genotyped in the patients. RESULTS: Paired t-test showed a significant difference in both NSAA (p=0.013) and 6MWT (p=0.03) between baseline and follow-up after 12 months in DMD patients carrying the G allele. The difference was not significant in the T subgroup. OPN was upregulated in DMD muscle. No significant differences in osteopontin mRNA or protein expression nor in TGFB and TGFBR2 mRNA between G and T genotype at rs28357094 were found. An increase in CD4+ and CD68 cells in the patients carrying the T allele at rs28357094 was observed. TGFBR2 rs4522809 polymorphism predicted SPP1 mRNA level. CONCLUSIONS: SPP1 genotype is a strong disease modifier in DMD and may be relevant as covariate in DMD clinical trials. OPN modulates inflammatory changes and TGFBR2 genotype predicts osteopontin in DMD muscle

7) [PD6.004] Overexpression of Human Alpha7 Integrin as a Potential Therapy for Duchenne Muscular Dystrophy

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

OBJECTIVE: To overexpress a7 integrin as a potential therapy for the treatment of Duchenne muscular dystrophy. BACKGROUND: Duchenne Muscular Dystrophy (DMD) is a severe muscle disease caused by mutations in the DMD gene. Dystrophin provides a link from the extracellular matrix to the actin cytoskeleton, stabilizing the sarcolemma during muscle activity. a7b1 integrin is a laminin receptor on skeletal muscle that serves a similar functional role. Transgenic double knockout mice (mdx/utr-/-) overexpressing a7 show significant improvement in life-span, kyphosis and muscle histology. We performed a pre-clinical study to address the potential of gene transfer of ha7 delivered by adeno-associated virus (AAV) under control of a muscle specific promoter (MCK) to avoid off targeted effects. Transfer of the a7 gene potentially avoids an immune response encountered with mini-dystrophin gene transfer (Mendell JR et al N Engl J Med 2010). DESIGN/METHODS: We have generated rAAV8.MCK.human
α7 integrin and delivered it to the lower limb muscle of mdx mice through isolated limb perfusion (Rodino-Klapac et al 2007). In mdx mice, we measured human (as opposed to mouse) α7 as a biomarker of gene transfer and assessed muscle histopathology, force generation and protection against eccentric contraction. RESULTS: We have demonstrated fifty-two percent of fibers in the TA and EDL overexpressing human α7 integrin. The increase in human α7 integrin in skeletal muscle significantly protected against eccentric contraction induced injury. Force generation was not increased. CONCLUSIONS: a7 integrin gene transfer protects the dystrophin-deficient muscle fiber against the deleterious effects of contraction induced injury. Overtime this reduces segmental necrosis and preserves muscle fibers. We are currently treating mdx/utrn-/- mice to determine if there is a similar improvement in muscle physiology and further investigating the mechanism that is involved in the protection from eccentric contraction induced injury.

8) [P04.086] Delayed Recognition of Dysphagia and Malnutrition among Teenaged Boys with Duchenne Muscular Dystrophy

Aimee Gasior, Douglas Sproule, New York, NY

OBJECTIVE: To evaluate the relationship between onset, recognition, and intervention for weight loss among teenaged boys with Duchenne muscular dystrophy (DMD) by treating physicians at a tertiary pediatric neuromuscular center. BACKGROUND: DMD is a neuromuscular disease leading to progressive weakness and loss of ambulation. While excessive weight is common early in the disease, gradual weight loss ensues in later stages. Weight loss in DMD has been attributed to muscle wasting and feeding difficulties, and malnutrition due to weakness of chewing and swallowing may anticipate frank dysphagia by years. We sought to ascertain the relationship and timing of weight loss and clinically recognized dysphagia in the DMD population. DESIGN/METHODS: This was a retrospective chart review of 60 boys with DMD, aged ≥10 years. Weight was obtained during routine clinical care and Z-scores were determined based on established normative values for age and gender. Age at malnourishment (defined as having weight Z-score ≤-2 or experiencing a drop in Z-score of ≥1 from baseline), and age at weight loss (drop in Z-score of ≥0.5) were determined. Ages at clinician-documented concern of malnutrition or weight loss, swallow evaluation, and gastrostomy were recorded. RESULTS: Malnourishment is common among older boys, affecting 5% of boys age 10-11 and 67.1% of boys 20 and older. Weight loss and malnourishment occurred at approximately the same age (174.45 and 174.6 months, respectively), reflecting a rapid decline in weight after long periods of relative stability. Initial clinician-documented concern of weight loss or malnutrition occurred at a mean age of 184.7 months, approximately 10 months after onset of weight loss. CONCLUSIONS: Our results suggest that boys with DMD enter into an energy deficit related to feeding dysfunction before the development of overt dyphagia and clinically obvious malnutrition, resulting in weight loss that persists for months to years before clinical recognition and management. Supported by: American Academy of Neurology.

9) [P02.001] Functional Outcome Measures Correlated with Strength in Becker Muscular Dystrophy

Lindsay Alfano, Linda Lowes, Laurence Viollet, Kevin Flanigan, Jerry Mendell, Columbus, OH

OBJECTIVE: To determine the relationship between quadriceps strength and performance on functional outcome measures in subjects with Becker muscular dystrophy (BMD). BACKGROUND: BMD is characterized by progressive muscle weakness. Upcoming treatment trials are designed to improve quadriceps muscle strength in these patients. However, the correlation of quadriceps strength to performance on functional outcomes has not been evaluated in BMD. Determination of the proper outcome measures is critical to the success of future interventions. DESIGN/METHODS: 24 subjects with BMD underwent strength testing of quadriceps and hamstrings muscles. Performance on the modified Timed Up & Go (mTUG), Berg Balance Scale (BBS), timed 4 stairs, curb steps, and 10 meter, 2 and 6 minute walk tests was recorded in one study session. RESULTS: Performance on the BBS was most highly correlated with quadriceps strength in BMD (r=0.648, p=0.012), followed by stepping up on curbs (r=0.612, p=0.02), timed 4 stairs (r=-0.554, p=0.026), and mTUG (r=-0.431, p=0.045). Distance walked in 2 (r=0.385, p=0.063) and 6 (r=0.378, p=0.068) minutes was not significantly correlated with quadriceps strength in this population, nor was performance on the 10 meter walk test (r=-0.294, p=0.571). CONCLUSIONS: Functional performance relates to distribution of muscle weakness and is disease specific. Selection of appropriate outcome measures should be investigated based on targeted intervention and must be established prior to clinical trials. In patients with BMD, performance on the BBS, stair climbing with and without upper extremity support, and the mTUG are most highly correlated with quadriceps strength, the target of current follistatin gene therapy. It is also of note that BMD function and strength correlations differ from those in DMD and sIBM. Supported by: Parent Project Muscular Dystrophy and The Myositis Association

 10) [S15.004] Results from a National Cross-Sectional Study of Disease-Burden in Facioscapulohumeral Muscular Dystrophy (FSHD)

Chad Heatwole, Rochester, NY, Rita Bode, Chicago, IL, William Martens, Michael McDermott, Richard Moxley, Christine Quinn, Alrabi Tawil, Rochester, NY, Nan Rothrock, Chicago, IL, Barbara Vickrey, Los Angeles, CA, David Victorson, Evanston, IL, Nicholas Johnson, Rochester, NY

OBJECTIVE: To assess: 1) The most critical symptoms and disease manifestations in FSHD patients; and, 2) The modifying factors that are associated with these symptoms. BACKGROUND: FSHD is a dominantly inherited multisystem disease capable of impairing the physical, mental, and social health of patients. The identification of the symptoms and disease manifestations that are most significant to patients' health and the relationship between the severity of these manifestations and other patient characteristics are currently lacking. These data are necessary to develop valid disease-specific patient-reported outcome measures for use in future FSHD clinical trials. DESIGN/METHODS: A cross-sectional study of 328 genetically or clinically confirmed adult FSHD patients from the National Registry of FSHD patients between September and December 2010. The prevalence and relative impact score of 250 critical symptoms and 15 disease themes previously identified through FSHD qualitative interviews were assessed. Responses were categorized by age, gender, education level, and duration of symptoms. RESULTS: FSHD participants from 46 states provided over 48,000 symptom rating responses to address the relative frequency and importance of each FSHD symptom. Problems with shoulders or arms (96.9%), inability to do activities (94.7%), fatigue (93.8%), back, chest, or abdomen weakness (93.8%), and limitations with mobility or walking (93.6%) were the symptomatic themes with the highest frequency in FSHD. Participants identified problems with shoulders or arms and limitations with mobility or walking as the symptomatic themes with the greatest impact on their lives. Significant associations were found between demographic data and the prevalence and relative impact of specific FSHD symptoms on patients' lives. CONCLUSIONS: In this national cross-sectional study patients with FSHD identified the symptoms of highest frequency and greatest impact. These symptoms, some under-recognized, vary based on patient age, gender, and duration of symptoms. Fortunately many, perhaps all symptoms, may be amenable to future therapeutic intervention. Supported by: The National Institute of Arthritis and Musculoskeletal and Skin Disorders (1K23AR055947), the NIH supported Senator Paul D. Wellstone Muscular Dystrophy Cooperative Research Center (U54NS48843-01), the Muscular Dystrophy Association, and the University of Rochester Clinical Translational Science Institute.

11) [P04.082] Focal Presentations of Facioscapulohumeral Dystrophy

Anhar Hassan, Gainesville, FL, Lyell Jones, Margherita Milone, Neeraj Kumar, Rochester, MN

OBJECTIVE: To identify additional focal presentations of FSHD. BACKGROUND: Facioscapulohumeral dystrophy (FSHD) is an autosomal dominant myopathy, classically presenting with facial and shoulder girdle weakness. Recently, monomelic lower limb presentations of genetically confirmed FSHD have been reported. DESIGN/METHODS: A retrospective review of our academic center medical record database of all FSHD cases seen from 1996 to 2011. Of 139 cases identified, 8 had DNA confirmation with a focal presentation. Demographic and clinical data was abstracted. RESULTS: There were 8 cases (4 male) with mean age at onset 39 years (range 18-63) and age at diagnosis 45 years (range 20-74). Presenting symptoms were monomelic lower limb atrophy (3); unilateral shoulder atrophy (3); and axial weakness (2). Examination showed isolated focal weakness in five. CK was normal or mildly elevated. All had a single family member with “weakness”. Co-existent unusual symptoms included dyspnea (2), S1 radicular pain with calf atrophy (2), peripheral neuropathy (1) and post-traumatic onset (1). EMG showed myopathic changes in all, apart from one case with severe denervation later followed by myopathic changes. DNA analysis showed D4Z4 EcoRI fragment size ranging from 20 to 37 kilobases. CONCLUSIONS: The clinical phenotype of FSHD should be expanded to include focal atrophy with myopathic EMG findings; axial weakness; or dyspnea at presentation. These cases are challenging due to lack of typical FSHD signs and coexistent symptoms suggestive of an alternative diagnosis.

12) [P05.186] Identifying High Impact Symptoms and Issues in Congenital and Juvenile Myotonic Dystrophy

Nicholas Johnson, Elizabeth Luebbe, Eileen Eastwood, Nancy Chin, Richard Moxley, Chad Heatwole, Rochester, NY

OBJECTIVE: To assess the symptoms impacting health related quality of life (HRQOL) in congenital and juvenile onset (CMD/JMD) Myotonic Dystrophy. BACKGROUND: Myotonic Dystrophy type-1 is a multisystemic, autosomal dominant disorder. Significant expansion of the trinucleotide repeat mutation may result in the onset of symptoms in infancy (CMD) or childhood (JMD). There is minimal data at this point with regards to HRQOL in CMD/JMD patients. DESIGN/METHODS: We conducted in-depth individual patient and parent interviews with 7 CMD patient-parent dyads and 5 JMD patient-parent dyads for a total of 20 children and 12 parents. Children were over the age of 8 and had a wide range of disability. Each interview focused on identifying the issues that have the greatest impact on patient HRQOL. Each interview was recorded, transcribed, coded, and analyzed using a qualitative framework technique, triangulation, and three investigator consensus approach. RESULTS: 32 patient-parent interviews were conducted. 195 critical symptoms of importance were identified. These symptoms were categorized into 20 themes that represent the physical, mental, social, and disease-specific HRQOL of the CMD/JMD populations. A disease-specific HRQOL model was created for CMD/JMD representing the most relevant issues for this population. Categories in this model included: 1) Mobility and ambulation; 2) Proximal leg weakness; 3) Truncal muscle weakness; 4) Fine motor and upper extremity function; 5) Emotional distress; 6) Cognitive impairment; 7) Social role dissatisfaction; 8) Social role limitation; 9) Genetic issues; 10) Activity impairment specific to CMD/JMD; 11) Sleep disturbance; 12) Fatigue; 13) Pain; 14) Myotonia; 15) Gastrointestinal dysfunction; 16) Problems swallowing or choking; 17) Central sensory impairment; 18)Communication difficulties; 19) Incontinence; and 20) Impaired body image. CONCLUSIONS: There are multiple themes and symptoms that impact CMD/JMD HRQOL. These issues must be carefully examined to develop a disease specific patient-reported outcome measure and functional rating scale that represents the most critical areas of CMD/JMD health.

13) [S15.005] Anoctamin 5 Mutations Are a Frequent Cause of Limb Girdle Muscular Dystrophy and of the Miyoshi-Type Muscular Dystrophy in the Netherlands

Marianne De Visser, Anneke J. van der Kooi, W. H. J. P. Linssen, Amsterdam, Netherlands, Ieke H. B. Ginjaar, Leiden, Netherlands, John Wokke, Utrecht, Netherlands, Pieter Van Doorn, Rotterdam, Netherlands

OBJECTIVE: To perform Anoctamin 5 (ANO) mutation analysis, neurological and cardiological examination in the genetically undiagnosed remainder of the Dutch limb girdle muscular dystrophy (LGMD) cohort encompassing 105 patients from 68 families and in a proportion of a cohort of 22 patients from 16 families with a Miyoshi-type muscular dystrophy (MMD) in which no dysferlin mutations were found. BACKGROUND: ANO 5 mutations have recently been found to cause LGMD2L, MMD3 and asymptomatic hyperCKemia. DESIGN/METHODS: Neurological examination, cardiological investigations (i.e. electrocardiography, Holter monitoring, echocardiography), serum creatine kinase (sCK) activity assessments and ANO 5 mutation analysis were performed in LGMD and MMD patients. RESULTS: 32 LGMD index patients and 12 MMD3 patients from 8 kindreds were examined. ANO 5 mutations were found in 13 sporadic LGMD cases (8 males). Age at onset ranged from 21-57 years, age at examination from 26-69. Symmetrical proximal leg muscle weakness had been the first manifestation. Rhabdomyolysis attacks were mentioned in one patient, and three had cardiological abnormalities, including intraventricular septum thickening (2) and hypertrophic cardiomyopathy (1). SCK was 10-25 times the upper limit of normal (ULN). In 8 patients (all male) from 6 MMD families mutations were identified. Age at onset ranged from 18-51 years and age at examination from 30-67. Uni- or bilateral calf muscle weakness had been the presenting symptom in 7 patients and one had been asymptomatic at time of diagnosis. SCK was 20-40 times ULN. None had associated symptoms and cardiological examination was normal. Exon 5 c.191dupA was the most frequently observed mutation and in addition novel mutations were identified. CONCLUSIONS: 1. LGMD 2L is found in 16% of the Dutch LGMD families and MMD3 in 40% of the MMD families.
2. In three LGMD2L patients heart involvement was found and in none of the MMD3 patients.

14) [S15.006] Limb Girdle Muscular Dystrophy Type 2L (Anoctamin 5 Deficiency): Cardiac Involvement and Characterization of the Skeletal Muscle Phenotype

John Vissing, Nanna Witting, Helle P. Lauritsen, Morten Duno, Copenhagen, Denmark

OBJECTIVE: To test the prevalence of the newly discovered limb girdle muscular dystrophy type 2L (LGMD2L) in Denmark, and further characterize the phenotype. BACKGROUND: Mutations in the anoctamin 5 gene causes LGMD2L, which was first described in 2010. Two existing reports on small LGMD2L cohorts, suggest that LGMD2L may be a relatively common cause of LGMD2. Less than 30 patients have been reported so far. It appears that the disease, like LGMD2B (dysferlinopathy) may present as either classical LGMD2 or as a distal myopathy (MMD3), primarily involving the posterior calf musculature. In this study, we determined the prevalence of anoctamin 5 deficiency in a cohort of patients with unclassified LGMD2, hyperCKemia or distal myopathy. DESIGN/METHODS: All unclassified LGMD2 patients (26 out of 161), unexplained hyperCKemia (15 patients) and distal myopathies (5 patients) were tested for mutations in ANO5. All mutation-positive patients underwent a clinical exam, echocardiography and 48-h Holter monitoring. RESULTS: Ten of 26 unclassified LGMD2 patients, 2 of the 15 hyperCKemia patients and 4 of 5 distal myopathy patients had LGMD2L. All patients carried the common c.191dupA mutation on at least one allele. Echocardiograhies were unremarkable, but Holter monitoring showed a 2.5-fold increase in premature ventricular contractions, which predicts an increased risk of cardiovascular disease. Only 2 of the 16 patients were women. In distal forms, the medial gastrocnemius muscle was the preferentially affected muscle, but the phenotype converges into a LGMD2 phenotype in all patients with time. Calpain 3 expression on Western blots was decreased in about half the patients. CONCLUSIONS: This study suggests that LGMD2L (16 patients) is the third commonest LGMD2 type in Denmark, only surpassed by LGMD2I (64 patients) and LGMD2A (22 patients). The study demonstrates for the first time that cardiac involvement may occur in this disease, and suggests a regular electrocardiographic follow-up of these patients.

15) [P04.083] Patient-Reported Disease Burden and Progression in Genetically Confirmed Participants in a National Facioscapulohumeral Muscular Dystrophy Registry

Jeffrey Statland, William Martens, Alrabi Tawil, Rochester, NY

OBJECTIVE: To determine disease progression in a genetically defined population of facioscapulohumeral muscular dystrophy (FSHD) patients. BACKGROUND: Recent breakthroughs in the molecular pathophysiology of FSHD have identified potential therapeutic targets. Consequently, an accurate understanding of disease progression in FSHD is crucial for the design of future clinical trials. DESIGN/METHODS: De-identified data from 343 genetically confirmed FSHD patients with an average 6 years of follow up (range 1-9) was analyzed from the National Registry of FSHD Patients and Family Members. Measures of disease included patient-reported symptoms, extra-muscular manifestations, use of assisted devices, pain, and a patient reported functional rating scale (4-8 mild, 9-13 moderate, 14-19 severe). RESULTS: Registry participants were 51% female, primarily Caucasian, non-Hispanic, with a mean age of 51 years. The median D4Z4 fragment size was 25 kb. The initial age of symptom onset was 22 years, and mean time until diagnosis 10 years. At baseline 91% had facial weakness, 70% arm involvement, and 67% leg involvement. 41% used an ankle brace, leg brace, cane, or walker. 24% used a wheelchair at some point during the day, with a mean age at first use of 42 years. 4% used a breathing machine, 18% reported hearing loss, 7% used hearing aids, and 1% reported a retinal hemorrhage or detachment, but no history of Coat's disease. The average self-reported total impairment score was 10.4 (moderately impaired). At follow up 11% of device-free patients started to use an ankle brace, leg brace, cane or walker. 22% started to use a wheelchair at some point during the day. And the self-reported total impairment score increased by 0.78. CONCLUSIONS: Genetically confirmed Registry participants are moderately impaired, demonstrate slow but steady progression of disease, and represent a valuable resource for future FSHD clinical trials. Supported by: NIH Experimental Therapeutics in Neurological Disorders grant #T32 NS07338-20 (PI, R. Griggs).

16) [P05.190] Is There a Difference in Gastric Emptying between Myotonic Dystrophy Patients with and without Gastrointestinal Symptoms? An Analysis Using the 13C-Acetate Breath Test

Yuji Tanaka, Tomohiro Kato, Hiroshi Nishida, Megumi Yamada, Akihiro Koumura, Takeo Sakurai, Akio Kimura, Isao Hozumi, Hisataka Moriwaki, Takashi Inuzuka, Gifu City, Japan

OBJECTIVE: To clarify gastric emptying of patients with myotonic dystrophy (MyD). BACKGROUND: Gastrointestinal symptoms are frequent complaint in patients with MyD and may be associated with reduced gastrointestinal motility as dysfunction of smooth muscle. However, it is few report of gastric emptying in MyD patients. In the present study, we investigated gastric emptying in MyD patients using the 13C-acetate breath test (13C-ABT). DESIGN/METHODS: We investigated gastric emptying in 19 MyD patients and 20 healthy volunteers. The MyD patients were divided into two groups: 7 patients with gastrointestinal symptoms and 12 patients without gastrointestinal symptoms. Gastric emptying was estimated by the 13C-ABT [the half emptying time (HET), the peak time of the 13C %-dose-excess curve (Tmax)], with expirations collected every 10 minutes for 4 hours after a test meal and analyzed for 13CO2 using an IR spectrophotometer. Informed consent was obtained from each subject prior to participation in this study. The study protocol was approved by the Ethical Committee of our institution and carried out in accordance with the 1975 Declaration of Helsinki. RESULTS: 1) The HET and Tmax of gastric emptying as assessed using the 13C-ABT was significantly delayed in MyD patients with and without gastrointestinal symptoms as compared to the controls (P<0.05). 2) The HET and Tmax were significantly delayed in MyD patients with gastrointestinal symptoms as compared to those without gastrointestinal symptoms (P<0.05). 3) There was a significant correlation between the HET and Tmax of MyD patients with gastrointestinal symptoms and duration of the disease. CONCLUSIONS: The results demonstrated that gastric emptying of the MyD patients was delayed, even those without gastrointestinal symptoms. The gastric emptying was delayed in MyD patients with gastrointestinal symptoms as compared to those without gastrointestinal symptoms. Delayed gastric emptying is one of constitutional symptoms of the MyD patients.

17) [P05.184] Symptoms of Myotonia as Reported by Patients with Myotonic Dystrophy Type 1: Location, Description and Severity- Implications for Clinical Trials

Shree Pandya, Katy Eichinger, Nuran Dilek, Jeanne Dekdebrun, William Martens, Chad Heatwole, Charles Thornton, Richard Moxley, Rochester, NY

OBJECTIVE: This study documents patient-reported descriptions of the location and severity of myotonia in patients with DM1. BACKGROUND: Myotonia is a cardinal disease manifestation in myotonic dystrophy type 1 (DM-1). Clinicians identify and grade myotonia based upon the tests they perform rather than asking patients to describe and rate their symptoms related to myotonia. DESIGN/METHODS: Data were gathered as part of a disease progression study. Patients were asked to fill out a survey that included questions about symptom/s of myotonia - Y/N, the body area affected, a description of the symptoms and the severity of the symptoms using a visual analog scale. Patients also filled out the Individualised Quality of Life (INQoL) instrument which includes questions about myotonia and the impact of myotonia on their health related quality of life. RESULTS: Of the 71 patients in the current cohort 65 (91.5%) report problems related to myotonia. Patients used terms like myotonia, cramps and tightness (51%). They also described important associated symptoms [weakness (30%); pain (11%); and, other (8%)]. The majority of myotonia related symptoms were associated with hand function (63%) and approximately 10% each described symptoms in face/tongue/jaw functions, calf/thighs/legs and back/neck. The severity scores for myotonia on the VAS were: back/neck 67; calf/thighs/legs 65; hands/fingers 57; and face/tongue/jaw 37. CONCLUSIONS: To our knowledge this is the first study that has directly asked DM1 patients to describe their symptoms related to myotonia. Along with symptoms related to myotonia, DM1 patients reported other associated symptoms. This suggests that many patients associate myotonia with other disease manifestations. For this reason prior to using any patient reported outcome measure of myotonia in clinical trials, it will be essential to assure that patients are able to differentiate the symptoms of myotonia from other manifestations of this multisystem disorder. Supported by: U54NS048843 from the National Institute of Neurological Disorders and Stroke.

18) [P05.187] Relationships between Upper Extremity Strength and the Purdue Pegboard Test and the Jebsen-Taylor Hand Function Test in Patients with Myotonic Dystrophy Type 1

Katy Eichinger, Nuran Dilek, Jeanne Dekdebrun, William Martens, Chad Heatwole, Charles Thornton, Richard T. Moxley, Shree Pandya, Rochester, NY

OBJECTIVE: To document the relationship between measures of upper extremity strength and hand function in patients with myotonic dystrophy type 1(DM1). BACKGROUND: Distal weakness is a common feature in patients with (DM1). The assessment of hand function in this patient population is just beginning to be explored. Measurements of upper extremity strength and hand function have demonstrated good reliability; however, the relationship between strength and function is unknown. DESIGN/METHODS: Patients with DM1 participating in a longitudinal study performed strength tests of the upper extremity including manual muscle testing (MMT) and quantitative muscles testing (QMT). MMT included shoulder abduction, elbow flexion and extension, wrist flexion and extension, and short and long finger flexors of the thumb and first two digits. QMT included shoulder abduction, elbow flexion, elbow extension and grip. Patients also performed the Purdue Pegboard Test (PPT) and the Jebsen-Taylor Hand Function Test (JTHFT). RESULTS: Data from 71 genetically confirmed patients with DM1 were analyzed using Spearman's rho. Statistically significant (p<.0001) relationships were documented between upper extremity MMT and the PPT (r=0.48); upper extremity QMT and overall JTHFT (r=-0.52). The relationships between QMT and the 3 of 7 total subtests of the JTHFT were found to be significant with lifting heavy objects being the most significant. (r= -0.58). CONCLUSIONS: Moderate relationships exist between upper extremity strength tests and hand function tests. The PPT correlates better with MMT which included several distal hand muscles and hence captures fine motor function deficits. The JTHFT correlated better with QMT and the most significant correlation was with lifting heavy objects and this may reflect more the proximal strength deficits. Patients need to be followed longitudinally to document which of these tests is able to reflect changes over time. Supported by: Award number U54NS048843 from the National Institute of Neurological Disorders and Stroke.

19) [PD6.003] Role of RNA and MBNL1 Nuclear Foci in Pathomolecular Mechanism in Myotonic Dystrophy Type 1 and Type 2

Giovanni Meola, Milan, Italy, Valentina Renna, Enrico Bugiardini, San Donato Mil., MI, Italy, Marzia Giagnacovo, Carlo Pellicciari, Pavia, Italy, Manuela Malatesta, Verona, Italy, Rosanna Cardani, Milan, Italy

OBJECTIVE: To study RNA and MBNL1 nuclear foci in myotonic dystrophies to elucidate their role in pathomolecular mechanism. BACKGROUND: Myotonic dystrophies (DM1 and DM2) are multisystemic RNA-mediated diseases. The presence of the CTG or CCTG expansion, leads to nuclear accumulation of expanded RNAs which interact with RNA binding proteins, such as MBNL1. The nuclear sequestration of MBNL1 appears to be involved in splicing defects of genes directly related to the DM phenotypes. However, the mechanisms underlying nuclear retention of expanded RNAs and their aggregation into nuclear foci are still not well understood. A recent study has revealed that CUG-foci are constantly aggregating and disaggregating structures, and that MBNL1 is directly involved in this process. DESIGN/METHODS: FISH in combination with MBNL1-immunofluorescence was performed on: 1) 3 DM2 skin fibroblast cultures to investigate the fate of MBNL1 foci in proliferating and in non-cycling cells; 2) muscle biopsies from 3 DM2 patients who underwent 2 biopsies at different years of age and from 3 DM1 patients for morphometric measurements of nuclear RNA and MBNL1 foci. RESULTS: Nuclear MBNL1 foci do not associate with chromosomes at mitosis, and remain in the cytoplasm at cytodieresis, being disassembled in early G1 and re-formed in the nucleus, at each cell cycle. In senescent fibroblasts the nuclear foci increase in number and size indicating that in non-dividing cells the sequestration of factors for RNA processing would be continuous and progressive. Interestingly, foci in DM2 muscle become larger with increasing patient's age thus suggesting that a relationship may exist between the amount of foci and the severe phenotype. CONCLUSIONS: However, despite DM1 phenotype is more severe than DM2 one, RNA and MBNL1 foci appear to be smaller in DM1 than in DM2 muscle. This suggests that DM pathomechanism is more complex and that other processes might be involved.
Supported by: by AFM, CMN and FMM.

20) [P05.189] Application of Digital Accelerometry for Swallow Imaging (DASI) for Dysphagia Evaluation and Treatment in a Series of Myotonic Dystrophy Type 1 (DM1) Patients

Lia-Ana Farkas, Lane Entrekin, Norma Milstead, Kristin Mosman, Kiera Berggren, Jacinda Sampson, Salt Lake City, UT

OBJECTIVE: To use digital accelerometry for swallow imaging (DASI) for evaluation and treatment of dysphagia in DM1 patients. BACKGROUND: Dysphagia is a potentially life threatening complication of DM1 patients. DESIGN/METHODS: Patients with DM1 and history of aspiration or dysphagia underwent a formal swallow evaluation +/- modified barium swallow (MBS). We used noninvasive DASI technology with software designed for swallow imaging and calculation of key parameters of swallow biomechanics. Trials included spontaneous and instructed dry and wet swallows with different consistencies. We analyzed shape, amplitude, timing, frequency and jitter looking for swallow movement dysfunction. DASI biofeedback: Patients observed swallow traces on the computer screen and were coached in improving strength, duration and timing of swallow. RESULTS: Case 1: A 24-year old man with congenital DM1, dysphagia, and repeated hospitalizations for aspiration pneumonia had a MBS showing aspiration of pyriform sinus residue. DASI evaluation showed excessive pre/post-swallow laryngeal movement, decreased pharyngeal phase control with multiple small amplitude peaks. DASI Treatment: Patient improved swallow strenght and control of pharyngeal movement. Case 2: A 46 year-old man with adult-onset DM1 and choking episodes was hospitalized with pneumonia and placed on a ventilator. MBS showed aspiration during/after the swallow with delayed cough. DASI evaluation showed low amplitude and frequency of swallow with increased jitter and frequent coughing. DASI Treatment: Patient improved swallow strength. Case 3: A 33 year-old female with adult-onset DM1 reported dysphagia. Clinical swallow evaluation showed oral residue, reduced hyolaryngeal excursion, and throat clearing. DASI evaluation showed reduced amplitude and increased jitter, with extraneous, inefficient lingual/hyolaryngeal movements. DASI Treatment: Reduced extraneous laryngeal movements and improved control of swallow. CONCLUSIONS: DASI is an adjunct in dysphagia evaluation in DM1. DASI biofeedback is helpful in gaining increased swallow function through Active Repetitive Motion Therapy which is instructive for reducing extraneous, inefficient laryngeal movements and can be personalized according to the patient physical and mental status.
Supported by: DASI equipment on loan from Elixir Therapeutics.

21) [P05.185] Correlation between Biomarkers and Surrogate Markers in Patients with Myotonic Dystrophy Type 1 (DM-1): Data from the Study of Pathogenesis and Progression in DM (STOPP DM)

Shree Pandya, Katy Eichinger, Nuran Dilek, Jeanne Dekdebrun, William Martens, Chad Heatwole, Charles Thornton, Richard Moxley, Rochester, NY

OBJECTIVE: To document correlations between biomarkers (muscle mass and strength measures) and surrogate markers (function and health related quality of life (HRQoL measures) in patients with DM-1. BACKGROUND: Research funding agencies and regulatory agencies are increasingly requiring investigators to validate and use outcome measures that patients feel have a direct relationship to their well being and performance. DESIGN/METHODS: Patients participating in the STOPP DM study at our institution undergo assessments of: muscle mass ( DXA), manual muscle testing (MMT) on 38 muscles, quantitative muscle tests (QMT) on 14 muscles, function testing – 6 and 2 minute walk tests (6MWT& 2MWT), run/walk 30', ascend/descend 4 stairs, rise from a chair, Purdue pegboard test (PPT), Jebsen Taylor Hand function test (JTHFT); and complete, HRQOL surveys (Short Form 36 & Individualised Quality of Life-INQoL). Data from patients who have undergone all the above evaluations at baseline were analyzed using Pearson correlation coefficients. Statistical significance was set at p<.001. RESULTS: To date 71(46F) patients average age 46.9yrs (18-69) have enrolled in the study. Significant correlations (p<.0001) were documented between muscle strength (MMT) and function tests- 6MWT (r=0.65), 2MWT(r=0.64), go 30' (r=-0.61), ascend/descend stairs (r=- 0.0.53/-0.51) and rise from chair (r=- 0.44). There were also significant correlations between strength (MMT/QMT) and INQoL weakness score (r=-0.65/r=-0.51) and SF-36 physical function score (r=0.58/r=0.46). Correlations were even higher if only the lower extremity strength scores were used. CONCLUSIONS: We have documented significant relationships between measures of muscle strength, function and quality of life in patients with DM-1. These correlations and subsequent longitudinal analyses of our findings at 12 and 36 months in the STOPP DM cohort will provide necessary guidance in selecting appropriate outcome measures for future therapeutic trial phases. Supported by: U54NS048843 from the National Institute of Neurological Disorders and Stroke.

22) [P05.188] Video Hand Opening Time (vHOT) in Myotonic Dystrophy Type 1 (DM1)

Araya Puwanant, Jeffrey Statland, Nuran Dilek, Richard Moxley, Charles Thornton, Rochester, NY

OBJECTIVE: To evaluate test-retest reliability of vHOT in DM1. BACKGROUND: Delayed release of the handgrip is a common symptom in DM1, reflecting myotonia plus a variable contribution of finger extensor weakness. Previous methods to quantify myotonia required sophisticated equipment and labor-intensive analysis. Video recording coupled with blinded review is an accepted procedure to evaluate motor performance in clinical trials. DESIGN/METHODS: vHOT recordings were made using a standardized procedure in 20 individuals with DM1. Subjects were asked to perform maximal handgrip for 5 seconds, followed by rapid extension of fingers without shaking of the hand. Each participant completed 3 trials with a 10-minute rest between trials. vHOT was recorded on 2 consecutive days by the same evaluator. vHOT recordings were later reviewed using the standard video editing software by 2 blinded raters. Time for maximal hand opening was measured separately for the thumb and long finger. RESULTS: The mean vHOT was 4.6 ± 5.6 seconds. vHOT was longer for the thumb than the long finger. The correlation of vHOT between trials on the same day (r = 0.94-0.97) was better than for trials on the separate days (r = 0.88). There is no evidence for “warm-up” in successive trials. The ICC for test-retest concordance for the same rater on 2 successive days was 0.71. The ICC for concordance between 2 raters was 0.96. CONCLUSIONS: vHOT provides a simple low-cost method to assess grip myotonia and finger extension. The inter-rater reliability is excellent and the test-retest reliability is moderate, reflecting the physiologic variability of myotonia over time. vHOT may provide a suitable endpoint for studies of disease progression and therapeutic response in DM1.
Supported by: NIH U54NS48843.

23) [S15.001] Defining Dystrophin-Specific T Cells in DMD Population

Laurence Viollet, Katie Campbell, William Bremer, Chelsea Rankin, Christopher Shilling, Kevin Flanigan, Christopher Walker, Jerry Mendell, Columbus, OH

OBJECTIVE: To determine the frequency of DMD patients with cellular immunity to dystrophin. BACKGROUND: Duchenne muscular dystrophy (DMD) is the most severe childhood form of muscular dystrophy. It is caused by mutation of the DMD gene. Studies attempting to replace the dystrophin gene using adeno-associated virus revealed pre-existing dystrophin-specific T cells that demonstrated an amnestic response upon gene transfer. Analysis revealed an HLA-restricted, exon specific response targeted to revertant fibers. DESIGN/METHODS: Seventy-six DMD patients were enrolled in this study. Six BMD (Becker MD, a milder form of DMD) and nine healthy subjects were used as controls. Age and corticosteroids (prednisone or deflazacort) regimen were recorded. Peripheral blood mononuclear cells were isolated and stimulated with peptide pools specific to dystrophin. Detection of IFN-
γ was measured by ELISpot assays. RESULTS: 52.9% (9/17) of DMD steroid-naïve patients exhibited an immune response. The response was blunted by steroid treatment: prednisone 25% (6/24); deflazacort 17.2% (5/29). In comparison, only one BMD subject had a response (16.6%, 1/6) and no response was found in normal controls. DMD patients and normal controls were around the same age (11.9 ± 4.6 years) while BMD subjects were older (20.5 ± 9.6 years). Thirty adult controls (age range 21-45) from a previous study did not show any immune response either. In three DMD groups, patients with an immune response were slightly older than the ones with no response. However, dosage of steroids and duration of treatment were not statistically different between responders or not. CONCLUSIONS: These results show that corticosteroids may have a protective effect on dystrophin-specific T cell response in DMD patients. This may be related to steroid responsiveness in this disease. In addition, immune response should be assessed prior and throughout therapeutic clinical trials since it may influence outcomes. Supported by: NIH.

24) [S49.005] Moderate Walking Distances and Velocity Correlate with Function Comparable to 6 Minute Walk Test

Linda Lowes, Lindsay Alfano, Laurence Viollet, Kevin Flanigan, Jerry Mendell, Columbus, OH

OBJECTIVE: To evaluate the utility of different walking tests in neuromuscular disease. BACKGROUND: Many different timed walking tests have been shown to be accurate, reproducible, and simple to administer (10 meter walk (10mw), and the 2-minute (2MWT), 6-minute (6MWT) and 12-minute walk distance). Recently the 6 MWT has emerged as the gold standard for use in clinical trials. DESIGN/METHODS: A total of 196 subjects (n= Duchenne Muscular Dystrophy (DMD) =85; Becker Muscular Dystrophy BMD=24; Inclusion Body Myositis (IBM) = 87) performed a variety of timed walking tests (10mw, distances walked in minute increments from 1-6 minutes). Walking ability was compared to quadriceps strength and the modified Timed Up & Go (mTUG), stepping up on curbs, stair climbing, and the North Star Ambulatory Assessment (NASA). RESULTS: Test/retest reliability is excellent for timed walking of any distance (range r =0. 898-0.972, p=.000). In BMD and IBM, the 2MWT was most highly correlated with performance on other strength and functional outcomes (BMD: stairs r=-0.836, Berg Balance Scale(BBS) r=0.891,; IBM: stairs r=-0.748, quadriceps strength r=0.585,(all p<0.001) compared to the 6MWT (BMD: stair climbing r=-0.836, BBS r=0.853; IBM: stairs r=-0.652, quadriceps strength r=0.558, (all p<0.001). In DMD, all distances were moderately to highly correlated with other functional outcomes (r=0.562-0.951, p<0.01), however, the 3MWT, 4MWT, and 5MWT were highly correlated consistently across outcomes (3MWT: 10mw r=-0.948, NASA r=0.871, stairs r=-0.768, p<0.001; 4MWT: 10mw r=-0.951, North Star r=0.879, stairs r=-0.766, p<0.001; 5MWT: 10mw r=-0.950, NASA r=0.891, stairs r=-0.784, p<0.001). CONCLUSIONS: A fixed walking distance such as 6 minutes is not necessary to assess function in patients with neuromuscular disease. Shorter distances enable more individuals to participate and require less time. Comparison to previously reported studies is possible by converting the distance walked to velocity.
Supported by: Parent Project Muscular Dystrophy. The Myositis Association.

25) [P01.116] Congenital Megaconial Myopathy Due to a Novel Defect in the Choline Kinase beta (CHKB) Gene

Purificacion Gutierrez Rios, New York, NY, Arun Asha Kalra, Shreveport, LA, Jon Wilson, Kurenai Tanji, H. Akman, Estela Area, Eric Schon, Salvatore DiMauro, New York, NY

OBJECTIVE: To describe the first American patient with mutations in the gene encoding choline kinase beta (CHKB), a novel cause of congenital muscular dystrophy with giant and displaced muscle mitochondria. BACKGROUND: Mutations in CHKB have been described in 15 children with congenital muscular dystrophy and mental retardation. The morphologic hallmark was the presence in muscle of giant mitochondria (megaconial myopathy) displaced to the periphery of the fibers (Mitsuhashi et al, 2011). DESIGN/METHODS: Here we describe a 2-year-old African American boy with weakness and psychomotor delay. At 22 months, he was not able to sit up and did not utter any intelligible word. He was severely hypotonic and had multiple dysmorphic features. Serum CK was elevated but lactate was normal. EMG showed a myopathic pattern and brain MRI revealed diffuse atrophy. RESULTS: Both histochemistry and electron microscopy of a muscle biopsy showed greatly enlarged mitochondria, which were pushed to the periphery of the fibers. Biochemical analysis revealed severely decreased cytochrome c oxidase activity (30% of normal). The patient harbored a novel homozygous CHKB mutation (E392X). CONCLUSIONS: Besides confirming the phenotype of CHKB mutations, we propose that this disorder affects the MAM (mitochondria-associated-membrane) and the impaired phospholipid metabolism in MAM causes both the abnormal size and the displacement of muscle mitochondria. Supported by: NIH Grant HD32062 and by the Marriott Mitochondrial Disorders Clinical Research Fund (MMDCRF).
EAS is supported by the Ellison Foundation.

26) [P04.081] Expression of microRNAs in the Histopathological Stages of LGMD2A (Calpainopathy)

Xiomara Rosales, Vinod Malik, Amita Sneh, Lei Chen, Janaiah Kota, Sarah Lewis, Julie Gastier-Foster, Caroline Astbury, Robert Pyatt, Shalini Reshmi, Louise Rodino-Klapac, Reed Clark, Jerry Mendell, Zarife Sahenk, Columbus, OH

OBJECTIVE: To examine expression of regulatory microRNAs in pathological stages of LGMD2A muscle to gain insight into disease pathogenesis. BACKGROUND: Recent in vitro studies have suggested that CAPN3 deficiency leads initially to accelerated myofiber formation followed by a depletion of the satellite cell pool. In normal muscle, upregulation of miR-1 facilitates transition from proliferating satellite cells to differentiating myogenic progenitors. DESIGN/METHODS: A fully characterized LGMD2A cohort (n = 45) had muscle biopsies (n = 39) with quantification of miR-1 expression (n = 12) and Pax 7, a satellite cell biomarker. RESULTS: Three groups of patients clustered by age, duration of disease and clinical severity. Group 1: prominent inflammation (n =8) with eosinophilia (n = 5). Group 2 (n=8): minimal inflammatory infiltrate, accompanied scattered fibers undergoing necrosis and regeneration with minimal focal perivascular fibrosis. Group 3 (n=23): prominent endomysial and perimysial fibrosis, rare necrosis/regeneration without inflammation. A continuum of changes was revealed by age and symptoms duration: group 1= age 13.4 ± 7.1; duration 4.6 ± 3.7 yrs; group 2 = age 25.9 ± 9.1; duration 10.9 ± 5.6 yrs; and group 3 = age 33.9 ± 11.8; duration 17.6 ± 9.3 yrs. Contrary to expectations, a significant increase of Pax7-positive satellite cells was seen in all samples with the greatest increase in the fibrotic group. Findings correlated with significant reduction in miR-1 in fibrotic group compared to others. CONCLUSIONS: The histopathological analysis identified 3 distinct stages of pathological changes representing a continuum of the disease correlating with age and disease duration. Pax7-positive satellite cell number was elevated even in the advanced dystrophic stage where reduced miR-1 failed to promote transition to satellite cell differentiation. This provides a potential entrée for treatment with miR-1 overexpression to facilitate satellite cell differentiation, skeletal muscle maturation, and promotion of normal muscle growth and regeneration. Supported by: NIH NIAMS U54 AR050733-05, Jesse's Journey, and the muscular Dystrophy Association.

27) [P04.089] Reducing Skeletal Muscle Fibrosis with AAV-Delivered miR-29

Eric Meadows, Janaiah Kota, Sarah Lewis, Zarife Sahenk, Reed Clark, Jerry Mendell, Louise Rodino-Klapac, Columbus, OH

OBJECTIVE: To demonstrate the degree of muscle fibrosis in mdx:utrophin heterozygote (mdx:utrn+/-) mice and in dystrophin-deficient patient-derived muscle biopsies, and correlate changes with microRNA-29 isoforms (miR-29a, miR-29b, and miR-29c). BACKGROUND: Duchenne muscular dystrophy (DMD) is a X-linked, inherited disease caused by dystrophin deficiency that results in progressive muscle weakness, endomysial inflammation, and fibrotic scarring. Muscle fibrosis impairs blood flow, excluding endomysial-derived constituents important for muscle repair and regeneration, anti-oxidation, and vascular-derived metabolites. In addition, fibrosis is a critical factor contributing to early loss of ambulation through limb contractures. Irrespective of the success of gene restoration using currently tested clinical approaches functional improvement for patients mandates reduction of muscle fibrosis. The gene regulator microRNA-29 is an ideal candidate for reducing muscle fibrosis. DESIGN/METHODS: Connective tissue was quantified from muscle biopsies of dystrophin-deficient patients and the mdx:utrn+/- mouse model, which exhibits increased endomysial fibrosis, using Sirius Red-stained sections analyzed by NIH ImageJ Software. 5'-nuclease assays were used to measure miR-29 expression levels and correlate with demonstrated levels of fibrosis. RESULTS: In the mdx:utrn+/- mouse, endomysial connective tissue levels were uniformly increased 2 to 4-fold in the diaphragm, gastrocnemius, and quadriceps muscles at 3 and 6-months of age. A 4-fold increase in fibrosis was also observed in muscle biopsies from dystrophin-deficient patients. We correlated these findings with the downregulation of miR-29a and miR-29c in the mouse disease model to approximately 60% of wild-type in most of the tested groups, and in patient biopsies to less than 50% of normal controls. CONCLUSIONS: Demonstration of increased fibrosis and decreased miR-29 expression in mdx:utrn+/- mice and dystrophin-deficient patients validates the mouse model as being representative of the human disease. In addition, these data provide a rationale for overexpression of these miR-29 isoforms to reduce fibrosis in translational, pre-clinical studies in mdx:utrn+/- mice in preparation for clinical trials.

28) [SC02.004] Adeno-Associated Viral (AAV)-Mediated Follistatin (FS) Gene Transfer Toxicology Studies in Preparation for Phase I Clinical Trial

Janaiah Kota, Christopher Shilling, Chrystal Montgomery, Sarah Lewis, Adam Bevan, Kim Shontz, Yuuki Kaminoh, Xiomara Rosales, Laurence Viollet, Kevin Flanigan, Reed Clark, Brian Kaspar, Zarife Sahenk, Jerry Mendell, Columbus, OH

OBJECTIVE: Establish safety of rAAV.CMV.FS gene transfer by intramuscular (i.m.) injection for improving quadriceps strength in preparation for phase I clinical trial. BACKGROUND: FS is a potent myostatin inhibitor that increases muscle size and strength in mice and non-human primates when secreted by muscle following delivery by AAV. Safety concerns have included impeded release of pituitary-gonadal axis hormones and general organ toxicity. In this study we used a non-tissue binding FS isoform (FS344) to address these safety concerns in mice at viral doses 10-fold greater than those planned for clinical use. DESIGN/METHODS: Mice were randomized to three experimental arms with bilateral i.m. injections to quadriceps muscle of rAAV1.CMV.FS344 equal to highest clinical dose (2.0e12 vg/kg) or 10-fold higher (2.0e13 vg/kg). Animals were followed to sacrifice time points at 6-,12-,24-, and 36-weeks (5 animals/sex/cohort/timepoint) post-injection (p.i.). Outcomes included: bodyweight, hematology, chemistry panels, pituitary-gonadal hormones; immune studies, and histopathology of 24 organs from each animal. RESULTS: No treatment-related adverse clinical or toxicology signs were observed and viral DNA in non-injected tissues dissipated by 24 weeks. Within 6 weeks, high dose animals showed scattered focal infiltrates of inflammatory cells corresponding to T-cell immunity to AAV1 identified by IFN-
γ ELISpot, but no other pathology was observed. Wet weights of quadriceps increased by 3.5-fold at 36 weeks p.i. in high dose animals. Muscle fiber hypertrophy was observed at 6 weeks and fiber diameter increased by 50% compared to controls by 36 weeks. Hypertrophic-multinucleated fibers were observed at 12 weeks p.i. with increased density of PAX7 positive nuclei. CONCLUSIONS: This study shows rAAV1.CMV.FS344 is safe and well tolerated at the proposed clinical doses and causes satellite cell proliferation and significant muscle hypertrophy by fusion with differentiated satellite cells. Early transient inflammation in muscle is related to immune response to AAV1. These results support this approach for clinical trial use. Supported by: Grants from The Myositis Association and Parent Project Muscular Dystrophy, Inc; and support from the Foundation at Nationwide Children's Hospital.

29) [PD6.005] Adeno-Associated Viral (AAV)-Mediated Follistatin (FS) Gene Transfer Toxicology Studies in Preparation for Phase I Clinical Trial

Janaiah Kota, Christopher Shilling, Chrystal Montgomery, Sarah Lewis, Adam Bevan, Kim Shontz, Yuuki Kaminoh, Xiomara Rosales, Laurence Viollet, Kevin Flanigan, Reed Clark, Brian Kaspar, Zarife Sahenk, Jerry Mendell, Columbus, OH

OBJECTIVE: Establish safety of rAAV.CMV.FS gene transfer by intramuscular (i.m.) injection for improving quadriceps strength in preparation for phase I clinical trial. BACKGROUND: FS is a potent myostatin inhibitor that increases muscle size and strength in mice and non-human primates when secreted by muscle following delivery by AAV. Safety concerns have included impeded release of pituitary-gonadal axis hormones and general organ toxicity. In this study we used a non-tissue binding FS isoform (FS344) to address these safety concerns in mice at viral doses 10-fold greater than those planned for clinical use. DESIGN/METHODS: Mice were randomized to three experimental arms with bilateral i.m. injections to quadriceps muscle of rAAV1.CMV.FS344 equal to highest clinical dose (2.0e12 vg/kg) or 10-fold higher (2.0e13 vg/kg). Animals were followed to sacrifice time points at 6-,12-,24-, and 36-weeks (5 animals/sex/cohort/timepoint) post-injection (p.i.). Outcomes included: bodyweight, hematology, chemistry panels, pituitary-gonadal hormones; immune studies, and histopathology of 24 organs from each animal. RESULTS: No treatment-related adverse clinical or toxicology signs were observed and viral DNA in non-injected tissues dissipated by 24 weeks. Within 6 weeks, high dose animals showed scattered focal infiltrates of inflammatory cells corresponding to T-cell immunity to AAV1 identified by IFN-
γ ELISpot, but no other pathology was observed. Wet weights of quadriceps increased by 3.5-fold at 36 weeks p.i. in high dose animals. Muscle fiber hypertrophy was observed at 6 weeks and fiber diameter increased by 50% compared to controls by 36 weeks. Hypertrophic-multinucleated fibers were observed at 12 weeks p.i. with increased density of PAX7 positive nuclei. CONCLUSIONS: This study shows rAAV1.CMV.FS344 is safe and well tolerated at the proposed clinical doses and causes satellite cell proliferation and significant muscle hypertrophy by fusion with differentiated satellite cells. Early transient inflammation in muscle is related to immune response to AAV1. These results support this approach for clinical trial use. Supported by: Grants from The Myositis Association and Parent Project Muscular Dystrophy, Inc; and support from the Foundation at Nationwide Children's Hospital.