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Manifesting carriers of DMD gene mutations may present diagnostic challenges, particularly in the absence of a family history of dystrophinopathy. We review the clinical and genetic features in fifteen manifesting carriers identified among 860 subjects within the United Dystrophinopathy Project, a large clinical dystrophinopathy cohort whose members undergo comprehensive DMD mutation analysis. We defined manifesting carriers as females with significant weakness, excluding those with only myalgias/cramps. DNA extracted from peripheral blood was used to study X chromosome inactivation patterns. Among these manifesting carriers, age at symptom onset ranged from 2 to 47 years. Seven had no family history and eight had male relatives with Duchene muscular dystrophy (DMD). Clinical severity among the manifesting carriers varied from a DMD-like progression to a very mild Becker muscular dystrophy-like phenotype. Eight had exonic deletions or duplications and six had point mutations. One patient had two mutations (an exonic deletion and a splice site mutation), consistent with a heterozygous compound state. The X chromosome inactivation pattern was skewed toward nonrandom in four out of seven informative deletions or duplications but was random in all cases with nonsense mutations. We present the results of DMD mutation analysis in this manifesting carrier cohort, including the first example of a presumably compound heterozygous DMD mutation. Our results demonstrate that improved molecular diagnostic methods facilitate the identification of DMD mutations in manifesting carriers, and confirm the heterogeneity of mutational mechanisms as well as the wide spectrum of phenotypes.

Hypokalemic periodic paralysis (hypoKPP) is an autosomal dominant or sporadic disorder characterized by periodic, reversible attacks of muscle weakness. Mutations in the skeletal muscle dihydropyridine receptor alpha 1-subunit that functions as a calcium channel (CACNL1A3) cause hypoKPP. We studied a group of 45 hypoKPP probands and demonstrated mutations in 30 of them. When compared with patients in whom CACNL1A3 mutations were not identified, those with mutations had an earlier age of onset and more often had a family history of hypoKPP. To date, three mutations have been identified. The R1239G mutation has only been found in one family. Of the 30 probands with recognized mutations, R528H accounted for 43% and R1239H was seen in 53%. Age of onset and potassium levels during attacks were lower in patients with the R1239H mutation than those with R528H. Cardiac dysrhythmias co-segregated with hypoKPP in one small kindred with the R528H mutation. No mutations were identified in exons of the gene encoding the S4 segments of domains one and three or the cytoplasmic loop between domains two and three. In addition to the 45 hypoKPP probands, an additional 11 probands with clinical variants of hypoKPP (three thyrotoxic hypoKPP and eight Andersen syndrome patients) were examined for CACNL1A3 mutations and none were found.

Muscle biopsy from two unrelated patients, a male aged 40 and a female aged 3, with relatively non-progressive limb weakness since infancy, revealed numerous subsarcolemmal glassy, hyaline appearing bodies present in 20–30% of the fibres. Type 1 fibre predominance was present, and the hyaline bodies were exclusive to type 1 fibres. The hyaline bodies were negative with oxidative enzyme and periodic acid-Schiff stains. Electron microscopy showed the hyaline bodies to contain amorphous granular material of unknown composition. No membrane separated the hyaline bodies from the surrounding sarcoplasm. Hyaline body myopathy most likely represents a distinct congenital myopathy because of its childhoot-onset, non-progressive course, and distinct morphological features.

Few clinical trials involve non-ambulatory boys and men with Duchenne muscular dystrophy. We previously tested clinical outcomes in 91 subjects and found Forced Vital Capacity (FVC), Brooke and Egen Klassifikation (EK) Scales, grip, key and pinch strength showed excellent intra-rater reliability and feasibility. We then followed subjects every six months. Over the two-year study, eight died (mean 18.7; range 33–12 years) and 33 missed at least one visit or dropped out. Four of the eight who died were taking corticosteroids and four were not. Sixty-six completed baseline and 12-month follow-up; 51 completed baseline and 24-month follow-up; 39 completed all 5 visits. FVC and and grip strength both declined significantly over one and two years (two tailed p for all

The success of clinical trials in infants with spinal muscular atrophy (SMA) requires careful trial planning and appropriate outcomes. Traditional tests are fatiguing for SMA infants, use non-linear ordinal scales and require highly trained evaluators to grade movement. Our study provides preliminary reliability and validity data on a video-based assessment, ACTIVE-mini, which uses the Microsoft Kinect to assess infant movement. The infant lies supine and spontaneous movements are recorded using a series of 3D depth map and color images. Algorithms are used to quantify infant movement speed (i.e. velocity, acceleration, and jerk), as well as total movement volume, and the complexity of infant movement within space. Weekly longitudinal and cross-sectional data were collected over 6 months on 6 SMA infants and 30 controls. Movement speed data are plotted as kernel density plots separated in 2-week age-matched bins. Differences are seen in each movement variable in every age bin. On average the SMA infants have an average jerk value of 57.8% of age-matched peers. Tracings of the extremities and the ratio of convex hull’s surface area to volume ratio, where higher numbers indicate decreased ability, illustrate that an SMA infant has a smaller total movement excursion as well as decreased spatial complexity (upper extremity average: 139–170; lower extremity average: 756–891) when compared to age-matched controls (all extremities average range: 43–68). Preliminary results indicate ACTIVE-mini differentiates movement differences related to speed, variability, quantity, and complexity of movement in SMA infants compared to controls in the first 2 weeks of life. ACTIVE-mini has both clinical and research potential to detect movement abnormalities and measure small changes in ability over time. Further research is needed in a larger sample of diverse diagnoses to refine the statistical models of normal movement and identify typical longitudinal changes in infants.

There is no cure for dysferlinopathies, a group of related disorders caused by absent or mutant dysferlin (DYSF). Lack of dysferlin leads to progressive dystrophy with chronic muscle fiber loss, inflammation, fat replacement and fibrosis leading to deteriorating muscle weakness. Dysferlin has multiple functions including T-tubule formation and membrane repair. Pre-clinical studies show that only delivery of full-length DYSF corrects the histopathological defects in DYSF−/− mice. As DYSF is too large to be accommodated with canonical packaging, we developed a unique dual vector system using AAV to deliver and express DYSF in muscle. This two vector system (AAV.DYSF.DV) packaged in the rh.74 serotype is defined by a 1 kb region of homology between the two vectors. We compared the efficiency and safety of DYSF.DV delivery in dysferlin deficient mice and in non-human primates (NHPs) in preparation for clinical trials. Dysf−/− mice were treated by intramuscular injection, isolated limb perfusion, and systemic injection with AAV.DYSF.DV. IM injections were performed in the flexor digitorum brevis (FDB) muscle to assess expression and restoration of membrane repair capacity using ascending vector doses (6e9–6e10vg). The FDB was extracted after 10 wks and an ex vivo laser injury membrane repair assay was performed. DYSF.DV restored repair capacity in a dose-dependent manner comparable to WT findings. DYSF.DV was also efficacious when delivered through arterial circulation to hind limbs or systemically via the tail vein. A systemic dose of 6e12vg resulted in widespread gene expression exceeding 50% of muscle fibers; restoring functional deficits in the diaphragm. In NHPs, DYSF.DV delivery was analyzed for gene expression and toxicity using FLAG tag. There was no evidence of toxicity in DYSF-null mice or NHPs showing overexpression of dysferlin. Findings showed clear evidence that functional dysferlin was translated without toxicity laying the foundation for clinical trial.

DMD is a rare, degenerative, genetic disease that results in progressive muscle loss and premature death and affects 1:5000 male births. Clinically manifest pulmonary dysfunction often occurs when DMD patients become non-ambulant and is preceded by subclinical deterioration of pulmonary function tests (PFTs). Specifically, MIP and MEP % predicted deteriorate by approximately 4 % per year between the ages of 8–19. Eteplirsen is an investigational drug designed to enable functional dystrophin production in boys who are amenable to skipping exon 51. When dosed for up to 120 weeks, stabilization of 6 min walk distance was demonstrated in a Phase 2b study. 12 boys were randomized 1:1:1 to 30/50 mg/kg or placebo. Upon completion of a 24-week double-blind, placebo-controlled phase, all patients were enrolled in an open-label extension and the placebo-treated patients initiated eteplirsen treatment. FVC, FVC % predicted, MIP, MEP and MIP and MEP % predicted were assessed every 12 to 24 weeks. For all 12 patients changes in function at Week 120 were examined from Week 1, and from last assessment pre-eteplirsen administration (Week 1 for 8 patients and Week 24 for 4 patients). One-sample t-test was used for statistical analysis. Reported here are the results for all 12 patients, including two patients who became non ambulant by Week 24. Median age at Week 120 was 12 years. The 120 week data for 5 of the 6 PFT parameters were not statistically different from baseline, with the exception of a statistically significant increase in MEP. Furthermore, individual patient values for all 12 patients continue to be in the age-adjusted normal ranges indicative of continued normal pulmonary function without the need for any ventilation assistance or respiratory failure. A PFT update after 3 years of treatment (Week 144) will be provided. Eteplirsen dosed for up to 120 weeks demonstrated stability on PFT measures contrary to a steady decline expected in DMD patients of this age.

DMD is a rare, degenerative, genetic disease that results in progressive muscle loss and premature death. Affecting 1 in 5000 male births, DMD is caused by the inability to produce the dystrophin protein. There are no approved drugs available to treat DMD, although glucocorticoids show a modest effect with long-term efficacy limited by side effects. Eteplirsen is an investigational therapy designed to enable functional dystrophin production in boys who are amenable to exon 51-skipping therapy (∼13%). Twelve boys aged 7–13 years with eligible genotypes were randomized 1:1:1 to 30 mg/kg, 50 mg/kg, or placebo. Upon completion of a 24-week double-blind, placebo-controlled study phase, all subjects were enrolled in an open-label extension and the placebo-treated subjects initiated eteplirsen treatment. The critical clinical endpoint was the change in 6-min walk test (6MWT) distance from baseline compared to the placebo/delayed-treatment cohort. Previously reported 48-week results showed a statistically significant increase in 6MWT distance for eteplirsen versus the placebo/delayed-treatment arm. At 62 weeks, a significant clinical benefit of 62 m was observed (p ⩽ 0.007) on the 6MWT for ambulatory-evaluable subjects in the combined eteplirsen-treated cohorts (n = 6) versus the placebo/delayed-treatment cohort (n = 4). The eteplirsen-treated cohorts showed less than a 5% decline in 6MWT distance from baseline. No clinically significant treatment-related adverse events were seen through 62 weeks. Through 62 weeks of treatment, eteplirsen demonstrated a significant clinical benefit on the 6MWT with no clinically significant treatment-related adverse events. Data on these outcomes will be reported through 2 years of treatment.

Clinical trials involving exon skipping, gene transfer, and small molecule development are currently underway for children with Duchenne muscular dystrophy. These experimental trials typically use timed walking as the primary measure of efficacy due to ease of test administration, ability to quantify distance walked, and a body of literature investigating the reliability and validity of these walking outcomes. However, a test lasting 6-min has inherent difficulties when testing young children in clinical trials. Ambulatory subjects with DMD (5–12 years old; mean = 9.6 ± 2.1 years) completed timed walking tests, strength testing, the North Star Ambulatory Assessment (NSAA), and timed 4 stairs. The 6-min walk test (6MWT) was compared to two shorter ambulatory assessments: 2-min walk test (2MWT), and 100 m walk/run test (100 m). The 2MWT was completed according to 6MWT guidelines but lasted only 2-min; whereas the 100 m allowed subjects to complete two 50-m laps as quickly as possible, including running if able. Maximum voluntary isometric strength of hip and knee muscles was assessed using a hand-held dynamometer. The 2MWT and 100 m were highly correlated with 6MWT (r = 0.827 and r = −0.827 respectively, p = 0.002). The 100 m was most highly correlated with lower extremity strength measures and other functional outcomes. A shorter timed walking or running test may be beneficial for use in clinical trials in children with Duchenne muscular dystrophy. The 2MWT or 100 m may decrease individual variability seen in the 6MWT due to shorter walking time or a concrete fixed distance. Further research is needed to determine which ambulatory assessments are most valid, reliable, and sensitive to change over time.

Clinical trials are being conducted for DMD using timed walking as a primary outcome measure due to the ease of administration, ability to quantify distance, and established validity. Unfortunately, participation in clinical trials is not equally accessible to all individuals due to the lack of a reliable, valid and sensitive upper extremity measure for the non-ambulatory population. It is essential that this gap in knowledge be filled, as upper body movement is required for many activities of daily living impacting quality of life across the lifespan and abilities in individuals with DMD. The purpose of this project was to develop a reliable and engaging upper extremity measure to quantify workspace volume, reaching velocity, and rate of arm fatigue using the Kinect system to capture data during a video virtual reality game across the spectrum of abilities. The Microsoft Kinect Windows 7 used for this project is a controller-free gaming device that projects an infrared reference matrix that is reflected off of the person and generates a high-resolution depth map of each pixel allowing for tracking of movement. Software is available to track 20 joints and provide positional data which can be used to calculate measurements such as workspace volume, reaching velocity, and rate of arm fatigue. Virtual gaming was developed as a motivational tool to be used during assessment. Work space volume gaming uses bubble popping game while velocity and fatigue use a disappearing image to encourage rapid movement. A prototype assessment system is being validated against a marker based motion capture system and traditional upper extremity assessments such as the Jebsen Hand Function Test, 9-hole peg test, and the Brooke Upper Extremity Rating Scale. Preliminary data suggests that this may be an engaging, reliable and valid upper extremity assessment measure for individuals with DMD across the lifespan.

Becker muscular dystrophy (BMD), a dystrophinopathy with milder skeletal muscle manifestations compared to Duchenne muscular dystrophy, characteristically demonstrates weakness of knee extensors (KE), accompanied by muscle atrophy and fibrosis. The weakness predisposes to gait impairment and loss of ambulation. Myostatin, a member of the TGF-β superfamily of signal peptides, is expressed in skeletal muscle, acting through the activin type IIB (ACTRIIB) receptor. Follistatin, a myostatin antagonist, inhibits this pathway resulting in muscle hypertrophy, and improved muscle strength. FS315 and FS288 are two isoforms of follistatin generated by alternative splicing. FS315 has no cell-surface affinity providing a safety margin avoiding pituitary–gonadal axis complications. FS344 is the precursor cDNA to FS315. When FS344 is delivered to mdx mice in adeno-associated virus (AAV1. CMV. FS344), muscle mass and strength increase. Similar findings are seen in non-human primates without safety concerns. Six BMD patients, 18 years and older with proven in-frame DMD gene mutations were enrolled in a clinical trial. The first 3 subjects received 12 intramuscular (IM) injections of AAV1. CMV. FS344 at a total dose of 6e11 vg/kg in the quadriceps muscles bilaterally. A second cohort received the same number of IM injections at higher total dose (1.2e12 vg/kg). Studies extending beyond one year in Cohort 1 and for six months in Cohort 2 showed no adverse events. The distance walked (meters) on the 6-min walk test (6MWT) was the primary functional outcome measure. At one year the distance walked by two subjects in Cohort 1 increased by 125 m and 58 m; subject 3 improved modestly by 9 m. In Cohort 2, one patient reaching the 6-month time point increased by 108 meters. For the entire group of BMD patients only a single patient declined in distance. Preliminary analysis of muscle biopsies favor increased muscle regeneration contributing to improved function.

Eteplirsen, SRP-4045 and SRP-4053 are three PMOs developed as treatments for DMD. DMD is mostly caused by mutations in the dystrophin gene that lead to a reading frame shift and premature translation termination. PMOs promote exon skipping during splicing of dystrophin pre-mRNA, restoring the reading frame and translation of internally truncated, but functional dystrophin protein. Eteplirsen, SRP-4045, and SRP-4053 are intended to treat patients with mutations amenable to skipping exon 51, 45 and 53, respectively. IND enabling preclinical studies have been completed for eteplirsen, SRP-4045, and SRP-4053. In vitro metabolism data and PK profiles in animals were similar for all three PMOs. No adverse responses were detected in safety pharmacology studies in non-human primates (NHPs). Genotoxicity studies were negative. NHPs given 12 weekly IV doses up to 320 mg/kg showed no significant sequence-specific toxicities. A clinical study of eteplirsen with doses of 30 and 50 mg/kg/wk reported no clinically significant treatment-related adverse events through 120 weeks of treatment. A few cases of transient, mild urine protein elevation resolved without intervention or drug interruption and one femur fracture was determined to be treatment-unrelated. There were no hospitalizations, discontinuations, or clinically significant treatment-related changes for lab parameters, including liver enzymes, kidney function, coagulation, or platelet counts. Plasma Cmax and AUC increased proportionally with dose, and half-life was approximately 3 h with no plasma accumulation observed. The consistent preclinical profiles and lack of significant, sequence-specific toxicities for eteplirsen, SRP-4045, and SRP-4053 demonstrate PMOs to be a well-tolerated therapeutic class, with potential applications in a wide variety of disease indications.

DMD, a rare, degenerative, genetic disease that results in progressive muscle loss and premature death affects 1:5000 male births. It is caused by deletions in the dystrophin gene, which prevents production of the dystrophin protein. There are no approved treatments available, although corticosteroids have shown some benefit. Eteplirsen is an investigational drug designed to enable functional dystrophin production in boys who are amenable to skipping exon 51. Twelve boys aged 7–13years with eligible genotypes were randomized 1:1:1 to eteplirsen 30mg/kg/wk, 50mg/kg/wk, or placebo IV for 24-weeks. All patients transitioned into an ongoing open-label extension with 30 or 50mg/kg eteplirsen. Clinical efficacy endpoints included the 6-min walk test (6MWT) and pulmonary function testing. Safety assessments included adverse event recording, EKG, ECHO, hematology, blood chemistry and urinalysis. After 120weeks of treatment, a significant clinical benefit of 65m was observed ( p =0.006) on the 6MWT for ambulatory-evaluable patients in the combined eteplirsen-treated cohorts ( n =6) versus the placebo/delayed-treatment cohort ( n =4). The eteplirsen-treated cohorts showed a decline of less than 14m in walking ability from baseline, which was not statistically significant. After a substantial decline in the first 36weeks of the study, the placebo/delayed-treatment cohort demonstrated stabilization in walking ability at week 48, i.e., 12weeks after initiation of treatment with eteplirsen when meaningful levels of dystrophin were likely produced, with

Limb-girdle muscular dystrophy (LGMD) type 2E results from mutations in the beta-sarcoglycan gene causing loss of functional protein. It is characterized by muscle weakness and progressive muscle wasting. The sarcoglycans (alpha, beta, gamma, and delta-SG) are structural proteins localized at the cell membrane of muscle fibers that together with dystrophin and other proteins make up the dystrophin-associated protein complex (DAPC). Loss of functional protein in this complex results in an unstable sarcolemma leading to eventual myofiber death. To date, no effective therapy exists to treat this debilitating disease. The goal of this study is to demonstrate efficacy of AAV-mediated beta-sarcoglycan gene transfer in beta-SG knock-out mice to provide proof-of-principle for gene replacement therapy for LGMD2E. A previous study by our group investigating AAV gene replacement therapy to treat LGMD2D led to a successful clinical trial in LGMD2D patients (alpha-sarcoglycan deficiency), which will provide a solid platform for this study. We generated a self-complementary AAVrh74 vector containing a codon optimized human beta-Sarcoglycan gene (hSGCB) driven by the muscle specific tMCK promoter. Following direct intramuscular injection to demonstrate potency of our vector, we treated beta-SG KO mice via a clinically relevant vascular delivery model to deliver AAV.tMCK.hSGCB in two doses (1x1011 and 5x1011 vg) to the lower limb muscles. We had clear demonstration of dose dependent beta-SG expression and improvement in dystrophic pathology with greater than 90% beta-SG expression in lower limb muscles following vascular delivery at high dose following 3 months of treatment. Functional outcome measures performed on the extensor digitorum longus (EDL) muscle from these mice showed an improvement in specific force generation and protection from eccentric contraction induced injury. This pre-clinical study is pivotal for establishing proof-of-principle for translation of AAV.hSGCB gene transfer in LGMD2E patients.

The need for a valid and reliable measure of upper extremity (UE) function in Duchenne muscular dystrophy (DMD) is critical as current tools focus on distal ability, use an ordinal scale, and/or require significant training to use reliably. ACTIVE-seated was developed using the Microsoft Kinect platform. This camera system and gaming interface gathers UE positional data while providing the platform for video games to improve consistency of performance. Our purpose was to determine the validity and test–retest reliability of ACTIVE-seated as an UE tool in DMD. A sample of 40 subjects (mean age 13 ± 3) with DMD (Brooke level 1–5) and 15 controls were tested. Eleven subjects returned for next-day testing. ACTIVE-seated uses skeletal tracking to quantify the UE excursion in the X (left, right), Y (overhead), and Z (forward) planes. The functional reaching volume (FRV) of the area around the subject, encompassing both UE and trunk movement, was reported. The raw data were converted into a percent predicted based on the subject’s UE length to standardize comparisons between subjects and accommodate growth. One hundred percent of predicted would equal the accessible FRV of solely the UE without leaning by the trunk. The FRV of subjects with DMD ranged from 6% to 150% (mean: 69%) of predicted FRV, with controls all reaching well over 100% (mean: 147%). ACTIVE-seated discriminately ranked subjects with DMD by Brooke level and from controls (p

Current trials in Duchenne muscular dystrophy (DMD) have focused on the distance traveled using the 6-min walk test (6MWT). As a primary efficacy outcome for clinical trials it can be quantified over a continuous scale. Limitations include a self-selected walking speed irrespective of encouragement and weakness predisposing to falls. We predicted that a monetary incentive of $50 could improve same-day 6MWT performance. Nine DMD boys (mean age 7.7) were instructed to walk quickly, not run. Encouragement was permitted throughout the test. Subjects were then stratified into an above or below 350 m group and randomly assigned to either the incentivized (IN) or non-incentivized (non-IN) group. A second 6MWT was completed after a rest period during which the following instructions were provided. The IN was told “If you walk faster on this repeat 6MWT you will receive $50. Remember to walk quickly and safely.” The non-IN were told “You are repeating the 6MWT and remember to walk quickly and safely.” The same evaluator conducted both tests, was blinded to group, and provided the same encouragement during each 6MWT. Current data is available in 9 boys, however data on a cohort of 36 will be presented. The boys in the IN (n = 5, mean age = 7.7) walked an average of 44 m (11%, max: 105 m) farther on the second 6MWT. The non-IN (n = 4, mean age = 7.6) walked an average of 5.1 m (2%, max: 16 m) farther. The increase in distance in IN was most distinct in the above 350 m (75 m or 17%) compared to the below 350 m (24 m or 7%) group. Our pilot data suggest that a financial incentive results in a measurable increase in within-day 6MWT distance in DMD. This variance is more pronounced in the above 350 m group which is the typical target for clinical trials. This data suggests that external motivators have a significant impact on the 6MWT and should be considered in the design and interpretation of clinical trials.

Duchenne Muscular Dystrophy (DMD) is the most common, severe neuromuscular disorder caused by mutations in the DMD gene. We investigated an alternative to dystrophin replacement by overexpressing ITGA7 using adeno-associated virus (AAV) delivery. ITGA7 is a laminin receptor in skeletal muscle, that like the dystrophin–glycoprotein complex, links the extracellular matrix (ECM) to the internal actin cytoskeleton. ITGA7 is expressed in DMD patients and thus overexpression will not elicit an immune response to the transgene. We delivered rAAVrh.74.MCK.ITGA7 systemically to the more severe mouse model of DMD, the mdx/utrn−/− mouse deficient for both dystrophin and utrophin at 2–4days of age. We demonstrated widespread expression of ITGA7 at the sarcolemma to multiple muscle groups following gene transfer eight weeks post injection. The increased expression of ITGA7 significantly extended longevity and reduced kyphosis and joint contractures, common to the mdx/utrn−/− mouse. The additional α 7 expression significantly protected against loss of force following contraction-induced damage and increased specific force in the diaphragm and EDL muscles eight weeks post gene transfer. Taken together, these results show that this therapeutic approach continues to demonstrate promise as a viable treatment for DMD.

Limb girdle muscular dystrophy type 2L (LGMD2L) is caused by mutations in the anoctamin 5 (ANO5) gene. ANO5 encodes a predicted 107 kDa protein containing 8 transmembrane domains and is part of a family that encodes at least 2 calcium-activated chloride channels; however the exact function of ANO5 has yet to be determined. The clinical phenotype overlaps with that of LGMD2B patients having mutations in dysferlin, a gene required for membrane repair. We have developed an ANO5 deficient mouse model (ANO5 KO) for LGMD2L for which no model currently exists. This model is necessary to study disease pathophysiology and test potential therapies. This deficient mouse model has been fundamental to characterize disease pathogenesis, discovering several proteins that may be associated with ANO5, and related functional incapacities. Based on the phenotypic overlap with dysferlin deficiency, we tested whether our ANO5 KO mouse model had a deficit in muscle membrane repair. Following laser induced injury, the flexor digitorum brevis muscles from ANO5 KO mice demonstrated reduced capacity to repair compared to WT strain controls. In addition, we also showed a loss of force compared to wild type mice after mechanical repetitive eccentric contraction in the extensor digitorum longus muscle.. Furthermore, we identified histopathological changes along with mislocalization or reduction in several muscle proteins which are linked and/or secondary to AN05. The ANO5 deficient mouse is novel and represents an important animal model for study of LGMD2L. It is the ideal model to develop proof of principle for gene replacement therapy or pharmacologic approaches and permits studies defining ANO5 function, a critical parameter for therapeutic intervention. The functional deficits we identified provide critical outcome measures to test efficacy of potential therapies and demonstrate feasibility of translation.

Clinical trials currently underway in Duchenne muscular dystrophy (DMD) almost always exclude infants and young boys because traditional outcome measures rely on cooperation. Despite this, it is well known that the pathogenesis of this progressive dystrophy starts in the first years of life. The Bayley-III Scales of Infant and Toddler Development (Bayley-III) have been validated in typically developing infants and children through age 42 months and recently in a cohort of 24 infants and boys with DMD. Clinical evaluators at six centers were trained and certified to perform the Bayley-III testing. Fourteen of these infants were young enough (1.6 ± 0.8 years) to be tested using the Bayley-III at baseline, 6, and 12 months later. Repeated measures analysis showed that cognitive and language comprehensive scores (normal 100 ± 15) remained stable, albeit low across one year (88.2 ± 9.7 and 81.5 ± 7.2 at baseline and 89.6 ± 9.2 and 83.5 ± 9.2 at 1 year). Fine motor scale scores (normal 10 ± 0.3) while low at baseline (8.1 ± 2.1), increased slightly over one year (8.9 ± 2.4). However, gross motor scaled scores which were low at baseline (6.8 ± 1.8) declined at six months (5.8 ± 2.0) and declined further at 12 months (5.5 ± 1.9). We show that gross motor development can be reliably measured in infants and young boys with DMD across one year. We further demonstrate that, relative to their peers, these boys are loosing ground, even in the so-called “honeymoon period”. We conclude that young boys with DMD can be tested across time and should be considered for clinical trials.

Infants with spinal muscular atrophy type I (SMA) demonstrate significantly decreased muscle strength and tone presenting before 6 months of age. Clinical trials are forthcoming which have the potential to improve outcomes. Traditional developmental assessments such as the Test of Infant Motor Performance (TIMP) tend to be fatiguing for infants with SMA. The CHOP Intend was developed specifically for this population to decrease this fatigue. However, both of these assessments grade infant movement on a Likert scale, which can be difficult to use to interpret treatment efficacy as the scale is not linear. Microsoft Kinect platform projects an infrared reference matrix that is reflected off of a subject and generates a high-resolution 3-dimensional depth map allowing for tracking of movement over time. A proof-of-concept assessment system, ACTIVE-mini (Ability Captured Through Interactive Video Evaluation-mini), has been created which measures total infant reaching and kicking movement excursion and frequency of movement of each extremity. Results from ACTIVE-mini have been compared to outcomes on traditional assessments such as the TIMP and CHOP Intend in infants with SMA and normal controls. Preliminary data suggests that this platform may capture fine-grained movement differences where traditional Likert-scale based assessments may not.

Sporadic inclusion body myositis (sIBM) causes progressive loss of ambulatory function due to declining muscle strength. Although the cause is currently unknown, clinical trials are underway with the goal of improving strength and function in individuals affected with this disease. Selection of appropriate outcome measures is critical for the success of these trials. The 6-min walk test (6MWT) has been the de facto standard for assessing function in neuromuscular disease, however, this test has not been well studied in subjects with sIBM. 55 individuals with sIBM (43 male, 12 female; age range: 46–85 years; mean age: 66.8 ± 8.4 years) completed a battery of strength and functional testing to determine the optimal walking assessment to demonstrate change throughout clinical trials for subjects with this diagnosis. Strength was measured using maximum voluntary isometric contraction testing of the hips, knees, and ankles using a fixed quantitative muscle analysis system. Functional testing included the 2-min walk test (2MWT), 6MWT, modified Timed Up and Go (mTUG), time to ascend and descend 4 stairs, and stepping up on curbs (SoC). Our results indicate that both the 2MWT and 6MWT are highly correlated (r = 0.975). Both tests are also similarly correlated with the other functional measures and lower extremity strength. Further research is needed to determine longitudinal changes on the 2MWT in individuals with sIBM, however preliminary evidence suggests that the 2MWT is a possible alternative to walking tests of longer duration.

Sporadic inclusion body myositis (sIBM) is the most common inflammatory myopathy in persons over the age of 50. Few studies have been completed to describe the natural history of the disease with both strength and functional outcome measures. 100 subjects were characterized in our initial study and 55 (43 male, 12 female; age range: 46–85 years; mean age: 66.8 ± 8.4 years) returned for longitudinal strength and functional outcomes testing. The average length of time between visits was 17.7 months (SD ± 7 mos.; range 1.1 months to 2.3 years). 5 subjects did not return because they were no longer ambulatory, 5 were enrolled in a clinical trial, 4 were lost to follow up, and the remaining 31 were unable to travel to Ohio during the testing period. Subjects reported falling up to 52 times per year, or weekly (mean = 5.4 ± 8.6 falls per year). Knee extension declined an average of 2% per month and knee flexion by 1.1% per month. Distance walked in 2 min decreased by 1.2 meters per month in this sample. The IBM functional rating scale and the PROMIS Health assessment questionnaire both captured patient perception of a decline in function (IBMFRS 0.32 points per month; PROMIS 1.1 points per month). Prediction models and rates of decline are also presented for walking distance, ability to get out of a chair and to step onto curbs. No significant difference in rate of change was found based on age of diagnosis. Understanding the rate of decline resulting from the natural history of a disease is essential to explaining the results of clinical trials. Expected rate of change over time can help patients and clinicians plan for the future needs and help researchers understand what changes are attributable to disease progression or to investigational agents. This is the most comprehensive longitudinal data reported in patients with sIBM to date.

Next generation sequencing technologies present opportunities for improved clinical molecular diagnosis of neuromuscular disorders (NMD). We sought to determine the feasibility of using whole exome sequencing to detect mutations in 347 isoforms of 143 NMD genes listed in the World Muscle Society Muscle Gene Table ( www.musclegenetable.org ). We initially tested DNA samples from eight subjects with limb-girdle muscular dystrophies (LGMD) and known mutations, using exome capture with one of three commercially available kits. We detected the same mutations as gene-specific Sanger sequencing had previously detected in seven of eight, missing 1 MYOT mutation due to a lack of representation of this specific exon in the capture kit. This led us to compare commercially available exome capture kits for coverage of NMD genes, by both predicted and by empiric coverage, in normal control genomic DNA. Based on manufacturer targeted region files, the three kits each targeted 92–94% of the known NMD exons; however, our actual capture results demonstrated that at best ∼60% of these exons obtained 100% coverage. In our hands the best performing kit, Agilent SureSelect v3, captures an average of 92.7% of the bases in the NMD gene exons, but only 58% of the NMD isoforms had all exons captured at 100% (e.g., 42% of the NMD genes had at least one exon with one base insufficiently captured to make a genotyping call). Illumina TruSeq captured 89.2% of exons but only 36.5% of genes had 100% coverage, and Nimblegen v2 captured 90.5% of genes but only 46.3% genes had 100% sequence coverage. We predict that by utilizing all three kits, ∼70% of the known NMD isoforms would have sufficient sequence coverage to make a genotyping call for 100% of all exons. When considering adoption of this approach clinically, this is an important limitation to be aware of but continued improvements in commercially available capture kits should allow economical and rapid NMD gene sequence analysis.

Limb-girdle muscular dystrophy refers to a group of 23 disorders characterized by progressive wasting and weakness of shoulder and hip girdle muscles. The onset and progression of LGMD varies among individuals and genetic subtypes. Pre-clinical studies support that gene therapy is a promising treatment approach for the LGMDs, and importantly one such strategy (for LGMD2D) was recently translated to human clinical trials. Despite the positive direction of LGMD-targeted gene therapies, all strategies to date focused on gene replacement approaches for recessive forms, while treatments for dominant LGMDs have been largely unexplored. This lack of focus on gene therapy for dominant LGMDs arose primarily because these disorders require disease gene knockdown, and the molecular tools to feasibly accomplish this did not exist until recently, with the emergence of RNA interference (RNAi). We hypothesized that patients with dominantly inherited LGMD would benefit from RNAi-mediated reduction of the pathogenic alleles underlying their disease. In this study, we developed the first RNAi-based, pre-clinical treatment for LGMD1A, caused by dominant mutation in one allele of the myotilin (MYOT) gene. To do this, we engineered and delivered MYOT-targeted artificial microRNA (miMYOT) vectors to knockdown mutant MYOT in muscles of an LGMD1A mouse model. Three months after treatment, miMYOT vectors significantly reduced soluble mutant MYOT protein to undetectable levels, and the protein aggregates that are characteristic of LGMD1A were either absent or very small in treated muscles. This reduction was associated with significantly improved muscle mass and whole muscle strength in LGMD1A mice. We are now assessing body wide improvements of miMYOT treatment following global vascular delivery. This work is an important first step toward translating targeted RNAi gene therapy approaches for LGMD1A, and our method could be adapted to impact a large class of dominant muscle disorders.

Evaluate the utility of timed walking tests in DMD. Many different timed walking tests have been shown to be accurate, reproducible, and simple to administer (10 m walk (10 mw), 2 min (2MWT), and 6 min (6MWT)). Recently the 6MWT has emerged as the gold standard for use in clinical trials. Thirty five subjects with DMD were evaluated 95 times. Tests include walking distance; isometric quadriceps strength testing and the North Star Ambulatory Assessment (NSAA). Mixed model analysis revealed that both the 2MWT and 6MWT are highly significant correlations to the NSAA (p < 0.0001) and quadriceps strength (p = 0.05). Boys walked a consistent speed throughout the entire test. Velocity during each minute of a single session ranged from 0.97 to 1.06 m/s, showing a high degree of correlation at each timed interval (r = 0.725 − 0.921; p < 0.001). Wilcoxon signed rank tests on walking test group data revealed no significant difference between distances on day 1 and day 2 for either the 2MWT (p = 0.8) or the 6MWT (p = 0.5). Individual variability between days, however, was quite large. For 6MWT distance walked between subsequent days ranged from 0–44 m or 0–59% (mean = 6.4% = /− 10.26) of the total distance. Variability for the 2MWT was similar, ranging from 0–33 m or 0–56% (mean = 7.9% = /−10.05). In DMD the 2MWT is comparable to the 6MWT. Both correlate with quadriceps strength and function. The caveat for the data set analyzed here is that each minute was part of a 6MWT. Future studies will need to test walking velocity in separate designated 6 and 2 min tests.

Historically, newborn screening for DMD has been challenging in part due to elevations in serum CK related to birth trauma, resulting in false-positive results when used as a screening test. Nevertheless, the rationale for newborn screening is supported by several lines of argument. One is that new techniques for molecular diagnostics hold promise for improved rapid mutational analysis from small amounts of starting material. Another is that new potential therapeutics are entering clinical trials, and these or future therapies may significantly impact disease course if offered in infancy. A complementary argument regards clinical trial readiness; studies addressing efficacy in children of this age cannot be performed if a reliable method of early detection is unavailable. We have established and validated a two-tier system of screening and molecular diagnosis that uses a dried blood spot obtained within the first 48 h to first assess CK via a fluorometric assay as the first step. Preliminary studies established a population-based range of CK in newborns using 30,547 deidentified anonymous dried blood spot samples. Mutation analysis used genomic DNA extracted from the dried blood spot for whole genome amplification followed by MLPA and/or direct sequencing methods. DMD gene mutations (all exonic deletions) were found in 6 of 37,649 newborn males, all of whom had CK levels >2000 U/l. In three newborns with CK >2000 U/l in whom DMD gene abnormalities were not found, we identified limb-girdle muscular dystrophy gene mutations affecting the DYSF, SGCB, and FKRP genes. This data is currently in press elsewhere, but here we will review this path for screening that fits the US health care system, minimizes false-positive testing, and uses predetermined levels of CK on dried blood spots to predict DMD gene mutations, and we will discuss prospects and challenges for widespread utilization.