Cobomarsen, an Oligonucleotide Inhibitor of miR-155, Slows DLBCL Tumor Cell Growth In Vitro and In Vivo.

PURPOSE: miRNA-155 is an oncogenic miRNA highly expressed in B-cell malignancies, particularly in the non-germinal center B-cell or activated B-cell subtype of diffuse large B-cell lymphoma (ABC-DLBCL), where it is considered a potential diagnostic and prognostic biomarker. Thus, miR-155 inhibition represents an important therapeutic strategy for B-cell lymphomas. In this study, we tested the efficacy and pharmacodynamic activity of an oligonucleotide inhibitor of miR-155, cobomarsen, in ABC-DLBCL cell lines and in corresponding xenograft mouse models. In addition, we assessed the therapeutic efficacy and safety of cobomarsen in a patient diagnosed with aggressive ABC-DLBCL. EXPERIMENTAL DESIGN: Preclinical studies included the delivery of cobomarsen to highly miR-155-expressing ABC-DLBCL cell lines to assess any phenotypic changes, as well as intravenous injections of cobomarsen in NSG mice carrying ABC-DLBCL xenografts, to study tumor growth and pharmacodynamics of the compound over time. To begin to test its safety and therapeutic efficacy, a patient was recruited who underwent five cycles of cobomarsen treatment. RESULTS: Cobomarsen decreased cell proliferation and induced apoptosis in ABC-DLBCL cell lines. Intravenous administration of cobomarsen in a xenograft NSG mouse model of ABC-DLBCL reduced tumor volume, triggered apoptosis, and derepressed direct miR-155 target genes. Finally, the compound reduced and stabilized tumor growth without any toxic effects for the patient. CONCLUSIONS: Our findings support the potential therapeutic application of cobomarsen in ABC-DLBCL and other types of lymphoma with elevated miR-155 expression.

Barriers and facilitators to clinical trial participation among parents of children with pediatric neuromuscular disorders.

BACKGROUND/AIMS: Pediatric rare disease presents a challenging situation of high unmet need and a limited pool of potential clinical trial participants. Understanding perspectives of parents of children who have not participated in trials may facilitate approaches to optimize participation rates. The objective of this study was to explore factors associated with parental interest in enrolling children with pediatric neuromuscular disorders in clinical trials. METHODS: Parents of individuals with Duchenne or Becker muscular dystrophy and spinal muscular atrophy were recruited through advocacy organizations, a registry, and clinics. These parents ( N = 203) completed a questionnaire including assessments of barriers and facilitators to clinical trial participation, parents' interest in trial participation, and their perceptions of others' views about participation in a clinical trial. RESULTS: Trial interest in participating parents was high (64% combined group). The most highly endorsed barrier to participation was the possibility of receiving placebo, followed by not having enough information on risks and trial procedures. Compared to parents of children with Duchenne or Becker muscular dystrophy, parents of children with spinal muscular atrophy endorsed significantly more information and knowledge barriers. The greatest facilitators of participation were (1) confidence in improving disease understanding and (2) guarantee to receive the treatment after a successful trial. A logistic regression model, χ2 (4, n = 188) = 80.64, p < .001, indicated that higher perceived barriers and more frequent trial communication by the provider were associated with lower interest, while positive trial perceptions by the child's providers and concordance in trial perceptions among those close to the decision-maker were associated with higher interest. CONCLUSION: We found high parental interest in pediatric neuromuscular trials that was tempered by concerns about the potential for randomization to a placebo arm. Participants perceived that their trial participation would be facilitated by additional education and guidance from their clinicians. Yet, intentions were negatively associated with frequency of provider communication, perhaps reflecting waning parental interest with a greater understanding of limitations in trial access, increased sophistication in their understanding of trial design, and appreciation of potential burden. To support parents' informed decisions, it is important to educate them to evaluate the quality of research, as well as providing lay information explaining the use of placebo, trial processes, and potential barriers to long-term drug access. Our findings should inform the development of targeted educational content, clinician training, and decision support tools.

Myostatin inhibitor ACE-031 treatment of ambulatory boys with Duchenne muscular dystrophy: Results of a randomized, placebo-controlled clinical trial.

INTRODUCTION: ACE-031 is a fusion protein of activin receptor type IIB and IgG1-Fc, which binds myostatin and related ligands. It aims to disrupt the inhibitory effect on muscle development and provide potential therapy for myopathies like Duchenne muscular dystrophy (DMD). METHODS: ACE-031 was administered subcutaneously every 2-4 weeks to DMD boys in a randomized, double-blind, placebo-controlled, ascending-dose trial. The primary objective was safety evaluation. Secondary objectives included characterization of pharmacokinetics and pharmacodynamics. RESULTS: ACE-031 was not associated with serious or severe adverse events. The study was stopped after the second dosing regimen due to potential safety concerns of epistaxis and telangiectasias. A trend for maintenance of the 6-minute walk test (6MWT) distance in the ACE-031 groups compared with a decline in the placebo group (not statistically significant) was noted, as was a trend for increased lean body mass and bone mineral density (BMD) and reduced fat mass. CONCLUSION: ACE-031 use demonstrated trends for pharmacodynamic effects on lean mass, fat mass, BMD, and 6MWT. Non-muscle-related adverse events contributed to the decision to discontinue the study. Myostatin inhibition is a promising therapeutic approach for DMD. Muscle Nerve 55: 458-464, 2017.

"Watching time tick by ": Decision making for Duchenne muscular dystrophy trials.

OBJECTIVE: This interview study explored clinicians' perspectives and parents' decision making about children's participation in Duchenne muscular dystrophy (DMD) clinical trials. METHODS: Data from semi-structured interviews conducted with clinicians and parents in U.S. or Canada were assessed using thematic analysis. RESULTS: Eleven clinicians involved in ten trials and fifteen parents involved in six trials were interviewed. Parents described benefit-risk assessments using information from advocacy, peers, professionals, and sponsors. Strong influence was attributed to the progressive nature of DMD. Most expected direct benefit. Few considered the possibility of trial failure. Most made decisions to participate before the informed consent (IC) process, but none-the-less perceived informed choice with little to lose for potential gain. Clinicians described more influence on parental decisions than attributed by parents. Clinicians felt responsible to facilitate IC while maintaining hope. Both clinicians and parents reported criticisms about the IC process and regulatory barriers. CONCLUSIONS: The majority of parents described undertaking benefit-risk assessments that led to informed choices that offered psychological and potential disease benefits. Parents' high expectations influenced their decisions while also reflecting optimism. Clinicians felt challenged in balancing parents' expectations and likely outcomes. Prognosis-related pressures coupled with decision making prior to IC suggest an obligation to ensure educational materials are understandable and accurate, and to consider an expanded notion of IC timeframes. Anticipatory guidance about potential trial failure might facilitate parents' deliberations while aiding clinicians in moderating overly-optimistic motivations. Regulators and industry should appreciate special challenges in progressive disorders, where doing nothing was equated with doing harm.

Electrical impedance myography in Duchenne muscular dystrophy and healthy controls: A multicenter study of reliability and validity.

INTRODUCTION: Electrical impedance myography (EIM) is a non-invasive, painless, objective technique to quantify muscle pathology. METHODS: We measured EIM in 8 arm and leg muscles in 61 boys with Duchenne muscular dystrophy (DMD) and 31 healthy boys, ages 3-12 years, at 5 centers. We determined the reliability of EIM and compared results in boys with DMD to controls and to 6-minute walk distance (6MWD), North Star Ambulatory Assessment (NSAA), timed functional tests (TFTs), and strength (hand-held dynamometry). RESULTS: EIM was well tolerated and had good inter- and intrarater reliability (intraclass correlation coefficient 0.81-0.96). The averaged EIM phase value from all muscles was higher (P < 0.001) in controls (10.45 ± 2.29) than boys with DMD (7.31 ± 2.23), and correlated (P ≤ 0.001) with 6MWD (r = 0.55), NSAA (r = 0.66), TFTs (r = -0.56), and strength (r = 0.44). CONCLUSION: EIM is a reliable and valid measure of disease severity in DMD. Longitudinal studies comparing EIM with other assessments over time in DMD are warranted.

The cooperative international neuromuscular research group Duchenne natural history study: glucocorticoid treatment preserves clinically meaningful functional milestones and reduces rate of disease progression as measured by manual muscle testing and other commonly used clinical trial outcome measures.

UNLABELLED: introduction: Glucocorticoid (GC) therapy in Duchenne muscular dystrophy (DMD) has altered disease progression, necessitating contemporary natural history studies. METHODS: The Cooperative Neuromuscular Research Group (CINRG) DMD Natural History Study (DMD-NHS) enrolled 340 DMD males, ages 2-28 years. A comprehensive battery of measures was obtained. RESULTS: A novel composite functional "milestone" scale scale showed clinically meaningful mobility and upper limb abilities were significantly preserved in GC-treated adolescents/young adults. Manual muscle test (MMT)-based calculations of global strength showed that those patients <10 years of age treated with steroids declined by 0.4 ± 0.39 MMT unit/year, compared with -0.4 ± 0.39 MMT unit/year in historical steroid-naive subjects. Pulmonary function tests (PFTs) were relatively preserved in steroid-treated adolescents. The linearity and magnitude of decline in measures were affected by maturational changes and functional status. CONCLUSIONS: In DMD, long-term use of GCs showed reduced strength loss and preserved functional capabilities and PFTs compared with previous natural history studies performed prior to the widespread use of GC therapy.

The cooperative international neuromuscular research group Duchenne natural history study--a longitudinal investigation in the era of glucocorticoid therapy: design of protocol and the methods used.

UNLABELLED: Contemporary natural history data in Duchenne muscular dystrophy (DMD) is needed to assess care recommendations and aid in planning future trials. METHODS: The Cooperative International Neuromuscular Research Group (CINRG) DMD Natural History Study (DMD-NHS) enrolled 340 individuals, aged 2-28 years, with DMD in a longitudinal, observational study at 20 centers. Assessments obtained every 3 months for 1 year, at 18 months, and annually thereafter included: clinical history; anthropometrics; goniometry; manual muscle testing; quantitative muscle strength; timed function tests; pulmonary function; and patient-reported outcomes/health-related quality-of-life instruments. RESULTS: Glucocorticoid (GC) use at baseline was 62% present, 14% past, and 24% GC-naive. In those ≥6 years of age, 16% lost ambulation over the first 12 months (mean age 10.8 years). CONCLUSIONS: Detailed information on the study methodology of the CINRG DMD-NHS lays the groundwork for future analyses of prospective longitudinal natural history data. These data will assist investigators in designing clinical trials of novel therapeutics.

Arginine butyrate: a therapeutic candidate for Duchenne muscular dystrophy.

As a strategy to treat Duchenne muscular dystrophy, we used arginine butyrate, which combines two pharmacological activities: nitric oxide pathway activation, and histone deacetylase inhibition. Continuous intraperitoneal administration to dystrophin-deficient mdx mice resulted in a near 2-fold increase in utrophin (protein homologous to dystrophin) in skeletal muscle, heart, and brain, accompanied by an improvement of the dystrophic phenotype in both adult and newborn mice (45 and 70% decrease in creatine kinase level, respectively; 14% increase in tidal volume, 30% decrease in necrotic area in limb and 23% increase in isometric force). Intermittent administration, as performed in clinical trials, was then used to reduce the frequency of injections and to improve safety. This also enhanced utrophin level around 2-fold (EC50=284 mg/ml) and alleviated the dystrophic phenotype (inverted grid and grip test performance near to wild-type values, creatine kinase level decreased by 50%). Skin biopsies were used to monitor treatment efficacy, instead of invasive muscle biopsies, and this could be done a few days after the start of treatment. A 2-fold increase in utrophin expression was also shown in cultured human myotubes. In vivo and in vitro experiments demonstrated that the drug combination acts synergistically. Together, these data constitute a proof of principle of the beneficial effects of arginine butyrate on muscular dystrophy.

CINRG pilot trial of coenzyme Q10 in steroid-treated Duchenne muscular dystrophy.

INTRODUCTION: Corticosteroid treatment slows disease progression and is the standard of care for Duchenne muscular dystrophy (DMD). Coenzyme Q10 (CoQ10) is a potent antioxidant that may improve function in dystrophin-deficient muscle. METHODS: We performed an open-label, "add-on" pilot study of CoQ10 in thirteen 5-10-year-old DMD patients on steroids. The primary outcome measure was the total quantitative muscle testing (QMT) score. RESULTS: Twelve of 16 children (mean age 8.03 ± 1.64 years) completed the trial. Target serum levels of CoQ10 (≥2.5 µg/ml) were shown to be subject- and administration-dependent. Nine of 12 subjects showed an increase in total QMT score. Overall, CoQ10 treatment resulted in an 8.5% increase in muscle strength (P = 0.03). CONCLUSIONS: Addition of CoQ10 to prednisone therapy in DMD patients resulted in an increase in muscle strength. These results warrant a larger, controlled trial of CoQ10 in DMD.

Liquid formulation of pentoxifylline is a poorly tolerated treatment for duchenne dystrophy.

INTRODUCTION: In this study we performed an open-label, pilot study of an orally administered liquid formulation of immediate-release pentoxifylline (PTX) on patients with Duchenne muscular dystrophy (DMD). Treatment efficacy, safety, and tolerability were assessed. METHODS: The tolerability and safety of PTX and measures of muscle strength and function were evaluated during 12 months of treatment. RESULTS: Seventeen boys with DMD, between 4 and 8 years of age, were enrolled at one of five Cooperative International Neuromuscular Research Group (CINRG) centers. Only 9 were able to complete the 12-month PTX treatment phase; the primary reason for discontinuation was adverse events. Intolerable gastrointestinal side effects were experienced by 65% of participants. Two participants had severe leukopenia that resolved with medication withdrawal. CONCLUSIONS: Open-label treatment with a liquid formulation of immediate-release PTX resulted in a high incidence of adverse events in boys with DMD. Poor tolerability of this PTX formulation precluded adequate assessment of efficacy.

Congenital muscular dystrophies.

Congenital muscular dystrophies (CMDs) are a heterogeneous group of disorders characterized by muscle weakness from birth, or shortly after, and variable clinical manifestations of the eye and central nervous system. Some of these disorders are fatal in the first years of life, whereas others have a milder course, with survival into adulthood. The CMDs were initially classified by clinical features and country of origin; however, with new molecular techniques it is now possible to classify these patients better. More than 10 genes have been identified to date that cause forms of CMD. However, even with current molecular diagnostic techniques, only approximately 25-50% of patients with CMD have an identifiable genetic mutation. In addition, some phenotypic classifications have been attempted. There is significant overlap between the phenotypic and molecular classifications, making diagnosis within this heterogeneous group of disorders difficult.

Novel LMNA mutations in patients with Emery-Dreifuss muscular dystrophy and functional characterization of four LMNA mutations.

Mutations in LMNA cause a variety of diseases affecting striated muscle including autosomal Emery-Dreifuss muscular dystrophy (EDMD), LMNA-associated congenital muscular dystrophy (L-CMD), and limb-girdle muscular dystrophy type 1B (LGMD1B). Here, we describe novel and recurrent LMNA mutations identified in 50 patients from the United States and Canada, which is the first report of the distribution of LMNA mutations from a large cohort outside Europe. This augments the number of LMNA mutations known to cause EDMD by 16.5%, equating to an increase of 5.9% in the total known LMNA mutations. Eight patients presented with either p.R249W/Q or p.E358K mutations and an early onset EDMD phenotype: two mutations recently associated with L-CMD. Importantly, 15 mutations are novel and include eight missense mutations (p.R189P, p.F206L, p.S268P, p.S295P, p.E361K, p.G449D, p.L454P, and p.W467R), three splice site mutations (c.IVS4 + 1G>A, c.IVS6 - 2A>G, and c.IVS8 + 1G>A), one duplication/in frame insertion (p.R190dup), one deletion (p.Q355del), and two silent mutations (p.R119R and p.K270K). Analysis of 4 of our lamin A mutations showed that some caused nuclear deformations and lamin B redistribution in a mutation specific manner. Together, this study significantly augments the number of EDMD patients on the database and describes 15 novel mutations that underlie EDMD, which will contribute to establishing genotype-phenotype correlations.

Functional and molecular effects of arginine butyrate and prednisone on muscle and heart in the mdx mouse model of Duchenne Muscular Dystrophy.

BACKGROUND: The number of promising therapeutic interventions for Duchenne Muscular Dystrophy (DMD) is increasing rapidly. One of the proposed strategies is to use drugs that are known to act by multiple different mechanisms including inducing of homologous fetal form of adult genes, for example utrophin in place of dystrophin. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we have treated mdx mice with arginine butyrate, prednisone, or a combination of arginine butyrate and prednisone for 6 months, beginning at 3 months of age, and have comprehensively evaluated the functional, biochemical, histological, and molecular effects of the treatments in this DMD model. Arginine butyrate treatment improved grip strength and decreased fibrosis in the gastrocnemius muscle, but did not produce significant improvement in muscle and cardiac histology, heart function, behavioral measurements, or serum creatine kinase levels. In contrast, 6 months of chronic continuous prednisone treatment resulted in deterioration in functional, histological, and biochemical measures. Arginine butyrate-treated mice gene expression profiling experiments revealed that several genes that control cell proliferation, growth and differentiation are differentially expressed consistent with its histone deacetylase inhibitory activity when compared to control (saline-treated) mdx mice. Prednisone and combination treated groups showed alterations in the expression of genes that control fibrosis, inflammation, myogenesis and atrophy. CONCLUSIONS/SIGNIFICANCE: These data indicate that 6 months treatment with arginine butyrate can produce modest beneficial effects on dystrophic pathology in mdx mice by reducing fibrosis and promoting muscle function while chronic continuous treatment with prednisone showed deleterious effects to skeletal and cardiac muscle. Our results clearly indicate the usefulness of multiple assays systems to monitor both beneficial and toxic effects of drugs with broad range of in vivo activity.

A randomized study of alglucosidase alfa in late-onset Pompe's disease.

BACKGROUND: Pompe's disease is a metabolic myopathy caused by a deficiency of acid alpha glucosidase (GAA), an enzyme that degrades lysosomal glycogen. Late-onset Pompe's disease is characterized by progressive muscle weakness and loss of respiratory function, leading to early death. We conducted a randomized, placebo-controlled trial of alglucosidase alfa, a recombinant human GAA, for the treatment of late-onset Pompe's disease. METHODS: Ninety patients who were 8 years of age or older, ambulatory, and free of invasive ventilation were randomly assigned to receive biweekly intravenous alglucosidase alfa (20 mg per kilogram of body weight) or placebo for 78 weeks at eight centers in the United States and Europe. The two primary end points were distance walked during a 6-minute walk test and percentage of predicted forced vital capacity (FVC). RESULTS: At 78 weeks, the estimated mean changes from baseline in the primary end points favored alglucosidase alfa (an increase of 28.1+/-13.1 m on the 6-minute walk test and an absolute increase of 3.4+/-1.2 percentage points in FVC; P=0.03 and P=0.006, respectively). Similar proportions of patients in the two groups had adverse events, serious adverse events, and infusion-associated reactions; events that occurred only in patients who received the active study drug included anaphylactic reactions and infusion-associated reactions of urticaria, flushing, hyperhidrosis, chest discomfort, vomiting, and increased blood pressure (each of which occurred in 5 to 8% of the patients). CONCLUSIONS: In this study population, treatment with alglucosidase alfa was associated with improved walking distance and stabilization of pulmonary function over an 18-month period. (ClinicalTrials.gov number, NCT00158600.)

Clinical features of late-onset Pompe disease: a prospective cohort study.

The objective of this 12-month study was to describe the clinical features of late-onset Pompe disease and identify appropriate outcome measures for use in clinical trials. Assessments included quantitative muscle testing (QMT), functional activities (FAA), 6-min walk test (6MWT), and pulmonary function testing (PFT). Percent predicted values indicated quantifiable upper and lower extremity weakness, impaired walking ability, and respiratory muscle weakness. Significant declines in arm and leg strength and pulmonary function were observed during the study period. The outcome measures were demonstrated to be safe and reliable. Symptom duration was identified as the best predictor of the extent of skeletal and respiratory muscle weakness.

A phase I/IItrial of MYO-029 in adult subjects with muscular dystrophy.

OBJECTIVE: Myostatin is an endogenous negative regulator of muscle growth and a novel target for muscle diseases. We conducted a safety trial of a neutralizing antibody to myostatin, MYO-029, in adult muscular dystrophies (Becker muscular dystrophy, facioscapulohumeral dystrophy, and limb-girdle muscular dystrophy). METHODS: This double-blind, placebo-controlled, multinational, randomized study included 116 subjects divided into sequential dose-escalation cohorts, each receiving MYO-029 or placebo (Cohort 1 at 1 mg/kg; Cohort 2 at 3 mg/kg; Cohort 3 at 10 mg/kg; Cohort 4 at 30 mg/kg). Safety and adverse events were assessed by reported signs and symptoms, as well as by physical examinations, laboratory results, echocardiograms, electrocardiograms, and in subjects with facioscapulohumeral dystrophy, funduscopic and audiometry examinations. Biological activity of MYO-029 was assessed through manual muscle testing, quantitative muscle testing, timed function tests, subject-reported outcomes, magnetic resonance imaging studies, dual-energy radiographic absorptiometry studies, and muscle biopsy. RESULTS: MYO-029 had good safety and tolerability with the exception of cutaneous hypersensitivity at the 10 and 30 mg/kg doses. There were no improvements noted in exploratory end points of muscle strength or function, but the study was not powered to look for efficacy. Importantly, bioactivity of MYO-029 was supported by a trend in a limited number of subjects toward increased muscle size using dual-energy radiographic absorptiometry and muscle histology. INTERPRETATION: This trial supports the hypothesis that systemic administration of myostatin inhibitors provides an adequate safety margin for clinical studies. Further evaluation of more potent myostatin inhibitors for stimulating muscle growth in muscular dystrophy should be considered.

CINRG pilot trial of oxatomide in steroid-naïve Duchenne muscular dystrophy.

The authors report a pilot open-label two-center therapeutic trial of oxatomide in 14 steroid-naive DMD boys aged 5-10 years. Comparison of linear evolutions between 3 months medication-free lead-in periods and 6 months treatment periods showed no significant differences in quantitative (QMT) and manual (MMT) measurements of muscle strength and timed functional tests. A modest mitigation of strength deterioration over time cannot be excluded.

Reliable surrogate outcome measures in multicenter clinical trials of Duchenne muscular dystrophy.

We studied the reliability of a series of endpoints in an evaluation of subjects with Duchenne muscular dystrophy (DMD). The endpoints included quantitative muscle tests (QMTs), timed function tests, forced vital capacity (FVC), and manual muscle tests (MMT). Thirty-one ambulatory subjects with DMD (mean age 8.9 years; range 5-16 years) were evaluated at eight sites by 15 newly trained evaluators as a test of interrater reliability of outcome measures. Both total QMT score [intraclass correlation coefficient (ICC) 0.96] and individual QMT assessments (ICC 0.85-0.96) were highly reliable. Forced vital capacity and all timed function tests were also highly reliable (ICC 0.97-0.99). MMT was the least reliable assessment method (ICC 0.61). These data suggest that primary surrogate outcome measures in large multicenter clinical trials in DMD should use QMT, FVC, or time function tests to obtain maximum power and greatest sensitivity.

Challenges in drug development for muscle disease: a stakeholders' meeting.

Current treatment benefits for patients with muscle disease are limited, but progress in legislative and scientific initiatives have set the stage for the development of new therapies. The MD-CARE Act (Public Law 107-84), which allocates federal resources to muscular dystrophy, was approved by Congress and signed into law by the President of the United States in 2001. This has shifted the emphasis toward translational research. To facilitate a push toward therapy for muscle disorders, the Muscular Dystrophy Association (MDA) sponsored a meeting with representatives from industry, the Food and Drug Administration (FDA), the National Institutes of Health (NIH), and other government agencies and academia. Each contributed in different ways. The FDA helped define the necessary data to support investigational new drug (IND) applications including the design of proof-of-principle studies, outcome measures for clinical trials, and the pathway for developing surrogate measures for fast-tracking promising new drugs. The NIH, other government agencies, and the MDA described potential funding sources for translational research. Industry delineated a complementary role with academia, and academic investigators elucidated the current strengths and weaknesses of available clinical endpoints. The meeting provided a format for communication for diverse disciplines that usually have no common meeting ground, helping to lay the foundation for bringing products to market in a timely fashion.

Fukutin gene mutations in steroid-responsive limb girdle muscular dystrophy.

OBJECTIVE: Defects in glycosylation of alpha-dystroglycan are associated with several forms of muscular dystrophy, often characterized by congenital onset and severe structural brain involvement, collectively known as dystroglycanopathies. Six causative genes have been identified in these disorders including fukutin. Mutations in fukutin cause Fukuyama congenital muscular dystrophy. This is the second most common form of muscular dystrophy in Japan and is invariably associated with mental retardation and structural brain defects. The aim of this study was to determine the genetic defect in two white families with a dystroglycanopathy. METHODS: The six genes responsible for dystroglycanopathies were studied in three children with a severe reduction of alpha-dystroglycan in skeletal muscle. RESULTS: We identified pathogenic fukutin mutations in these two families. Affected children had normal intelligence and brain structure and shared a limb girdle muscular dystrophy (LGMD) phenotype, had marked elevation of serum creatine kinase, and were all ambulant with remarkable steroid responsiveness. INTERPRETATION: Our data suggest that fukutin mutations occur outside Japan and can be associated with much milder phenotypes than Fukuyama congenital muscular dystrophy. These findings significantly expand the spectrum of phenotypes associated with fukutin mutations to include this novel form of limb girdle muscular dystrophy that we propose to name LGMD2L.