RESEARCH THAT WILL BE PRESENTED IN AMERICAN ACADEMY OF NEUROLOGY MEETING - APRIL 2011

[P02.059] A Randomized, Placebo-Controlled, Multiple-Ascending Dose Study of ACE-031 (ActRIIB-IgG1) in Duchenne Muscular Dystrophy (DMD)

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

OBJECTIVE: To evaluate the safety, tolerability, pharmacokinetics (PK), and pharmacodynamic (PD) effects of ACE-031 in DMD. BACKGROUND: DMD is caused by dystrophin deficiency resulting in muscle weakness. ACE-031 is a fusion protein comprised of the extracellular domain of activin receptor type IIB and IgG1 Fc domain, which inhibits myostatin and other negative muscle regulators. PRe-clinically, ACE-031 treatment resulted in increased muscle mass, strength and utrophin expression, and decreased intramuscular fibrosis and CK levels. DESIGN/METHODS: 36-48 ambulatory, steroid-treated DMD boys, age 4 years, will be randomized to escalating doses of ACE-031 or placebo (3:1), SC, every 2-4 weeks for 12 weeks, with 12 weeks follow-up. Safety assessments include adverse events, physical examination, laboratory and cardiac monitoring, anti-drug antibodies. PD endpoints include body composition and bone density by DXA, muscle and fat volumes by MRI, muscle strength and function, pulmonary function, bone biomarkers, quality of life. RESULTS: Preliminary baseline data for 24 patients are presented (all correlations p<0.0001 unless indicated). Mean (SD) age: 10.33.1 years; height z-score: -1.91.3; total body %lean mass 65.811.5%; %fat mass 31.511.8%. Distances walked in 1, 2, or 3 minutes correlated with 6-minute walk distance (6MWD) (r>0.92). 6MWD was inversely correlated with time to perform 10-meter walk/run (10MWR) (r=-0.80) and 4-stair climb (4SC) (r=-0.78). 10MWR correlated with 4SC (r=0.90) and Gower (r=0.86); 4SC correlated with Gower (r=0.68, p=0.003, n=17). Lower extremity (LE) %lean mass correlated with LE strength (r=0.62, p=0.003, n=20), 6MWD (r=0.60,p=0.003), 10MWR(r=-0.76), 4SC (r = -0.75), and Gower (r=-0.73,p=0.001,n=16). Total body %fat mass correlated with time to perform 10MWR (r=0.58,p=0.004) and 4SC (r=0.64,p=0.001). CONCLUSIONS: DMD patients on steroids demonstrate baseline correlations of % lean and fat mass with measures of muscle strength and function. This study will provide safety and exploratory outcomes data for ACE-031 in boys with DMD.

[S34.004] Forty-Eight Week Follow-Up Data from a Phase I/IIa Extension Study of PRO051/GSK2402968 in Subjects with Duchenne Muscular Dystrophy

Nathalie Goemans, Leuven, Belgium, Mar Tulinius, Gothenburg, Sweden, Rosamund Wilson, Marlborough, Wiltshire, United Kingdom, Judith van Deutekom, Sjef de Kimpe, Giles Campion, Leiden, The Netherlands

OBJECTIVE: To evaluate the efficacy and safety of 48 weeks of treatment with PRO051/GSK2402968 in boys with Duchenne muscular dystrophy (DMD). BACKGROUND: DMD patients suffer from progressive muscle degeneration due to mutations in the DMD gene and resulting absence of functional dystrophin in the muscle cell wall. PRO051/GSK2402968 is an antisense oligonucleotide compound which induces exon 51 skipping during pre-mRNA splicing and produces novel dystrophin expression in a subpopulation of DMD patients. DESIGN/METHODS: Twelve DMD patients with mutations correctable by skipping exon 51, (11 ambulatory, 1 non-ambulatory at study entry) completed a dose-escalation Phase I/IIa study (Netherlands Trial Register #NTR124) and entered the open-label extension study. Subjects were to receive weekly subcutaneous injections of 6mg/kg of PRO051/GSK2402968 in the extension study, regardless of earlier dose. All subjects were at stable steroid doses during the study. Assessments were performed at baseline and 4-week intervals thereafter. RESULTS: All boys reported treatment-emergent AEs. The most common AEs were increased urinary ₁-microglobulin (100%), proteinuria (92%) and injection site reactions (100%). However, the majority of AEs were considered to be mild; there were no severe treatment-related AEs. There was some evidence of mild proteinuria, confirmed as greater than the upper limit of normal range (>0.15g<0.3g/24hrs), in four boys at subsequent 24-hr collection. Increases in some renal and hepatic parameters were observed but none were considered progressive. Four serious AEs (not related to treatment) were reported. There was an improvement in 6-minute walking distance test (6MWD) from 12 weeks; this was maintained until week 48, when the mean (SD) improvement in 6MWD was +29 (80)m. CONCLUSIONS: PRO051/GSK2402968 6mg/kg administered weekly by subcutaneous injection was generally well tolerated across 48 weeks of treatment. Renal and hepatic function warrant further monitoring. As 6MWD is expected to deteriorate over 48 weeks, the improvements observed in this functional measurement are encouraging.

[S34.002] Safety, Pharmacokinetic and Exploratory Efficacy of AVI-4658 a Phosphorodiamidate Morpholino Oligomer (PMO) To Skip Exon 51 in Duchenne Muscular Dystrophy Patients

Stephen B. Shrewsbury, Bothell, WA, Sebahattin Cirak, London, United Kingdom, Michela Guglieri, Newcastle, United Kingdom, Kate Bushby, Newcastle Upon Tyne, United Kingdom, Francesco Muntoni, London, United Kingdom

OBJECTIVE: This study investigated safety, pharmacokinetics (PK), exploratory biological and clinical efficacy of the first systemic administration of AVI-4658 over 12 weekly IV injections at 0.5, 1.0, 2.0, 4.0, 10.0 and 20.0 mg/kg. BACKGROUND: DMD is the commonest inherited muscular dystrophy in children, affecting 1 in every 3,500 boys, presenting with muscle weakness by age 5, wheelchair confinement by age 12; later respiratory insufficiency requiring mechanical ventilation and cardiomyopathy, placing a huge burden on patient (who rarely survive into their 30s) and parents/caregivers. Out of frame exon deletions in the dystrophin gene are the commonest mutations. DESIGN/METHODS: Open label, dose escalation study in ambulant DMD patients aged 5-15 years; muscle biopsy to assess dystrophin expression at baseline and 14 weeks. Clinical parameters were followed for 26 weeks: safety (adverse events (AEs), physical examinations, laboratory tests), skeletal muscle, pulmonary and cardiac function, PK at 1^st, 6^th and 12^th doses. RESULTS: All 219 doses of AVI-4658 were well tolerated; no dose related increase in AEs, change in vital signs, laboratory tests (or other investigations). PK showed expected short plasma half life, rapid renal clearance. Biologically, dose related exon skipping demonstrated, with consistent increase in dystrophin positive fibers in the higher two doses, reaching 55% in one patient. Proper assembly of Dystroglycan Protein Complex observed with reductions in various inflammatory T cell counts and no immune response to new dystrophin. Clinically, general functional stability shown over 26 weeks. CONCLUSIONS: The first PMO for DMD was well tolerated at all doses in all 19 patients; predictable PK with systemic delivery demonstrating exon skipping, new dystrophin expression and resulting restoration of associated proteins and reduction of inflammatory infiltrate. Clinical benefit remains to be characterized in higher dose and/or longer duration studies.

[P02.066] Clinical and Preclinical Comparison of the Pharmacokinetic Parameters of AVI-4658, a Phosphorodiamidate Morpholino Oligomer for the Treatment of Duchenne Muscular Dystrophy

Peter Sazani, Stephen Shrewsbury, Bothell, WA

OBJECTIVE: Compare the pharmacokinetics of intravenous AVI-4658 in Duchenne muscular dystrophy (DMD) patients (0.5 to 20mg/kg) with the toxicokinetic parameters of intravenous dosing in, cynomolgus monkeys (5 to 320 mg/kg). BACKGROUND: DMD is a degenerative neuromuscular disease resulting from dystrophin gene mutations, and affects 1 of 3500 male newborns. AVI-4658 is a splice switching oligomer (SSO) designed to skip dystrophin exon 51, restore the translation reading frame, and enable an internally truncated but functional dystrophin protein. DESIGN/METHODS: Ambulant DMD patients were dosed intravenously with AVI-4658 once weekly for 12 weeks with 0.5, 1.0, 2.0, 4.0, 10.0 or 20.0mg/kg. Following the 1^st, 6^th and 12^th doses, blood was taken between 5 minutes and 24 hours postdose. Cynomolgus monkeys were dosed intravenously once weekly for 12 weeks at 0, 5, 40 or 320mg/kg, with blood taken from 0.25 to 36 hours postdose. All samples were analyzed using a validated hybridization-based bioanalytical method. Pharmacokinetic/toxicokinetic parameters were then calculated, including C_max, AUC, Tmax, V_ss, Cl, and T. RESULTS: Pharmacokinetically, AVI-4658 behaved in a manner consistent with other PMO drugs, in both DMD patients and cynomolgus monkeys. Total exposure, as well as the C_max achieved in plasma, for AVI-4658 increased proportionally with dose, with no apparent saturation at higher doses. In general, the plasma elimination half-life was short (up to 3.6hours), and consistent across dose levels and species. In cynomolgus monkeys, increase in total exposure was proportional with the instance and intensity of histological findings in the kidney, which did not alter the function of the kidneys at any dose. No dose limiting toxicities were observed in either patients or monkeys. CONCLUSIONS: Across cynomolgus monkeys and DMD patients, AVI-4658 demonstrated a favorable pharmacokinetic profile, in which total exposure was proportional to the dose administered, up to the maximum feasible dose of 320mg/kg in cynomolgus monkeys.
 

[P02.058] The Health-Related Needs of Patients and Families Affected by Duchenne and Becker Muscular Dystrophy (DBMD): Data from the Muscular Dystrophy Surveillance, Tracking and Research Network (MDSTARNet)

Alexis M. Smirnow, Shree Pandya, Helena Temkin-Greener, James Dolan, Rochester, NY, Katherine Mathews, Christina Trout, Iowa City, IA, Christopher Cunniff, Tucson, AZ, Charlotte Druschel, Troy, NY, Paul Romitti, Iowa City, IA, Lisa Miller, Denver, CO, MDSTARnet, Rochester, NY

OBJECTIVE: To develop a profile of healthcare, psychosocial, and information related needs of patients with DBMD and their caregivers. BACKGROUND: Previous research exploring the experience of individuals diagnosed with a chronic childhood illness, such as DBMD, and their families has revealed a wide range of needs, many of which exceed the scope of traditional medical care. DESIGN/METHODS: The MDSTARNet is a multistate collaboration that conducts population-based surveillance for DBMD and annual surveys with their caregivers. A Needs Assessment survey was conducted between September 2008 and June 2009 to evaluate the needs of patients with DBMD and their caregivers. The survey included 76 questions related to 3 domains - healthcare services, psychosocial issues and information related to DBMD care. RESULTS: Responses were received from 53 adults with DBMD and 145 primary caregivers from four states (Arizona, Colorado, Iowa and Western NY). At least one unmet need was reported by 87% of affected adults and 96% of caregivers. Among affected adults, the most frequent unmet needs reported were: making and maintaining romantic adult relationships (36%), finding funding for vehicle modifications (38%) and managing joint contractures and muscle weakness (17%). Among caregivers, counseling to help emotional adjustment (39%), information about jobs/future planning for the male with DBMD (49%) and accessing a nurse coordinator/health team advocate (26%) were unmet needs most frequently reported. Overall, among affected adults and caregivers, the number of unmet needs reported for psychosocial issues and information exceeded those reported for healthcare. CONCLUSIONS: The survey results demonstrate a broad range of unmet needs for patients and families affected by DBMD. Developing a profile of health-related needs for those affected by DBMD will help identify gaps in care and guide the development of interventions to facilitate optimal health outcomes.

[P02.060] Health Care Services Received by Patients with Duchenne/Becker Muscular Dystrophy (DBMD): Data from the Muscular Dystrophy Surveillance, Tracking and Research Network (MDSTARNet)

Shree Pandya, Rochester, NY, Kim Campbell, Troy, NY, Jennifer Andrews, John Meaney, Chris Cunniff, Tucson, AZ, Charlotte Druschel, Troy, NY, Lisa Miller, Denver, CO, Paul Romitti, Iowa City, IA, MDSTARNet, Rochester, NY

OBJECTIVE: To document the type, frequency and setting of health care services received by patients with DBMD in a population based surveillance program covering four states. BACKGROUND: Anecdotal data from families and health care providers suggests a wide variation in services received by patients with DBMD. DESIGN/METHODS: The MDSTARNet is a multistate collaboration that conducts population based surveillance for DBMD and annual surveys with their primary caregivers. From April 2007-May 2008, 372 eligible caregivers from four states (AZ, CO, IA and western NY) were identified. 200 (53.7%) completed a telephone interview. The interview included questions about the type of provider seen (primary care, neuromuscular specialist, orthopedist, pulmonologist, cardiologist, physical therapist (PT), occupational therapist(OT), speech therapist (ST) and other) and the type of services received (respiratory, dietary, mental health, social work, pain management, pastoral and case management) over the past 12 months, and setting (clinic, school, home) for these visits. Frequencies with which these services were received were compared among states using Chi square tests. Statistical significance was set at p<0.05. RESULTS: The care providers reported seeing primary care clinicians (85%), neuromuscular specialists (86%), cardiologists (72%), pulmonologists (55%), orthopedists (30%) and PT (72%), OT (39%), ST (17%) and other (17%). Services received included case management (62%), social work (26%), respiratory (27%), dietary (19%), mental health (16%), pastoral (16%) and pain management (5%). There were significant differences among states in regards to the cardiac, pulmonary, and orthopedic specialists seen and in the location of receipt of physical therapy services. CONCLUSIONS: The results of this study support the anecdotal data regarding the variation in services perceived by parents and providers.

[S34.003] Safety and Feasibility of Transvenous Limb Perfusion with Saline in Human Muscular Dystrophy

Zheng Fan, Keith Kocis, Robert Valley, James Howard, Manisha Chopra, Hongyu An, Weili Lin, Joseph Muenzer, William Powers, Chapel Hill, NC

OBJECTIVE: Assess the safety and feasibility of the transvenous limb perfusion gene delivery method in human muscular dystrophy. BACKGROUND: High-pressure retrograde transvenous limb perfusion has been successfully used to deliver gene therapies into skeletal muscles in experimental animals. Translating this promising technique to humans with muscular dystrophy requires addressing multiple safety and logistical aspects separate from the therapeutic agent. DESIGN/METHODS: A dose escalation study of transvenous single limb perfusion with 0.9% saline in adults with Becker or limb-girdle muscular dystrophies starting with 5% of limb volume. Cardiac, vascular, renal, muscle and nerve function were monitored. Anesthesia was provided with fentanyl, midazolam and propofol. An 18 or 20 g intravenous catheter was inserted into the distal great saphenous vein. A single cuff tourniquet was placed just above the knee with pressure of 310 mmHg. Infusion of normal saline was carried out with a Belmont FMS 2000 Rapid Infuser at a maximum line pressure of 300 mmHg with a goal infusion rate of 80ml/min. RESULTS: Infusion volume was escalated stepwise to 20% limb volume in six subjects. No subject complained of any post procedure pain other than due to needle punctures. Safety warning boundaries were exceeded only for transient depression of limb tissue oximetry and transient elevation of muscle compartment pressures; these were not associated with nerve, muscle or vascular damage within 72 hours post procedure. Muscle MRI demonstrated fluid accumulation in muscles of the perfused leg at 20% limb volume. CONCLUSIONS: High-pressure retrograde transvenous limb perfusion with saline up to 20% of limb volume at these infusion parameters is safe and feasible in human muscular dystrophy. These studies will serve as a basis for future gene therapy clinical trials.

[P04.023] Systemic Gene Delivery for Therapy of Neuromuscular Disorders

Jeffrey Chamberlain, Seattle, WA

OBJECTIVE: To develop pre-clinical methods for gene therapy of neuromuscular disorders. BACKGROUND: We are developing methods for systemic delivery of therapeutic genes to muscles throughout the body using animal models of Duchenne muscular dystrophy (DMD), which lack dystrophin. We have previously shown that adeno-associated virus type 6 (AAV6) vectors are able to systemically deliver genes throughout the body of adult mice when injected intravascularly, resulting in highly efficient gene expression that persists for the lifespan of the mouse. However, the AAV shuttles have a limited carrying capacity, and as a result we have also been developing functional yet truncated versions of the dystrophin gene. A single injection of an AAV6/micro-dystrophin vector into the vasculature of adult, dystrophic mice results in elimination of dystrophic histopathology for the lifespan of the mouse. DESIGN/METHODS: We have been exploring whether systemic gene transfer can be used in larger animal models and whether AAV-mediated transfer of dystrophin elicits an immune response. RESULTS: Delivery of AAV6 vectors into various veins and arteries of the dog results in efficient gene transfer to downstream muscles, but does not lead to whole body gene transfer. Instead, it appears that vector will need to be delivered into multiple vascular sites to target muscles body wide. Delivery of AAV vectors encoding micro-dystrophin in dogs results in a cellular immune response against the AAV capsid. However, this immune response can be blocked with transient immune suppression, resulting in dystrophin expression for up to 2 years. To address concerns about whether dystrophin could be immunogenic in patients with dystrophin null-alleles, we have developed a micro-utrophin gene. When delivered to dystrophic mice, micro-utrophin is nearly as effective as dystrophin in alleviating pathology. CONCLUSIONS: Intravascular AAV delivery of micro-utrophin or micro-dystrophin coupled with transient immune suppression could lead to an effective therapy for DMD or other neuromuscular disorders.

[P04.022] Treatment with a Soluble Activin Receptor Type IIB Fusion Protein Reduces Serum Creatine Kinase Levels in Treadmill-Exercised mdx Mice

Jennifer Lachey, Abigail Pullen, Alan Koncarevic, Jasbir Seehra, Cambridge, MA

OBJECTIVE: This study evaluates the potential benefit of treatment with a soluble activin receptor type IIB (ActRIIB) on myofiber integrity in the mdx model of Duchenne muscular dystrophy (DMD). BACKGROUND: DMD is characterized by the absence of functional dystrophin protein which results in reduced sarcolemmal integrity. Creatine kinase (CK), normally localized to muscle cytoplasm, is found at high levels in serum of DMD patients and considered a biomarker of myofiber integrity. Multiple ligands signal through ActRIIB to negatively regulate muscle growth. Treatment with a soluble ActRIIB fusion protein (RAP-031) to inhibit endogenous ActRIIB signaling increased muscle mass and body weight, and restored muscle strength in mdx mice. To determine possible benefits to myofiber stability, we assessed the effect of RAP-031 on serum CK levels in treadmill-exercised mdx mice. DESIGN/METHODS: Mdx mice were divided into 4 groups: unexercised for 4 weeks (Control), exercised for 4 weeks (4 WEEK), exercised for 8 weeks/vehicle-treated (VEH) and exercised for 8 weeks/RAP-031-treated (RAP; 10mg/kg s.c. twice weekly). The VEH and RAP groups were exercised for 4 weeks without treatment, then dosed and exercised for an additional 4 weeks. Serum CK was assayed after the last exercise exposure. RESULTS: We have previously demonstrated that RAP-031 increased lean mass and body weight in mdx mice. Consistent with this, the RAP cohort was significantly heavier than the VEH group (p<0.001) at study termination. CK levels were elevated in the 4 WEEK group compared to the Control group and further increased in the VEH group. In contrast, mice in the RAP group had significantly lower CK values (p<0.05) compared to all other groups. CONCLUSIONS: RAP-031 treatment prevented increases in CK due to exercise in mdx mice. Furthermore, RAP-031 appeared to reverse the increases in CK from pre-dose exercise. These data suggest that RAP-031 improves dystrophic muscle by, at least in part, improving myofiber stability.
 

[P02.063] Assessment of Muscle Strength in Healthy Children and Ambulant Boys with Duchenne Muscular Dystrophy at Ages 6 through 11 Years of Age

Richard Abresch, Davis, CA, Craig McDonald, Sacramento, CA, Jay J. Han, Folsom, CA, Erik Henricson, Sacramento, CA

OBJECTIVE: To quantify the disease progression of knee and elbow extensor and flexor muscles of ambulant boys with Duchenne muscular dystrophy (DMD) using an isometric dynamometer and to develop predictive equations by comparing these values to healthy controls (CTRL). BACKGROUND: The primary impairment resulting from DMD is progressive loss of muscle tissue and strength. However, the clinical significance of quantitative myometry data is not well understood. DESIGN/METHODS: Isometric strength of knee and elbow extensor and flexor muscles was obtained using a Lido dynamometer in 125 DMD and 61 CTRL boys, ages 6 11 years. RESULTS: Extension of the knee and elbow of the DMD (14.7 and 6.4NM, respectively) was 40% and 42% of CTRL (36.6 and 15.1 NM, respectively) at age 6 years and decreased linearly to 15.8% and 10.3% of CTRL at age 11. Flexion of the knee and elbow of the DMD (11.6 and 9.6 NM, respectively) was 52% and 62% of CTRL (22.2 and 15.5 NM, respectively) at age 6 years and decreased linearly to 25.8% and 33.4% of CTRL at age 10. CONCLUSIONS: This study quantifies patterns of muscle weakness in DMD and compares them to percent predictive strength equations of healthy controls using height, age, and weight as predictors.

[P02.064] Impairment of Excitation-Contraction Coupling in Patients with Duchenne Musclar Dystrophy

Takayasu Fukudome, Higashisonogigun, Nagasaki, Japan

OBJECTIVE: To investigate whether excitation-contraction (E-C) coupling of muscle is impaired in patients with Duchenne muscular dystrophy (DMD). BACKGROUND: The dystrophyn-associated glycoprotein complex is associated with E-C coupling and abnormal E-C coupling has been reported in dystrophic muscle fibers from mdx mouse. Impaired E-C coupling was assumed in patients with DMD. DESIGN/METHODS: In 3 patients with Duchenne muscular dystrophy (DMD), 2 patients with myotonic dystrophy (DM) and 10 normal subjects, compound muscle action potentials (CMAPs) of the abductor pollicis brevis and movement-related potentials using an accelerometer placed at the thumb tip were simultaneously recorded after median nerve stimulation at the wrist. The E-C coupling time (ECCT) was estimated by a latency difference between CMAP and movement-related potential. RESULTS: The mean ECCT was 3.6 0.44 msec (mean SEM) in normal controls (4 men and 6 women; mean age,37 years old, range, 24-49 years old). The ECCT in 3 patients with DMD (3 men; ages,15, 16 and 17 years old) were 4.7 msec, 14.6 msec and 6.0 msec. The ECCT in 2 patients with DM (one man; aged 61 years and one woman; aged 58 years old) were 6.6 msec and 5.0 msec. CONCLUSIONS: ECCT was prolonged in both DMD and DM patients compared to control subjects. For one of the patients with DMD, the ECCT was significantly prolonged. To evaluate the relationship between the ECCT and muscle weakness, impairment of motor function or gene mutation of the dystrophyn protein is needed. ECCT may be useful for evaluating the effects of DMD genetic therapy clinical trials.
 

[P02.062] A Canadian Survey on Lung Volume Recruitment (LVR) in Children with Duchenne Muscular Dystrophy

Jean Mah, Calgary, AB, Canada, Douglas McKim, Lynda Hoey, Nick Barrowman, Sherri Katz, Ottawa, ON, Canada

OBJECTIVE: (1) To describe the current respiratory management of children with Duchenne Muscular Dystrophy (DMD); and (2) To compare the clinical practice of Pediatric Respirologists and Neuromuscular specialists across Canada. BACKGROUND: DMD is the most common muscular dystrophy of childhood. Recent improvement in survival until the third or fourth decade of life is partly related to early detection and management of respiratory complications. Previous observational studies have indicated that regular LVR, a technique to increase lung volumes and cough flows, may improve respiratory outcomes. DESIGN/METHODS: A web-based survey was sent to all 56 practicing Pediatric Respirologists in Canada and to all 24 Neuromuscular specialists of the Canadian Pediatric Neuromuscular Group (CPNG), who follow children with neuromuscular diseases. RESULTS: Responses were received from 38 (66%) Pediatric Respirologists and 17 (66%) CPNG members. Among 38 Respirologists, 33 see DMD patients under the age of 18. Of these 33, only 6 (18%) practicing at 3 centers reported using LVR in about one-third of their patients regularly during clinical stability. Among 17 CPNG members, all of whom see DMD patients under the age of 18, only 2 (12%) practicing at a single center reported using LVR in about half of their patients routinely. The majority of the Respirologists (61%) and Neuromuscular specialists (64%) felt that manual LVR between respiratory exacerbations is an intervention that warrants further study, with decline in forced vital capacity (percent predicted) and health-related quality of life being important clinical outcome measures. CONCLUSIONS: There is variability in the respiratory management of pediatric DMD patients across Canada. Few Pediatric Respirologists and Neuromuscular specialists use LVR at least twice daily during periods of clinical stability. The majority believe that LVR should be the intervention for a larger clinical trial in this population. Decline in forced vital capacity and quality of life are considered the most important outcomes of interest.
 

[P02.065] Use of Optical Coherence Tomography for Idiopathic Intracranial Hypertension in Boys with Duchenne Muscular Dystrophy

Jean K. Mah, Fiona Costello, Calgary, AB, Canada

OBJECTIVE: (1) To report the association between idiopathic intracranial hypertension (IIH) and Duchenne muscular dystrophy (DMD); (2) To describe the use of optical coherence tomography (OCT) for the detection of IIH in DMD. BACKGROUND: DMD is an X-linked recessive form of muscular dystrophy, with progressive weakness leading to quadriparesis, cardiomyopathy, respiratory insufficiency, and death around the third decade of life. Corticosteroid is often used as a disease-modifying therapy to prolong the duration of independent ambulation and to delay the onset of secondary complications. Side effects of corticosteroid include weight gain, behavioral change, short stature, and increase risk of infection or fractures. To our knowledge, there has been no reported association between IIH and DMD. DESIGN/METHODS: This was a retrospective case review from a tertiary paediatric neuromuscular center in Calgary. RESULTS: Two boys (age 11 and 15 years, BMI>95% for age) with DMD on long-term deflazacort treatment developed IIH. The first boy had asymptomatic papilloedema found during routine eye exam; the second boy presented with a two-week history of severe bifrontal pulsatile headache with bilateral sixth nerve palsy. Fundoscopic exam in both cases confirmed bilateral papilloedema, and lumbar puncture revealed elevated opening pressure without any evidence of infection or inflammation. Cerebral sinovenous thrombosis was excluded by brain MRI/MRV or cranial CTV. Treatment with lumbar puncture, acetozolamide, and weight control measures resulted in complete resolution of symptoms. OCT showed marked thickening of the retinal nerve fiber layer thickness consistent with papilloedema in both eyes acutely, which improved in follow-up. CONCLUSIONS: IIH is a rare complication of boys with DMD. Potential risk factors include young age, use of corticosteroid, obesity, and physical inactivity due to underlying weakness. Early recognition, exclusion of other secondary causes of IIH, and prompt treatment is needed to avoid permanent visual loss. OCT is useful for detecting IIH and monitoring the response to treatment.
 

[S34.006] Predictors of Ambulation Loss in Boys with Duchenne Muscular Dystrophy

Richard Abresch, Davis, CA, Craig McDonald, Sacramento, CA, Jay Han, Folsom, CA

OBJECTIVE: The purpose of this study was to predict which variables could predict loss of ambulation and reduction in walking velocity in ambulant boys with Duchenne muscular dystrophy (DMD) over a one-year time period. BACKGROUND: The FDA has recently specified that clinical trials conducted in support of drug approvals must incorporate a primary endpoint that objectively measures a clinically meaningful life-changing milestones. DMD clinical trials need short-term surrogate outcome measures that are predictive of alterations of these life-changing milestones. DESIGN/METHODS: Height, weight, timed motor performance, strength, and functional grade were assessed at baseline and approximately one-year time later in 63 ambulatory DMD subjects, ages 5 -13 years. Stepwise discriminant analysis was used to identify the significant predictors of (a) loss of ambulation and (b) a minimum 40% reduction in walking velocity. RESULTS: Age, weight, and time to stand from supine at the initial evaluation were significant predictors of one-year ambulation status (Wilks' = 0.573, F=19.36, p<0.0001). Age and time to stand from supine at initial evaluation were significant predictors of reduction in walking velocity and ambulation approximately one-year later (Wilks' = 0.753, F=4.93, p<0.005). No other variables significantly entered into the equation. Baseline assessment of age, weight and time to stand correctly classified one-year post-baseline ambulation status 78% of the time, and non-ambulating status 89% of the time. Baseline assessment of age and time to stand correctly classified patients who exhibited a 40% reduction in walking speed and those who did not 73% and 89% of the time, respectively. CONCLUSIONS: Findings from this study suggests that time to rise from supine and age can effectively predict ambulation status and reductions in walking speed in boys with DMD. This information will be useful for anticipatory guidance, development of outcome measures, and translational studies of DMD.

[P02.067] Novel Pointmutation in R1 Domain of Dystrophin Gene Causes Missfolding of Dystrophin and Becker's Muscular Dystrophy

Gyula Acsadi, Detroit, MI, Steven A. Moore, Iowa City, IA, Angélique Cheron, Rennes, France, Lindsey Bennett, William Kupsky, Detroit, MI, Elisabeth Le Rumeur, Jean-François Hubert, Rennes, France

OBJECTIVE: To determine structural and functional characteristics of the mutant dystrophin protein found in mild Becker's muscular dystrophy (BMD). BACKGROUND: Dystrophin provides a structural link between the sarcolemma and cytoskeletal f-actin. Missense mutations in the dystrophin gene often lead to BMD but a genotype/phenotype correlation is difficult to establish. Depending on the mutation locus, a disruption of various binding capacities even in the repeat region may have a major impact on the functionality of dystrophin. DESIGN/METHODS: We have identified a family with two young brothers having very mild proximal weakness, recurrent abdominal pain and moderately elevated serum creatine kinase levels (3,000 IU). An immunostaining approach to evaluating dystrophin expression was performed on a muscle biopsy. After determining the mutation in the dystrophin gene, we carried out in vitro biophysical analysis of a two-repeat portion of the dystrophin rod domain bearing the mutation. RESULTS: Dystrophin immunofluorescence showed absent to weak staining for dystrophin regions encoded by exons 7 to 9. A T>C transition at codon 427 causing a Leu>Pro amino acid change in the first spectrin-like repeat (R1) was identified as a novel mutation. There was no change in the lipid binding capacity of R1, R2 dystrophin peptides containing the patient's mutation. However, significant alteration in thermal and chemical folding behavior was observed. CONCLUSIONS: In frame mutations in the R1 spectrin-like domain can affect the folding and binding properties of dystrophin protein and lead to a mild forms of BMD.
 

[P06.268] Prevalence and Characteristics of Pain in Myotonic Dystrophy Type 2: A Postal Survey in Finland

Aki J. Hietaharju, Kimmo I. Suokas, Tampere, Finland, Maija Haanpaa, Espoo, Finland, Bjarne Udd, Vasa, Finland

OBJECTIVE: The objective of this study was to assess the prevalence and clinical characteristics of pain in Finnish myotonic dystrophy type 2 (DM2) patients. BACKGROUND: DM2 is an autosomal dominant multisystem disease caused by the expansion of tetranucleotide repeats (CCTG) in the zinc finger protein 9 gene. In addition to muscle weakness and myotonia, fluctuating or episodic musculoskeletal pain is regarded as one of the key symptoms of DM2. However, only a few studies have addressed the occurrence of pain and its clinical features in this disorder. DESIGN/METHODS: Postal survey was carried out for all patients with genetically confirmed DM2 and available contact information (n=132). The cohort was estimated to represent 85 % of Finnish DM2 patients. The respondents (n=93) answered a specific questionnaire including pain charts, 11-point pain intensity numerical rating scale (PI-NRS) and Brief Pain Inventory (BPI). Health-related quality of life was assessed by using a Finnish validated version of the RAND 36-Item Short Form Health Survey (RAND-36). Depression was screened using the short form of Beck Depression Inventory (RBDI). RESULTS: Prevalence of pain at the time of the survey was 53,8 %. In addition, 22,6 % had experienced pain previously. Majority of the patients (48,0 %) reported daily pain at the time of the survey, and in 14 % of the responders pain was constant. Pain was increased by exercise in 57, 9 % and by cold in 56,1 % of the responders. Quality of life scores were significantly lower in DM2 patients reporting pain than in those with no pain (p< 0.01). In 18,3 % of the responders, the RBDI score was suggestive of moderate or severe depression. CONCLUSIONS: Pain is a very common feature of DM2 and is experienced on a daily basis by most of the patients. It significantly reduces quality of life and is related with depression.
 

[P06.275] Myotonic Dystrophy Type-2 (DM2): Patient-Reported Highest-Impact Symptoms and Issues

Chad Heatwole, Rochester, NY, Rita Bode, Elmwood Park, IL, Nicholas Johnson, Christine Quinn, William Martens, Richard Moxley, Rochester, NY, Barbara Vickrey, Los Angeles, CA, David Victorson, Evanston, IL

OBJECTIVE: To identify, quantify, and analyze the symptoms and issues most relevant to DM2 patients. BACKGROUND: DM2 is an autosomal dominant multisystemic disease capable of impairing the physical, mental, and social health of patients. Patient-centered studies identifying the symptoms and issues of greatest importance to this population are currently limited. DESIGN/METHODS: We conducted structured interviews with fifteen genetically confirmed adult DM2 patients. Each interview focused on identifying the symptoms and issues that exert the most critical impact on patients' health related quality-of-life (HRQOL). We recorded, transcribed, coded, and analyzed each interview using a qualitative framework technique and three investigator approach. RESULTS: Results: We coded 943 participant quotes which were sorted into 244 themes and 19 subdomains that represent the physical, mental, social, and disease-specific HRQOL of DM2 patients. We designed a disease-specific HRQOL model. Categories in this model include: 1) mobility and ambulation; 2) proximal leg weakness; 3) fine motor and upper extremity dysfunction; 4) proximal upper extremity and shoulder limitations; 5) truncal weakness; 6) activity impairment specific to DM2; 7) emotional distress; 8) cognitive impairment; 9) impaired body image specific to DM2; 10) social role dissatisfaction; 11) social role limitations; 12) offspring and genetic issues; and DM2 specific symptoms of: 13) sleep disturbance; 14) fatigue; 15) pain; 16) myotonia; 17) gastrointestinal dysfunction; 18) vision/hearing impairment; and 19) communication difficulties. CONCLUSIONS: There are multiple themes, some previously under-recognized, that impact DM2 HRQOL. Obtaining the patient's point of view is a critical first step in the future development of valid patient-reported outcome measures and functional rating scales for this population.

[P06.270] Test-Retest Reliability of Strength Measurements of the Long Finger Flexors (LFF) in Patients with Myotonic Dystrophy Type 1 (DM1)

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

OBJECTIVE: To document the test-retest reliability of strength measurements of the LFF in patients with DM1. BACKGROUND: Patients with DM1 have distal upper extremity weakness which begins in the long finger flexors. Grip strength using a Jamar dynamometer has been used in the past to document hand strength. Grip strength however, is a measure of intrinsic and extrinsic hand muscle strength and does not isolate the LFF. Testing procedures to specifically examine the LFF have not been established. DESIGN/METHODS: Strength of the LFF was measured by manual muscle testing (MMT) and quantitative muscle testing (QMT) in 22 patients with DM1. MMT was performed on the LLF of the thumb, first and second digits using standard MRC procedures and grading. QMT was performed using a Jamar hand dynamometer and a pinch gauge (B&L Engineering). The LFF were tested using the Jamar hand dynamometer on the last setting, and the palmar pinch was measured using the pinch gauge. These testing procedures were performed twice in a 24 hour period. RESULTS: The interclass correlation coefficient (ICC) for MMT of the LFF for the right and left hands were .971 and .943 respectively. The ICC for the LFF using the Jamar dynamometer was .919 for the right hand and.847 for the left hand. The ICC for palmar pinch was .957 and .954 for the right and left hands respectively. CONCLUSIONS: Strength testing of the LFF demonstrated excellent test-retest reliability over a 24 hour period. Tests of sensitivity to change over longer periods of time will need to be performed prior to using these tests as outcome measures for clinical trials.

[P06.260] Cardiomyopathy in Limb Girdle Muscular Dystrophy 2B and 2I

Xiomara Q. Rosales, Sean J. Moser, Tam Tran, Beth McCarthy, Nicholas Dunn, Philip Habib, Orlando P. Simonetti, Jerry R. Mendell, Subha V. Raman, Columbus, OH

OBJECTIVE: To characterize the myocardium in LGMD2I and 2B patients using cardiac magnetic resonance (CMR). BACKGROUND: Limb girdle muscular dystrophies (LGMD) are inclusive of 7 autosomal dominant and 14 autosomal recessive disorders featuring progressive muscle weakness and atrophy. Two of the most common forms of LGMD include deficiencies of dysferlin (LGMD2B) and fukutin related protein (LGMD2I). Considering the importance of cardiac function as a predictor of prognosis, we used CMR as highly sensitive measure of cardiac function to define systolic dysfunction, myocardial fibrosis, and diastolic dysfunction. DESIGN/METHODS: Consecutive patients with genetically-proven LGMD types 2I (n= 7) and 2B (n=9) were enrolled. All subjects underwent cardiac magnetic resonance (CMR) on a standard 1.5 Tesla clinical scanner with cine imaging for left ventricular (LV) volume and ejection fraction (EF) measurement, vector velocity analysis of cine data to calculate myocardial strain, and late post-gadolinium enhancement imaging (LGE) to assess for myocardial fibrosis. RESULTS: Sixteen LGMD patients (7 LGMD2I, 9 LGMD2B), and 8 control subjects completed CMR. All but one patient had normal LV size and systolic function; one (type 2I) had severe dilated cardiomyopathy. Of 15 LGMD patients with normal systolic function, LGE imaging revealed myocardial fibrosis in 7 (47%). Peak systolic circumferential and radial strain rates were similar in patients vs. controls. Five of 7 LGE-positive patients had mild diastolic dysfunction (3 type 2B, 2 type 2I) that was not present in any LGE-negative patients or controls. CONCLUSIONS: The prevalence of advanced cardiomyopathy in patients with LGMD2I and LGMD2B appears to be limited, but subclinical fibrosis and diastolic dysfunction do occur and may warrant consideration of cardioprotective therapies. Longitudinal studies will be necessary to define the natural history of cardiomyopathy in LGMD2B and 2I.
 

[P06.278] Diminished PGC-1 Contributes to Mitochondrial Dysfunction and Elevated Oxidative Stress in Myotonic Dystrophy Type 1 (DM1)

Xiang Fang, Rui Gao, Qinghua Liang, Galveston, TX, Analisa Botta, Rome, Italy, Robert Smith, Partha Sarkar, Galveston, TX

OBJECTIVE: To understand the molecular basis of mitochondrial dysfunction and oxidative stress in DM1 skeletal muscle. BACKGROUND: DM1 is an autosomal dominant dystrophic muscle disease caused by the expansion of CTG repeats in the 3' UTR of the DMPK gene. Massive expansion of CTG repeats within the 3' UTR results in dystrophic and degenerative skeletal muscle defects and associated mitochondrial dysfunction. The mechanism by which expanded CTG repeats leads to mitochondrial abnormalities is not yet established. Because PGC-1 is a key regulator of mitochondrial biogenesis/respiration and oxidative stress, we tested whether PGC-1 is involved in dysregulation of mitochondrial function and oxidative stress in DM1 skeletal muscle. DESIGN/METHODS: DNA and protein from control and DM1 patient skeletal muscle were isolated and extracted. The expression of PGC-1 and genes regulating mitochondrial biogenesis, oxidative phosphorylation and reactive oxygen species (ROS) detoxification were assessed by Western-blot and real-time quantitative PCR analyses. RESULTS: Steady-state mRNA levels of PGC-1 were significantly down-regulated in DM1 skeletal muscle. Similarly, expression of PGC-1 target genes regulating mitochondrial OXPHOS was significantly decreased in DM1 skeletal muscle. TFA1, 2, ATP5A1, COX2, 3, and 4, and TFAM expression was decreased by 40-75% from DM1 muscle samples as compared with those of controls. Consistent with these findings, levels of the ROS detoxifying genes e.g. superoxide dismutase, glutathione peroxidase and catalase were also markedly decreased in DM1 skeletal muscle, concomitantly associated with elevated tissue ROS levels. CONCLUSIONS: PGC-1 plays a critical role in the pathogenesis of DM1, altering mitochondrial function and oxidative stress.
 

[P06.267] Steroid Response in Patients with Dystroglycanopathy

Katherine D. Mathews, Carrie M. Stephan, Karla S. Laubenthal, Steven A. Moore, Iowa City, IA

OBJECTIVE: Retrospective review of response to corticosteroids in cohort of patients with dystroglycanopathies. BACKGROUND: The dystroglycanopathies are a group of autosomal recessive disorders that share a deficiency in glycosylation of a-dystroglycan. Mutations in six genes are known to result in disease. There is a wide range of clinical severity from Walker-Warburg syndrome, a severe form of congenital muscular dystrophy, to limb girdle muscular dystrophy. Case reports and anecdotal clinical experience suggest that some individuals respond to steroids. DESIGN/METHODS: All patients participating in a natural history study of genetically confirmed dystroglycanopathies were included. Information about current and previous medications used for muscular dystrophy, along with their responses to the medications was systematically collected. Medical records were reviewed when possible. RESULTS: Thirteen of 32 (40.6%) participants have used corticosteroids due to muscle disease. Nine patients with mutations in FKRP, FKTN, or POMT2 remain on steroids at varied doses due to reported beneficial clinical response. Four discontinued steroids due to lack of effect or side-effects. Steroids were started in five patients prior to their dystroglycanopathy diagnoses, in three for suspicion of myositis and in two for presumed Duchenne muscular dystrophy. In two families, younger affected siblings are on steroids due to the dramatic response seen in the older sibling. Four patients resumed steroids due to worsening weakness after discontinuation and they remain on steroids. CONCLUSIONS: This is the first systematic report of steroid use in a cohort of dystroglycanopathy patients. The reason for steroid responsiveness is not clear, but modulation of inflammation has been suggested. While retrospective, these data together with published case reports suggest that corticosteroid treatment modifies the course of disease in some individuals with dystroglycanopathy, and provide support for proceeding with a prospective trial in this patient group.

[P06.265] Psychometric Evaluation of Manual Muscle Testing in Adult Patients with Muscular Dystrophies

Reza Sadjadi, West Vancouver, BC, Canada, Michael Rose, London, United Kingdom

OBJECTIVE: To investigate psychometric properties of Manual Muscle Testing in Muscular Dystrophies. BACKGROUND: There is an increased interest in psychometric evaluation and validation of outcome measures (OM) in different diseases. Rasch analysis allows us to compare psychometric properties of items in an outcome measure and for MMT it could tell us which muscles are the most informative and how well any expanded MRC gading scale works. We investigated psychometric properties of the Manual Muscle Testing (MMT) protocol used during a clinical trial investigating the safety and efficacy of the myostatin inhibitor MYO-029 in the treatment of three types of muscular dystrophy. DESIGN/METHODS: 86 patients with Becker, Limb-Girdle or Facio-Scapulo-Humeral Muscular Dystrophy had 345 MMT assessments. A total of 24 muscle groups (22 pairs and neck flexion/extension) were rated using an expanded 10 level Medical Research Council (MRC) scale (0,1,2,3-,3,3+,4-,4,4+,5). We used Rasch model to compare item fit, separation and difficulty for different muscle groups and for the expanded MRC scale. RESULTS: Overall, MMT showed relatively reliable psychometric properties (Outfit MNSQ 1.03 Reliability 0.91). Relative distributions of item and person location estimates showed a relatively good coverage of muscle strength by continuous MMT items. Most muscle groups had disordered category thresholds and reducing detailed MRC scale to less detailed 6-level scale (0,1,2,3,4,5) improved psychometric properties of MMT. Hip muscle group form a small sub-dimension behaving slightly different from other muscle groups in MMT. CONCLUSIONS: Overall, most muscle groups were useful for reliable differentiation between different levels of overall muscle strength in muscular dystrophies. The original MRC scale (6-points) has better measurement properties than the expanded (10-point) MRC scale for MMT. Hip muscle group MMT sub-scores may need to be analysed as a potentially independent outcome measure in future trials.
 

[P06.273] Natural History of DM2/PROMM in Italy: Neuromuscular and Multisystem Progression over Time in 88 Patients

Alice Zanolini, Valeria A. Sansone, Enrico Bugiardini, Barbara Fossati, San Donato Milanese (Milan), Italy, Rosanna Cardani, Milan, Italy, Luigi De Ambroggi, Giovanni Meola, San Donato Milanese (Milan), Italy

OBJECTIVE: Quantify progression of muscle weakness, myotonia and multiorgan involvement in a cohort of Italian patients with myotonic dystrophy type 2 (DM2-PROMM phenotype) over time. BACKGROUND: Progression of muscle weakness and multisystem involvement in DM2 is thought to be slower and less severe than that of myotonic dystrophy type 1 (DM1). However, history data in DM2 are still limited. DESIGN/METHODS: Muscle involvement (MegaMRC, clinical and EMG myotonia tests, biceps muscle biopsy), cardiac (AV/IV blocks, RR interval, ejection fraction, PM/ICD implantation) and respiratory involvement (daytime sleepiness, sleep apnoeas, NIV support) were retrospectively analyzed in 88 patients with DM2 (56 males, 32 females) admitted from January 1994 to September 2010, mean follow-up 8.3 2.1. RESULTS: Age at onset was in the 3^rd and 4^th decade (65%, mean age 39.3 11.1); 15% developed symptoms over 60 (range 5-15 years). Progression of muscle weakness (mean MMRC from 139.7 to 137.2, p=0.002) occurred at a rate of 0.4 MRC points/year. 12% of patients became wheelchair bound during observation (all after age 70); 3% required PM implantation and 4% NIV support. Clinical and EMG myotonia was detectable in 25% of patients at onset and in an additional 20% at follow-up EMG. Muscle biopsy showed type II atrophy, ribonuclear and muscle-blind inclusions at baseline and with a size increase over time in repeat muscle biopsy (6 patients). CONCLUSIONS: Progression of muscle, cardiac and respiratory involvement occur in DM2 although at a later age and with less frequency than in DM1. Respiratory insufficiency seems rarer in DM2. EMG may fail to detect myotonia at onset whereas immunohistochemistry and FISH are abnormal at the very onset of symptoms. This suggests that muscle biopsy rather than EMG may be of greater support to the clinical diagnosis of DM2.

[P06.276] Myotonic Dystrophy Type-1: Patient Validation Study of the Highest-Impact Symptoms and Issues

Chad Heatwole, Rochester, NY, Rita Bode, Elmwood Park, IL, Nicholas Johnson, Christine Quinn, William Martens, Richard Moxley, Rochester, NY, Barbara Vickrey, Los Angeles, CA, David Victorson, Evanston, IL

OBJECTIVE: To determine the prevalence and relative impact of the symptoms and issues most important to the myotonic dystrophy type-1 (DM1) population's heath related quality-of-life (HRQOL). BACKGROUND: DM1 is an autosomal dominant multisystemic disease capable of impairing the physical, mental, and social health of patients. A previous pilot study of 20 DM1 patients identified 221 high priority symptoms and issues to DM1 patients. The relative importance and frequency of these themes in an extended DM1 population is not known. DESIGN/METHODS: 550 DM1 patients from the National Registry of Myotonic Dystrophy Patients and Family Members were sent a survey designed to identify the most critical DM1-specific HRQOL issues. Participants were asked to identify the relative impact on their lives of 221 previously identified high-impact themes. A relative impact score for each theme was determined (0=no impact on DM1 patients; 100=severe impact on all DM1 patients). RESULTS: 278 DM1 participants completed the surveys. Prevalence and relative impact of all 221 themes were determined. Problems with hands or arms (93.5%), fatigue (90.8%), myotonia (90.4%), impaired sleep (87.9%), and limitations in mobility (85%) were the subdomains with the highest prevalence. Fatigue (62.3), limitations in mobility (60.6), inability to do specific activities (58.7), and impaired sleep (56.2) were the subdomains with the highest relative impact scores. Participant responses varied based on age, CTG repeat length, employment, education, and time since symptom onset. CONCLUSIONS: DM1-specific HRQOL is defined by multiple themes of varying importance and prevalence. Identifying the most important issues for patients is a critical step in selecting optimal therapeutic targets and for the development of a valid, DM1-specific, patient-reported outcome measure for use in clinical trials. Supported by: NIH, MDA, Registry of Myotonic Dystrophy and Facioscapulohumeral Muscular Dystrophy Patients and Family Members.
 

[P06.262] Infantile Muscular Dystrophy in Canadian Aboriginals Is an B-Crystallinopathy

Duygu Selcen, Rochester, MN, Marc R. Del Bigio, Albert E. Chudley, Winnipeg, MB, Canada, Harvey Sarnat, Seattle, WA, Craig Campbell, Sharan Goobie, London, ON, Canada, Bernard N. Chodirker, Winnipeg, MB, Canada

OBJECTIVE: To determine the genetic basis of a recessively transmitted fatal infantile muscular dystrophy. BACKGROUND: A recessively transmitted fatal infantile muscular dystrophy associated with hypertonia has been described in Canadian Aboriginals. The affected infants present with progressive limb and axial muscle stiffness, develop severe respiratory insufficiency, and most die in the first year of life. DESIGN/METHODS: We performed histochemical, immunocytochemical, electron microscopy and molecular genetic studies in a cohort of 12 patients affected by this disease. RESULTS: Conventional histochemical and electron microscopy studies suggested myofibrillar myopathy (MFM). Therefore we searched for ectopic expression of multiple proteins typical of MFM. B-crystallin (BC) expression was absent from all fibers using a monoclonal antibody raised against the entire protein. However, a monoclonal antibody directed against the first 10 residues of BC immunostained portions of abnormal fibers. Pursuing this clue, we searched for mutations in the gene for BC (CRYAB) in available DNA samples of 8 patients. All harbor a homozygous deletion, c.60C, predicting a Ser to Ala change at codon 21 and a stop codon after 23 missense residues (p.Ser21AlafsX24). Clinically unaffected parents are heterozygous for this mutation. CONCLUSIONS: The homozygous c.60delC in CRYAB pinpoints the genetic basis of the fatal infantile hypertonic muscular dystrophy of Canadian Aboriginals. MFMs are typically transmitted by dominant inheritance but in this disease the parental phenotype is rescued by limited expression of the highly truncated nonfunctional mutant gene product. The severe patient phenotype is due to homozygosity for the markedly hypomorphic allele

[P06.277] Bent Spine Syndrome as an Early Manifestation of Myotonic Dystrophy Type 1

Vivek Krishna, William Arnold, Columbus, OH, Victoria Lawson, Lewis Center, OH

OBJECTIVE: To describe two cases of myotonic dystrophy type 1 exhibiting early weakness of spinal extensor muscles. BACKGROUND: Bent spine syndrome (BSS) is a posture of forward flexion of the trunk or neck, commonly associated with dystonia of the axial muscles in extrapyramidal disease. The syndrome may additionally result from spinal extensor muscle weakness. A variety of muscle diseases are associated with BSS including fascioscapulohumeral dystrophy, nemaline rod myopathy, inflammatory myopathies, and proximal myotonic myopathy (PROMM), but only a few reports exist describing BSS in association with type 1 myotonic dystrophy (DM1). We report two cases with early, prominent spinal extensor weakness. DESIGN/METHODS: A chart review was performed. RESULTS: Case 1: A 63 year-old female presented with flexed posture, immobility, and back pain, prompting her evaluation 10 years after the onset of symptoms. Examination showed nearly 90 degrees of forward flexion of the thoracic spine with standing and walking. She had mild facial and limb weakness with no apparent grip or percussion myotonia. Genetic testing revealed 130 trinucleotide repeats in the dystrophia myotonicaprotein kinase (DMPK) gene. Case 2: A 66 year-old female presented with a 13-year history of progressive proximal weakness and forward flexion of the spine. Examination showed facial, distal upper and proximal lower limb weakness, grip myotonia, and marked forward flexion of the upper thoracic spine and nearly complete head drop. Genetic testing revealed 283 CTG repeats in the DMPK gene. CONCLUSIONS: Patients presenting with spinal extensor muscle weakness present a diagnostic challenge. BSS has rarely been reported as an early or distinctive feature of DM1. These cases emphasize the phenotypic heterogeneity of DM1 and underscore the importance of considering DM1 in the differential diagnosis of BSS.
 

[P04.255] Sleep Apnea and Other Sleep Dysfunction in Myotonic Dystrophy Type 2

Amtul Farheen, Woodbridge, NJ, Sudhansu Chokroverty, Edison, NJ, Howard Sander, New York, NY, Raji Grewal, Cranbury, NJ, Qi Rui Yang, West Orange, NJ, Kamran Kabolizadeh, Edison, NJ, Sombabu Maganti, Metuchen, NJ, David Rosen, Edison, NJ, Vinay Chaudhry, Baltimore, MD

OBJECTIVE: To describe sleep dysfunction in myotonic dystrophy type 2 (DM2). BACKGROUND: DM2 is a recently described hereditary myotonic dystrophy which is differentiated from myotonic dystrophy type 1 (DM1) by different genetic mutations. Sleep apnea has been described in DM1 but is a novel finding in four of our six patients with DM2. DESIGN/METHODS: Four women (51, 53, 62, 63) and two men (57, 63) presented with difficulty raising their shoulders and climbing stairs. Major sleep complaints included chronic insomnia, snoring, and excessive daytime somnolence. One patient (63 F) also gave a history of dream-enacting behavior. Examination revealed diffuse symmetric muscle weakness involving proximal more than distal muscles and thenar eminence percussion myotonia. EMG/NCV and polysomnographic (PSG) studies were performed. RESULTS: Genetic testing was consistent with a CCTG expansion in the ZNF9 gene on chromosome 3. Two sisters (51, 53) had normal chromosome 19 CTG repeat size which excluded DM1 and the DM2 genetic testing was not available when they were investigated. EMG revealed myotonia with myopathic findings. PSG results in four patients (57M, 63M, 63 F, 62 F) were consistent with obstructive sleep apnea (OSA). One patient (63 F) during CPAP titration had a dream-enacting behavior (someone was trying to strangle her) and PSG showed REM without atonia and excessive phasic muscle bursts (consistent with REM behavior disorder). In two sisters (51, 53) PSG findings showed absent REM sleep in one (51) and alpha-REM-spindle mixture in the other who also showed paradoxical movements in the effort channels suggesting increased upper airway resistance. CONCLUSIONS: These sleep abnormalities including REM behavior disorder in one and sleep apnea in four patients with DM2 are unique observations and to our knowledge have not been described before.
 

[S34.005] Systemic Delivery of Targeted RNA Interference as a Treatment for Myotonic Dystrophy

Joel R. Chamberlain, Eva Stepniak, Seattle, WA

OBJECTIVE: To develop a treatment for dominantly inherited disorders. BACKGROUND: Therapy for dominant genetic disease requires elimination of the disease causing entity. We have chosen to target the trinucleotide repeat-expanded mRNA that causes myotonic dystrophy (DM1) through coercion of the RNA interference pathway. DM1 is the most common adult form of muscular dystrophy and is characterized by an inability of skeletal muscles to relax after contraction (myotonia), as well as the seemingly unrelated manifestations of hyperinsulinemia, somnolence, endocrine disturbances, and cognitive deficits. Evidence relating the varied effects of expressing the repeat-expanded mRNA emerged from studies showing that proteins involved in splicing were disrupted in their ability to dictate the adult splicing pattern of a subset of genes in the muscle cell. Reversion to the fetal pattern of splicing of certain genes appears to be related to particular phenotypic disturbances in DM. A mouse model of DM1, the HSA^LR mouse, was created by placing 250 CTG repeats in the 3'UTR of the human HSA gene and displays the characteristic myotonia and defects in splicing as observed in the human disease. DESIGN/METHODS: Artificial expression cassettes were designed and constructed to direct sequence specific degradation of HSA^LR mRNA using the mouse U6 promoter driving expression of short hairpin RNAs. Knockdown of HSA^LR mRNA was assayed in vitro and effective expression cassettes to trigger RNA interference (RNAi) were selected for use in adeno-associated virus (AAV) shuttles, which were used to carry the RNAi expression cassettes to the muscles of the HSA^LRmice. RESULTS: Systemic injection of the AAV6 RNAi cassettes targeting the HSA^LR mRNA showed wide-spread transduction of muscles. We assayed quadriceps muscle for changes in the splicing pattern of the SERCA1 gene and observed a reversion in splicing in a population of mRNAs after injection of select HSA^LR mice. CONCLUSIONS: Further development of targeted RNAi could lead to therapy for myotonic dystrophy and other dominant diseases of muscle.

[P06.271] Laboratory Abnormalities in Patients with Myotonic Dystrophy Type 2

Nicholas Johnson, Chad Heatwole, Rochester, NY, Bradley S. Goldberg, Tel Aviv, Israel, William Martens, Richard Moxley, Rochester, NY

OBJECTIVE: To study, identify, and describe the clinical laboratory abnormalities common to myotonic dystrophy type-2 (DM2). BACKGROUND: Myotonic dystrophy type 2, similar to myotonic dystrophy type-1, is an autosomal dominant, multi-system disorder. Patients commonly experience progressive weakness, early onset cataracts, and clinical myotonia; however, other body systems are frequently involved including the brain, endocrinologic, hematologic, hepatic, and renal systems. Although clinical laboratory abnormalities are frequently observed in DM2, large scale studies of these abnormalities in this relatively rare disease are limited. DESIGN/METHODS: 83 DM2 patients previously evaluated through a university medical center were identified. Laboratory results from this population were compiled, averaged, and compared to university standard reference ranges. RESULTS: 1442 total DM2 studies representing sixty-eight different types of laboratory tests were evaluated. Results for 359 (24.9%) of these studies were outside of their standard reference range. The relative frequency of abnormally elevated laboratory values was greater than 50% in several tests, including creatine kinase, total cholesterol, lipase, alanine aminotransferase (ALT), and lactate dehydrogenase. In addition, serum levels of immunoglobulin G (IgG) and absolute lymphocyte count were found to be low in 75% and 54% of all DM2 patients tested. CONCLUSIONS: There is a high frequency of laboratory abnormalities in DM2. These abnormalities are referable to multiple organ systems, indicating the widespread pathological manifestations of DM2. These abnormalities may provide a foundation for clinical monitoring and disease screening in this population.

[S24.004] Reducing Bodies and Myofibrillar Myopathy Features in FHL1 Dystrophy

Duygu Selcen, Rochester, MN, Mark Bromberg, Steven S. Chin, Salt Lake City, UT, Andrew G. Engel, Rochester, MN

OBJECTIVE: To search for mutations in FHL1 in patients with myofibrillar myopathy (MFM). BACKGROUND: Mutations in the FHL1 have been associated with a diverse chronic myopathies including late onset X-linked axial and scapuloperoneal myopathy with bent spine syndrome, reducing body (RB) myopathy, X-linked dominant scapuloperoneal muscular dystrophy, rigid spine syndrome, and contractures and cardiomyopathy mimicking Emery-Dreifuss muscular dystrophy. MFMs are morphologically distinct but genetically heterogeneous muscular dystrophies in which disintegration of Z-disks and then of myofibrils is followed by ectopic accumulation of multiple proteins. Because some pathologic features of the FHL1-muscular dystrophies and the MFMs overlap, we searched for mutations in FHL1 in 50 MFM patients. DESIGN/METHODS: Histochemical, immunocytochemical, and ultrastructural studies; mutation analysis. RESULTS: We detected two novel and one previously identified missense mutation in 5 MFM patients. Two mutations are in the second LIM domain and one outside the LIM domains of FHL1. The age of onset was 6-75 years and the age at diagnosis 10-77 years. All but one patient presented with progressive muscle weakness; one had hypertrophied muscles, rigid spine, and joint contractures, and one also had peripheral neuropathy. Patients harboring LIM2 domain mutations had typical MFM features and also displayed menadione-NBT positive RBs whereas the patient with the mutation outside LIM domains had no RBs. High resolution EM showed the RBs composed of 13 nm tubulofilaments that emanated from Z-disks. This was followed by myofibrillar disintegration, accumulation of myriad RBs in cytoplasm and many nuclei and of cytoplasmic degradation products, and aggregation of endoplasmic reticulum and sarcotubular profiles. CONCLUSIONS: Light microscopy of FHL1 dystrophies can show MFM features with or without RBs. Presence of RBs points to a mutation in LIM2 domain of FHL1. Mutation outside the LIM domains resulted in a milder phenotype with late onset.

[P06.274] Factors Related to Quality of Life in Patients with Myotonic Dystrophy Type 1

Stojan Peric, Vidosava Rakocevic Stojanovic, Sanja Pavlovic, Ivana Basta, Zorica Tasic, Dragana Lavrnic, Belgrade, Serbia, Serbia and Montenegro

OBJECTIVE: To assess potentially treatable factors that might influence self-reported QoL in patients with adult form of DM1. BACKGROUND: Health-related quality of life (QoL) measures obtained through patient-oriented tools are now considered essential in the evaluation of neurological diseases, especially in those that may affect the general health status of patients such as myotonic dystrophy type 1 (DM1). Quality of life assessments contribute to greater understanding of disease's consequences, the effects of its treatment and palliative care. DESIGN/METHODS: A cross-sectional study was applied in 114 consecutively recruited DM1 patients. The severity of muscular involvement in DM1 was measured by MIRS scale. Multisystemic affection was thoroughly investigated in all patients. The following assessment instruments were also used: The Fatigue Severity Scale (FSS), The Daytime Sleepiness Scale (DSS), The Hamilton Depression Scale, Visual Pain Scale and The SF-36 questionnaire. RESULTS: The mean total SF-36 score was 45.423.9 indicating that QoL was significantly affected in patients with DM1. Considering demographic features, younger patients (p<0.01), married patients and patients without children had better QoL (p<0.05). Duration of disease and severity of muscular involvement significantly affected QoL in DM1 patients (p<0.01). Among multisystemic affections, we found constipation and snoring (p<0.05), as well as presence of fainting and gallstones (p<0.01) to have a significant influence on QoL. Central symptoms of DM1 including depression, anxiety and fatigue significantly affected QoL (p<0.01), while daytime sleepiness did not show correlation with QoL (p>0.05). QoL was also affected by presence and intensity of pain (p<0.01). CONCLUSIONS: QoL was significantly affected in patients with DM1. We identified potentially treatable factors that influenced QoL in DM1 patients, including constipation, snoring, cardiac abnormalities, gallstones, depression, anxiety, fatigue and pain. We assume that treatment of these symptoms may significantly improve palliative care and QoL in patients with DM1.
 

[P06.269] Test-Retest Reliability of the Purdue Pegboard Test and the Jebsen-Taylor Hand Function Test in Patients with Myotonic Dystrophy Type 1 (DM1)

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

OBJECTIVE: To document the test-retest reliability of Purdue Pegboard Test and the Jebsen -Taylor Hand Function Test in patients with DM1. BACKGROUND: Hand weakness has been well documented in patients with DM1; however hand function using standardized tests like the Purdue Pegboard Test and the Jebsen-Taylor Hand Function Test has not been examined in patients with DM1. Reliable and sensitive measures of hand function are essential for therapeutic trials in DM-1. DESIGN/METHODS: The Purdue Pegboard Test and the Jebsen-Taylor Hand Function Test were performed twice within a 24 hour period in 22 patients using standardized instructions. The Purdue Pegboard Test consists of having the patient place as many pegs as they can in the board in 30 seconds. The Jebsen-Taylor Hand Function Test consists of 7 subtests: writing, picking up small objects, turning cards over, simulated feeding, stacking checkers, lifting light cans, and lifting heavy cans. The tests were performed sequentially. RESULTS: The intraclass correlation coefficient (ICC) for the Purdue Pegboard Test was .835. The ICC for the overall Jebsen-Taylor Hand Function Test was .896, with ICC's ranging from .394 to .931 on the subtests. The subtests of writing had excellent test-retest reliability with the majority of the other subtests demonstrating moderate test-retest reliability. CONCLUSIONS: These tests of hand function demonstrate good reliability in patients with DM-1 when repeated over a 24 hour time period. Tests of sensitivity to change over longer periods of time will need to be performed prior to using these tests as outcome measures for clinical trials.

[S34.001] A Phase I Dose-Escalating Study of AAV1-Gamma-Sarcoglycan Gene Therapy for Limb Girdle Muscular Dystrophy Type 2C

Olivier Benveniste, Paris, France, Faycal Hentati, Tunis, Tunisia, Bruno Eymard, Paris, France, Didier Caizergues, Evry, France, Thomas Voit, Serge Herson, Paris, France

OBJECTIVE: Safety of dose-escalating AAV1-gamma-sarcoglycan gene therapy (Phase I) for Limb Girdle Muscular Dystrophy type 2C (LGMD 2C). BACKGROUND: Gamma-sarcoglycanopathy or LGMD 2C is an as yet untreatable disease caused by autosomal recessively inherited mutations of the gamma-sarcoglycan gene (SGC). DESIGN/METHODS: Nine non-ambulant LGMD 2C patients (2 M, 7 F, age 27 y [16 to 38]), with a del525t homozygous mutation on the SGC gene and absence of SGC immunostaining in muscle biopsy were enrolled. They were divided into 3 groups to receive 3 escalating doses of an AAV1 vector expressing human SGC gene under the control of the desmin promoter, by local intramuscular injection into the extensor carpi radialis muscle. The first group received a single injection of 3x10⁹ viral genome (vg) in 100 l, the second a single injection of 1.5x10¹⁰ vg in 100 l and the third group received 3 concomitant 100 l injections at the same site (a total of 4.5x10¹⁰ vg). RESULTS: No serious side effects were observed. All patients became seropositive for AAV1 and one developed a cytotoxic response again AAV1 capsid. In biopsy specimens taken 30 days after the injection, immunohistochemistry showed SGC expression in 5/9 patients, with 4.7 to 10.5% of positively stained fibers and detection of SGC mRNA by RT-PCR in the 3 patients who received the highest dose. The SGC protein became also detectable by Western blot in one patient. CONCLUSIONS: Exogenous SGC expression can be obtained in LGMD2C patients following AAV1 gene transfer, without adverse effects, paving the way for future developments in gene therapy of hereditary muscle diseases.