Russell J. Butterfield

Ataluren in patients with nonsense mutation Duchenne muscular dystrophy (ACT DMD): a multicentre, randomised, double-blind, placebo-controlled, phase 3 trial

BACKGROUND Duchenne muscular dystrophy (DMD) is a severe, progressive, and rare neuromuscular, X-linked recessive disease. Dystrophin deficiency is the underlying cause of disease; therefore, mutation-specific therapies aimed at restoring dystrophin protein production are being explored. We aimed to assess the efficacy and safety of ataluren in ambulatory boys with nonsense mutation DMD. METHODS We did this multicentre, randomised, double-blind, placebo-controlled, phase 3 trial at 54 sites in 18 countries located in North America, Europe, the Asia-Pacific region, and Latin America. Boys aged 7-16 years with nonsense mutation DMD and a baseline 6-minute walk distance (6MWD) of 150 m or more and 80% or less of the predicted normal value for age and height were randomly assigned (1:1), via permuted block randomisation (block size of four) using an interactive voice-response or web-response system, to receive ataluren orally three times daily (40 mg/kg per day) or matching placebo. Randomisation was stratified by age (<9 years vs ≥9 years), duration of previous corticosteroid use (6 months to <12 months vs ≥12 months), and baseline 6MWD (<350 m vs ≥350 m). Patients, parents and caregivers, investigational site personnel, PTC Therapeutics employees, and all other study personnel were masked to group allocation until after database lock. The primary endpoint was change in 6MWD from baseline to week 48. We additionally did a prespecified subgroup analysis of the primary endpoint, based on baseline 6MWD, which is reflective of anticipated rates of disease progression over 1 year. The primary analysis was by intention to treat. This study is registered with ClinicalTrials.gov, number NCT01826487. FINDINGS Between March 26, 2013, and Aug 26, 2014, we randomly assigned 230 patients to receive ataluren (n=115) or placebo (n=115); 228 patients comprised the intention-to-treat population. The least-squares mean change in 6MWD from baseline to week 48 was -47·7 m (SE 9·3) for ataluren-treated patients and -60·7 m (9·3) for placebo-treated patients (difference 13·0 m [SE 10·4], 95% CI -7·4 to 33·4; p=0·213). The least-squares mean change for ataluren versus placebo in the prespecified subgroups was -7·7 m (SE 24·1, 95% CI -54·9 to 39·5; p=0·749) in the group with a 6MWD of less than 300 m, 42·9 m (15·9, 11·8-74·0; p=0·007) in the group with a 6MWD of 300 m or more to less than 400 m, and -9·5 m (17·2, -43·2 to 24·2; p=0·580) in the group with a 6MWD of 400 m or more. Ataluren was generally well tolerated and most treatment-emergent adverse events were mild to moderate in severity. Eight (3%) patients (n=4 per group) reported serious adverse events; all except one event in the placebo group (abnormal hepatic function deemed possibly related to treatment) were deemed unrelated to treatment. INTERPRETATION Change in 6MWD did not differ significantly between patients in the ataluren group and those in the placebo group, neither in the intention-to-treat population nor in the prespecified subgroups with a baseline 6MWD of less than 300 m or 400 m or more. However, we recorded a significant effect of ataluren in the prespecified subgroup of patients with a baseline 6MWD of 300 m or more to less than 400 m. Baseline 6MWD values within this range were associated with a more predictable rate of decline over 1 year; this finding has implications for the design of future DMD trials with the 6-minute walk test as the endpoint. FUNDING PTC Therapeutics.

Position of Glycine Substitutions in the Triple Helix of COL6A1, COL6A2, and COL6A3 is Correlated with Severity and Mode of Inheritance in Collagen VI Myopathies

Glycine substitutions in the conserved Gly‐X‐Y motif in the triple helical (TH) domain of collagen VI are the most commonly identified mutations in the collagen VI myopathies including Ullrich congenital muscular dystrophy, Bethlem myopathy, and intermediate (INT) phenotypes. We describe clinical and genetic characteristics of 97 individuals with glycine substitutions in the TH domain of COL6A1, COL6A2, or COL6A3 and add a review of 97 published cases, for a total of 194 cases. Clinical findings include severe, INT, and mild phenotypes even from patients with identical mutations. INT phenotypes were most common, accounting for almost half of patients, emphasizing the importance of INT phenotypes to the overall phenotypic spectrum. Glycine substitutions in the TH domain are heavily clustered in a short segment N‐terminal to the 17th Gly‐X‐Y triplet, where they are acting as dominants. The most severe cases are clustered in an even smaller region including Gly‐X‐Y triplets 10–15, accounting for only 5% of the TH domain. Our findings suggest that clustering of glycine substitutions in the N‐terminal region of collagen VI is not based on features of the primary sequence. We hypothesize that this region may represent a functional domain within the triple helix.

Congenital lethal motor neuron disease with a novel defect in ribosome biogenesis

Objective: We describe a novel congenital motor neuron disease with early demise due to respiratory insufficiency with clinical overlap with spinal muscular atrophy with respiratory distress (SMARD) type 1 but lacking a mutation in the IGHMBP2 gene. Methods: Exome sequencing was used to identify a de novo mutation in the LAS1L gene in the proband. Pathogenicity of the mutation was validated using a zebrafish model by morpholino-mediated knockdown of las1l. Results: We identified a de novo mutation in the X-linked LAS1L gene in the proband (p.S477N). The mutation is in a highly conserved region of the LAS1L gene predicted to be deleterious by bioinformatic analysis. Morpholino-based knockdown of las1l, the orthologous gene in zebrafish, results in early lethality and disruption of muscle and peripheral nerve architecture. Coinjection of wild-type but not mutant human RNA results in partial rescue of the phenotype. Conclusion: We report a patient with a SMARD phenotype due to a mutation in LAS1L, a gene important in coordinating processing of the 45S pre-rRNA and maturation of the large 60S ribosomal subunit. Similarly, the IGHMB2 gene associated with SMARD type 1 has been suggested to have an important role in ribosomal biogenesis from its role in processing the 45S pre-rRNA. We propose that disruption of ribosomal maturation may be a common pathogenic mechanism linking SMARD phenotypes caused by both IGHMBP2 and LAS1L.

Genetics of experimental allergic encephalomyelitis supports the role of T helper cells in multiple sclerosis pathogenesis.

The major histocompatibility complex (MHC) is the primary genetic contributor to multiple sclerosis (MS) and experimental allergic encephalomyelitis (EAE), but multiple additional interacting loci are required for genetic susceptibility. The identity of most of these non‐MHC genes is unknown. In this report, we identify genes within evolutionarily conserved genetic pathways leading to MS and EAE.

Methylenetetrahydrofolate reductase gene polymorphism and childhood stroke.

Genotyping for the methylenetetrahydrofolate reductase gene (MTHFR) has been recommended for part of the evaluation for underlying prothrombotic state in childhood stroke; however, studies are inconclusive regarding the role of this gene and also the role of hyperhomocysteinemia, which is the putative mechanism by which MTHFR polymorphism is related to stroke. The prevalence of MTHFR polymorphism in childhood arterial ischemic stroke and cerebral sinovenous thrombosis was compared with that of a reference population, and prevalence of hyperhomocysteinemia was reviewed. In arterial ischemic stroke, the prevalence of at-risk methylenetetrahydrofolate reductase genotypes was 27%, and in cerebral sinovenous thrombosis it was 13%; the population prevalence was 26%. The odds ratio for at-risk genotype in childhood arterial ischemic stroke was 1.06 (95% confidence interval, 0.22-4.0); in cerebral sinovenous thrombosis, it was 0.42 (95% confidence interval, 0.01-3.6). No tested cases had hyperhomocysteinemia. MTHFR polymorphism and hyperhomocysteinemia were not risk factors in childhood arterial ischemic stroke or cerebral sinovenous thrombosis in the Intermountain West region.

Mosaicism for dominant collagen 6 mutations as a cause for intrafamilial phenotypic variability.

Collagen 6‐related dystrophies and myopathies (COL6‐RD) are a group of disorders that form a wide phenotypic spectrum, ranging from severe Ullrich congenital muscular dystrophy, intermediate phenotypes, to the milder Bethlem myopathy. Both inter‐ and intrafamilial variable expressivity are commonly observed. We present clinical, immunohistochemical, and genetic data on four COL6‐RD families with marked intergenerational phenotypic heterogeneity. This variable expression seemingly masquerades as anticipation is due to parental mosaicism for a dominant mutation, with subsequent full inheritance and penetrance of the mutation in the heterozygous offspring. We also present an additional fifth simplex patient identified as a mosaic carrier. Parental mosaicism was confirmed in the four families through quantitative analysis of the ratio of mutant versus wild‐type allele (COL6A1, COL6A2, and COL6A3) in genomic DNA from various tissues, including blood, dermal fibroblasts, and saliva. Consistent with somatic mosaicism, parental samples had lower ratios of mutant versus wild‐type allele compared with the fully heterozygote offspring. However, there was notable variability of the mutant allele levels between tissues tested, ranging from 16% (saliva) to 43% (fibroblasts) in one mosaic father. This is the first report demonstrating mosaicism as a cause of intrafamilial/intergenerational variability of COL6‐RD, and suggests that sporadic and parental mosaicism may be more common than previously suspected.

A novel form of juvenile recessive ALS maps to loci on 6p25 and 21q22

We describe a novel form of juvenile recessive ALS (JRALS) affecting four of six offspring from a consanguineous first cousin marriage. The syndrome is characterized by early and prominent upper motor neuron signs, along with striking amyotrophy of the upper and lower limbs and bulbar involvement. After excluding linkage to loci with known association to ALS and other motor neuron diseases, we used a homozygosity mapping approach to identify loci on chromosomes 6p25 and 21q22, each with an equal probability of linkage to the trait (with a LOD score=3.1, the maximum possible given the family structure). Mutation analysis of seven candidate genes that are expressed in the CNS or have roles in neuronal function did not reveal any pathogenic mutations. Identification of additional families will help to distinguish between which of the two autosomal loci contains the disease-causing gene, or whether this is a digenic trait.

Developing standardized corticosteroid treatment for Duchenne muscular dystrophy

Despite corticosteroids being the only treatment documented to improve strength and function in boys with Duchenne muscular dystrophy (DMD) corticosteroid prescription is inconsistent and in some countries, corticosteroids are not prescribed. We are conducting a clinical trial that (1) compares the 3 most frequently prescribed corticosteroid regimes; (2) standardizes treatment of DMD complications; and (3) standardizes prevention of corticosteroid side effects. Investigators at 38 sites in 5 countries plan to recruit 300 boys aged 4-7 who are randomly assigned to one of three regimens: daily prednisone; daily deflazacort; or intermittent prednisone (10days on/10days off). Boys are followed for a minimum of 3years to assess the relative effectiveness and adverse event profiles of the different regimens. The primary outcome is a 3-dimensional variable consisting of log-transformed time to rise from the floor, forced vital capacity, and subject/parent satisfaction with treatment, each averaged over all post-baseline visits. The study protocol includes evidence- and consensus-based treatment of DMD complications and of corticosteroid side effects. This study seeks to establish a standard corticosteroid regimen for DMD. Since all new interventions for DMD are being developed as add-on therapies to corticosteroids, defining the optimum regimen is of importance for all new treatments.

Disease burden and functional outcomes in congenital myotonic dystrophy A cross-sectional study

Objective: Herein, we describe the disease burden and age-related changes of congenital-onset myotonic dystrophy (CDM) in childhood. Methods: Children with CDM and age-matched controls aged 0 to 13 years were enrolled. Participants were divided into cohorts based on the following age groups: 0–2, 3–6, and 7–13 years. Each cohort received age-appropriate evaluations including functional testing, oral facial strength testing, neuropsychological testing, quality-of-life measurements, and ECG. Independent-samples t test or Wilcoxon 2-sample test was used to compare the differences between children with CDM and controls. Probability values less than 0.05 are reported as significant. Results: Forty-one participants with CDM and 29 healthy controls were enrolled. The 6-minute walk was significantly different between CDM (258.3 m [SD 176.0]) and control participants (568.2 m [SD 73.2]). The mean lip force strength was significantly different in CDM (2.1 N [SD 2.8)] compared to control participants (17.8 N [SD 7.6]). In participants with CDM, the mean IQ (65.8; SD 18.4) was 3 SDs below the mean compared to standardized norms. Measurements of grip strength, sleep quality, and quality of life were also significantly different. Strength measures (oral facial strength, grip strength, and 6-minute walk) correlated with each other but not with participant IQ. Conclusions: This work identifies important phenotypes associated with CDM during childhood. Several measures of strength and function were significantly different between participants with CDM and controls and may be useful during future therapeutic trials.

Physical Function and Mobility in Children with Congenital Myotonic Dystrophy

Congenital myotonic dystrophy (CDM) occurs when symptoms of myotonic dystrophy present at birth. In this study we evaluated the relationship between physical function, muscle mass, and age to provide an assessment of the disease and help prepare for therapeutic trials.