Evaluation of safety and early efficacy of AAV gene therapy in mouse models of vanishing white matter disease.

Leukoencephalopathy with vanishing white matter (VWM) is a progressive incurable white matter disease that most commonly occurs in childhood and presents with ataxia, spasticity, neurological degeneration, seizures, and premature death. A distinctive feature is episodes of rapid neurological deterioration provoked by stressors such as infection, seizures, or trauma. VWM is caused by autosomal recessive mutations in one of five genes that encode the eukaryotic initiation factor 2B complex, which is necessary for protein translation and regulation of the integrated stress response. The majority of mutations are in EIF2B5. Astrocytic dysfunction is central to pathophysiology, thereby constituting a potential therapeutic target. Herein we characterize two VWM murine models and investigate astrocyte-targeted adeno-associated virus serotype 9 (AAV9)-mediated EIF2B5 gene supplementation therapy as a therapeutic option for VWM. Our results demonstrate significant rescue in body weight, motor function, gait normalization, life extension, and finally, evidence that gene supplementation attenuates demyelination. Last, the greatest rescue results from a vector using a modified glial fibrillary acidic protein (GFAP) promoter-AAV9-gfaABC(1)D-EIF2B5-thereby supporting that astrocytic targeting is critical for disease correction. In conclusion, we demonstrate safety and early efficacy through treatment with a translatable astrocyte-targeted gene supplementation therapy for a disease that has no cure.

Exome sequencing in the pediatric neuromuscular clinic leads to more frequent diagnosis of both neuromuscular and neurodevelopmental conditions.

INTRODUCTION/AIMS: Exome sequencing (ES) has proven to be a valuable diagnostic tool for neuromuscular disorders, which often pose a diagnostic challenge. The aims of this study were to investigate the clinical outcomes associated with utilization of ES in the pediatric neuromuscular clinic and to determine if specific phenotypic features or abnormal neurodiagnostic tests were predictive of a diagnostic result. METHODS: This was a retrospective medical record review of 76 pediatric neuromuscular clinic patients who underwent ES. Based upon clinical assessment prior to ES, patients were divided into two groups: affected by neuromuscular (n = 53) or non-neuromuscular (n = 23) syndromes. RESULTS: A diagnosis was made in 28/76 (36.8%), with 29 unique disorders identified. In the neuromuscular group, a neuromuscular condition was confirmed in 78% of those receiving a genetic diagnosis. Early age of symptom onset was associated with a significantly higher diagnostic yield. The most common reason neuromuscular diagnoses were not detected on prior testing was due to causative genes not being present on disease-specific panels. Changes to medical care were made in 57% of individuals receiving a diagnosis on ES. DISCUSSION: These data further support ES as a powerful diagnostic tool in the pediatric neuromuscular clinic and highlight the advantages of ES over gene panels, including the ability to identify diagnoses regardless of etiology, identify genes newly associated with disease, and identify multiple confounding diagnoses. Rapid and accurate diagnosis by ES can not only end the patient's diagnostic odyssey, but often impacts patients' medical management and genetic counseling of families.

Intron mutations and early transcription termination in Duchenne and Becker muscular dystrophy.

DMD pathogenic variants for Duchenne and Becker muscular dystrophy are detectable with high sensitivity by standard clinical exome analyses of genomic DNA. However, up to 7% of DMD mutations are deep intronic and analysis of muscle-derived RNA is an important diagnostic step for patients who have negative genomic testing but abnormal dystrophin expression in muscle. In this study, muscle biopsies were evaluated from 19 patients with clinical features of a dystrophinopathy, but negative clinical DMD mutation analysis. Reverse transcription-polymerase chain reaction or high-throughput RNA sequencing methods identified 19 mutations with one of three pathogenic pseudoexon types: deep intronic point mutations, deletions or insertions, and translocations. In association with point mutations creating intronic splice acceptor sites, we observed the first examples of DMD pseudo 3'-terminal exon mutations causing high efficiency transcription termination within introns. This connection between splicing and premature transcription termination is reminiscent of U1 snRNP-mediating telescripting in sustaining RNA polymerase II elongation across large genes, such as DMD. We propose a novel classification of three distinct types of mutations identifiable by muscle RNA analysis, each of which differ in potential treatment approaches. Recognition and appropriate characterization may lead to therapies directed toward full-length dystrophin expression for some patients.

Automated immunofluorescence analysis for sensitive and precise dystrophin quantification in muscle biopsies.

AIMS: Dystrophin, the protein product of the DMD gene, plays a critical role in muscle integrity by stabilising the sarcolemma during contraction and relaxation. The DMD gene is vulnerable to a variety of mutations that may cause complete loss, depletion or truncation of the protein, leading to Duchenne and Becker muscular dystrophies. Precise and reproducible dystrophin quantification is essential in characterising DMD mutations and evaluating the outcome of efforts to induce dystrophin through gene therapies. Immunofluorescence microscopy offers high sensitivity to low levels of protein expression along with confirmation of localisation, making it a critical component of quantitative dystrophin expression assays. METHODS: We have developed an automated and unbiased approach for precise quantification of dystrophin immunofluorescence in muscle sections. This methodology uses microscope images of whole-tissue sections stained for dystrophin and spectrin to measure dystrophin intensity and the proportion of dystrophin-positive coverage at the sarcolemma of each muscle fibre. To ensure objectivity, the thresholds for dystrophin and spectrin are derived empirically from non-sarcolemmal signal intensity within each tissue section. Furthermore, this approach is readily adaptable for measuring fibre morphology and other tissue markers. RESULTS: Our method demonstrates the sensitivity and reproducibility of this quantification approach across a wide range of dystrophin expression in both dystrophinopathy patient and healthy control samples, with high inter-operator concordance. CONCLUSION: As efforts to restore dystrophin expression in dystrophic muscle bring new potential therapies into clinical trials, this methodology represents a valuable tool for efficient and precise analysis of dystrophin and other muscle markers that reflect treatment efficacy.

Effect of Different Corticosteroid Dosing Regimens on Clinical Outcomes in Boys With Duchenne Muscular Dystrophy: A Randomized Clinical Trial.

Importance: Corticosteroids improve strength and function in boys with Duchenne muscular dystrophy. However, there is uncertainty regarding the optimum regimen and dosage. Objective: To compare efficacy and adverse effects of the 3 most frequently prescribed corticosteroid regimens in boys with Duchenne muscular dystrophy. Design, Setting, and Participants: Double-blind, parallel-group randomized clinical trial including 196 boys aged 4 to 7 years with Duchenne muscular dystrophy who had not previously been treated with corticosteroids; enrollment occurred between January 30, 2013, and September 17, 2016, at 32 clinic sites in 5 countries. The boys were assessed for 3 years (last participant visit on October 16, 2019). Interventions: Participants were randomized to daily prednisone (0.75 mg/kg) (n = 65), daily deflazacort (0.90 mg/kg) (n = 65), or intermittent prednisone (0.75 mg/kg for 10 days on and then 10 days off) (n = 66). Main Outcomes and Measures: The global primary outcome comprised 3 end points: rise from the floor velocity (in rise/seconds), forced vital capacity (in liters), and participant or parent global satisfaction with treatment measured by the Treatment Satisfaction Questionnaire for Medication (TSQM; score range, 0 to 100), each averaged across all study visits after baseline. Pairwise group comparisons used a Bonferroni-adjusted significance level of .017. Results: Among the 196 boys randomized (mean age, 5.8 years [SD, 1.0 years]), 164 (84%) completed the trial. Both daily prednisone and daily deflazacort were more effective than intermittent prednisone for the primary outcome (P < .001 for daily prednisone vs intermittent prednisone using a global test; P = .017 for daily deflazacort vs intermittent prednisone using a global test) and the daily regimens did not differ significantly (P = .38 for daily prednisone vs daily deflazacort using a global test). The between-group differences were principally attributable to rise from the floor velocity (0.06 rise/s [98.3% CI, 0.03 to 0.08 rise/s] for daily prednisone vs intermittent prednisone [P = .003]; 0.06 rise/s [98.3% CI, 0.03 to 0.09 rise/s] for daily deflazacort vs intermittent prednisone [P = .017]; and -0.004 rise/s [98.3% CI, -0.03 to 0.02 rise/s] for daily prednisone vs daily deflazacort [P = .75]). The pairwise comparisons for forced vital capacity and TSQM global satisfaction subscale score were not statistically significant. The most common adverse events were abnormal behavior (22 [34%] in the daily prednisone group, 25 [38%] in the daily deflazacort group, and 24 [36%] in the intermittent prednisone group), upper respiratory tract infection (24 [37%], 19 [29%], and 24 [36%], respectively), and vomiting (19 [29%], 17 [26%], and 15 [23%]). Conclusions and Relevance: Among patients with Duchenne muscular dystrophy, treatment with daily prednisone or daily deflazacort, compared with intermittent prednisone alternating 10 days on and 10 days off, resulted in significant improvement over 3 years in a composite outcome comprising measures of motor function, pulmonary function, and satisfaction with treatment; there was no significant difference between the 2 daily corticosteroid regimens. The findings support the use of a daily corticosteroid regimen over the intermittent prednisone regimen tested in this study as initial treatment for boys with Duchenne muscular dystrophy. Trial Registration: ClinicalTrials.gov Identifier: NCT01603407.

Longitudinal MRI brain volume changes over one year in children with mucopolysaccharidosis types IIIA and IIIB.

OBJECTIVE: To quantify changes in segmented brain volumes over 12 months in children with mucopolysaccharidosis types IIIA and IIIB (MPS IIIA and IIIB). METHODS: In order to establish suitable outcome measures for clinical trials, twenty-five children greater than 2 years of age were enrolled in a prospective natural history study of MPS IIIA and IIIB at Nationwide Children's Hospital. Data from sedated non-contrast brain 3 T MRIs and neuropsychological measures were reviewed from the baseline visit and at 12-month follow-up. No intervention beyond standard clinical care was provided. Age- and sex-matched controls were gathered from the National Institute of Mental Health Data Archive. Automated brain volume segmentation with longitudinal processing was performed using FreeSurfer. RESULTS: Of the 25 subjects enrolled with MPS III, 17 children (4 females, 13 males) completed at least one MRI with interpretable volumetric data. The ages ranged from 2.8 to 13.7 years old (average 7.2 years old) at enrollment, including 8 with MPS IIIA and 9 with MPS IIIB. At baseline, individuals with MPS III demonstrated reduced cerebral white matter and corpus callosum volumes, but greater volumes of the lateral ventricles, cerebellar cortex, and cerebellar white matter compared to controls. Among the 13 individuals with MPS III with two interpretable MRIs, there were annualized losses or plateaus in supratentorial brain tissue volumes (cerebral cortex -42.10 ± 18.52 cm3/year [mean ± SD], cerebral white matter -4.37 ± 11.82 cm3/year, subcortical gray matter -6.54 ± 3.63 cm3/year, corpus callosum -0.18 ± 0.62 cm3/yr) and in cerebellar cortex (-0.49 ± 12.57 cm3/year), with a compensatory increase in lateral ventricular volume (7.17 ± 6.79 cm3/year). Reductions in the cerebral cortex and subcortical gray matter were more striking in individuals younger than 8 years of age. Greater cerebral cortex volume was associated with higher fine and gross motor functioning on the Mullen Scales of Early Learning, while greater subcortical gray matter volume was associated with higher nonverbal functioning on the Leiter International Performance Scale. Larger cerebellar cortex was associated with higher receptive language performance on the Mullen, but greater cerebellar white matter correlated with worse adaptive functioning on the Vineland Adaptive Behavioral Scales and visual problem-solving on the Mullen. CONCLUSIONS: Loss or plateauing of supratentorial brain tissue volumes may serve as longitudinal biomarkers of MPS III age-related disease progression compared to age-related growth in typically developing controls. Abnormally increased cerebellar white matter in MPS III, and its association with worse performance on neuropsychological measures, suggest the possibility of pathophysiological mechanisms distinct from neurodegeneration-associated atrophy that warrant further investigation.

The Genotypic and Phenotypic Spectrum of BICD2 Variants in Spinal Muscular Atrophy.

The bicaudal D cargo adaptor 2 (BICD2) gene encodes a conserved cargo adaptor protein required for dynein-mediated transport. Inherited and de novo variants in BICD2 cause SMALED2 (spinal muscular atrophy lower extremity dominant 2), and a subset have recently been reported to cause severe, often lethal disease. However, a true genotype-phenotype correlation for BICD2 has not been performed, and cases described to date are scattered among at least 14 publications. In this review, we identify the characteristics of disease-causing variants in BICD2 that distinguish them from benign variation and perform genotype-phenotype correlations for 99 BICD2 variant carriers from 35 families. ANN NEUROL 2020;87:487-496.

Evaluating longitudinal therapy effects via the North Star Ambulatory Assessment.

INTRODUCTION/AIMS: In comparative studies, treatment effects are typically evaluated at a specific time point. When data are collected periodically, an alternative, clinically meaningful approach could be used to assess the totality of treatment effects. We applied a well-developed analytical procedure for evaluating longitudinal treatment effects using North Star Ambulatory Assessment (NSAA) data for illustration. METHODS: The NSAA comprises 17 scorable items/outcomes that measure changes in motor function. Using NSAA data from the published ataluren phase 3, randomized, placebo-controlled trial (NCT01826487), cumulative counts of failures to perform each item (transition from 2/1 [able/impaired] to 0 [unable]) were collected at specified time points for each patient over 48 wk. Treatment group-wise mean cumulative item failure count curves were constructed, comparing ataluren versus placebo and deflazacort versus prednisone/prednisolone among placebo-treated patients. The steeper the curve, the worse the outcome. A clinically meaningful summary of the between-group difference was provided for each comparison. RESULTS: The curve was uniformly steeper for placebo than ataluren after 16 wk and for prednisone/prednisolone than deflazacort after 8 wk. The two curves in each comparison continued to diverge thereafter, indicating sustained treatment benefits over time. Using a unique analytical approach, cumulative failure rates were reduced, on average, by 27% for ataluren versus placebo (rate ratio, 0.73; 95% confidence interval [CI], 0.55-0.97; p = .027) and 28% for deflazacort versus prednisone/prednisolone (rate ratio, 0.72; 95% CI, 0.53-0.96; p = .028). DISCUSSION: Unlike fixed-time analyses, this analytical approach enabled demonstration of cumulative, longitudinal treatment effects over time using repeatedly measured NSAA observations.

Truncating variants in UBAP1 associated with childhood-onset nonsyndromic hereditary spastic paraplegia.

Hereditary spastic paraplegia (HSP) is a group of disorders with predominant symptoms of lower-extremity weakness and spasticity. Despite the delineation of numerous genetic causes of HSP, a significant portion of individuals with HSP remain molecularly undiagnosed. Through exome sequencing, we identified five unrelated families with childhood-onset nonsyndromic HSP, all presenting with progressive spastic gait, leg clonus, and toe walking starting from 7 to 8 years old. A recurrent two-base pair deletion (c.426_427delGA, p.K143Sfs*15) in the UBAP1 gene was found in four families, and a similar variant (c.475_476delTT, p.F159*) was detected in a fifth family. The variant was confirmed to be de novo in two families and inherited from an affected parent in two other families. RNA studies performed in lymphocytes from one patient with the de novo c.426_427delGA variant demonstrated escape of nonsense-mediated decay of the UBAP1 mutant transcript, suggesting the generation of a truncated protein. Both variants identified in this study are predicted to result in truncated proteins losing the capacity of binding to ubiquitinated proteins, hence appearing to exhibit a dominant-negative effect on the normal function of the endosome-specific endosomal sorting complexes required for the transport-I complex.

Homozygous variants in AMPD2 and COL11A1 lead to a complex phenotype of pontocerebellar hypoplasia type 9 and Stickler syndrome type 2.

Pontocerebellar hypoplasia type 9 (PCH9) is an autosomal recessive neurodevelopmental disorder caused by pathogenic variants in the AMPD2 gene. We evaluated the son of a consanguineous couple who presented with profound hypotonia and global developmental delay. Other features included sensorineural hearing loss, asymmetric astigmatism, and high myopia. Clinical whole-exome sequence analysis identified a homozygous missense variant in AMPD2 (NM_001257360.1:c.2201C > T, p.[Pro734Leu]) that has not been previously reported. Given the strong phenotypic overlap with PCH9, including the identification of the typical "Figure 8" appearance of the brainstem on neuroimaging, we suspect this variant was causative of the neurodevelopmental disability in this individual. An additional homozygous nonsense variant in COL11A1 (NM_001854.4:c.1168G > T, p.[Glu390Ter]) was identified. Variants in this alternatively spliced region of COL11A1 have been identified to cause an autosomal recessive form of Stickler syndrome type 2 characterized by sensorineural hearing loss and eye abnormalities, but without musculoskeletal abnormalities. The COL11A1 variant likely also contributed to the individual's phenotype, suggesting two potentially relevant genetic findings. This challenging case highlights the importance of detailed phenotypic characterization when interpreting whole exome data.

Association Study of Exon Variants in the NF-κB and TGFß Pathways Identifies CD40 as a Modifier of Duchenne Muscular Dystrophy.

The expressivity of Mendelian diseases can be influenced by factors independent from the pathogenic mutation: in Duchenne muscular dystrophy (DMD), for instance, age at loss of ambulation (LoA) varies between individuals whose DMD mutations all abolish dystrophin expression. This suggests the existence of trans-acting variants in modifier genes. Common single nucleotide polymorphisms (SNPs) in candidate genes (SPP1, encoding osteopontin, and LTBP4, encoding latent transforming growth factor ß [TGFß]-binding protein 4) have been established as DMD modifiers. We performed a genome-wide association study of age at LoA in a sub-cohort of European or European American ancestry (n = 109) from the Cooperative International Research Group Duchenne Natural History Study (CINRG-DNHS). We focused on protein-altering variants (Exome Chip) and included glucocorticoid treatment as a covariate. As expected, due to the small population size, no SNPs displayed an exome-wide significant p value (< 1.8 × 10-6). Subsequently, we prioritized 438 SNPs in the vicinities of 384 genes implicated in DMD-related pathways, i.e., the nuclear-factor-κB and TGFß pathways. The minor allele at rs1883832, in the 5'-untranslated region of CD40, was associated with earlier LoA (p = 3.5 × 10-5). This allele diminishes the expression of CD40, a co-stimulatory molecule for T cell polarization. We validated this association in multiple independent DMD cohorts (United Dystrophinopathy Project, Bio-NMD, and Padova, total n = 660), establishing this locus as a DMD modifier. This finding points to cell-mediated immunity as a relevant pathogenetic mechanism and potential therapeutic target in DMD.

Update in Duchenne and Becker muscular dystrophy.

PURPOSE OF REVIEW: The purpose of this review is to highlight updates in the standard of care recommendations for DMD, and to describe approaches to and recent advances in genetic therapies for DMD. RECENT FINDINGS: Treatment of DMD patients with the corticosteroids prednisone or deflazacort remains the standard of care, and recent data shows that early treatment (as young as 5 months) with a weekend dosing regimen results in measurable improvement in motor outcomes. A mutation-specific therapy directed at restoring an open reading frame by skipping exon 51 is FDA-approved, and therapies directed at other exons are in trials. Gene replacement therapy shows significant promise in animal models, and trials are underway. Genome editing has received significant attention because of results in animal models, but challenges to implementation in humans remain. SUMMARY: The mainstay of treatment remains meeting well defined standards of care that have been shown to influence morbidity and mortality. These include use of systemic steroids, early nocturnal ventilatory support, appropriate cardiac care and prophylaxis, and wherever appropriate, scoliosis surgery. Early and accurate molecular diagnosis, along with appropriate and multidisciplinary care, provides the best opportunity for maximum benefit of both current standard and upcoming novel therapies for boys with DMD. Among the most promising of these is AAV-based gene replacement therapy, which is currently in clinical trials.

Correction: Variants in MED12L, encoding a subunit of the Mediator kinase module, are responsible for intellectual disability associated with transcriptional defect.

In the Acknowledgements section of the paper the authors neglected to mention that the study was supported by a grant from the National Human Genome Research Institute (NHGRI) UM1HG007301 (S.H., M.L.T.). In addition, the award of MD was associated with the authors Michelle L. Thompson and Susan Hiatt instead of PhD. The PDF and HTML versions of the Article have been modified accordingly.

Variants in MED12L, encoding a subunit of the mediator kinase module, are responsible for intellectual disability associated with transcriptional defect.

PURPOSE: Mediator is a multiprotein complex that allows the transfer of genetic information from DNA binding proteins to the RNA polymerase II during transcription initiation. MED12L is a subunit of the kinase module, which is one of the four subcomplexes of the mediator complex. Other subunits of the kinase module have been already implicated in intellectual disability, namely MED12, MED13L, MED13, and CDK19. METHODS: We describe an international cohort of seven affected individuals harboring variants involving MED12L identified by array CGH, exome or genome sequencing. RESULTS: All affected individuals presented with intellectual disability and/or developmental delay, including speech impairment. Other features included autism spectrum disorder, aggressive behavior, corpus callosum abnormality, and mild facial morphological features. Three individuals had a MED12L deletion or duplication. The other four individuals harbored single-nucleotide variants (one nonsense, one frameshift, and two splicing variants). Functional analysis confirmed a moderate and significant alteration of RNA synthesis in two individuals. CONCLUSION: Overall data suggest that MED12L haploinsufficiency is responsible for intellectual disability and transcriptional defect. Our findings confirm that the integrity of this kinase module is a critical factor for neurological development.

Evaluation of biomarkers for Sanfilippo syndrome.

Sanfilippo syndrome or mucopolysaccharidosis type III (MPS III) is a childhood metabolic disorder marked by neuropathology arising due to impaired heparan sulphate (HS) catabolism. Consequently, partially degraded HS accumulates in the lysosomes of affected cells and is excreted in the urine. The measurement of HS in urine has long been considered a biomarker of Sanfilippo syndrome although it is largely non-specific. Using blood, urine and CSF collected from a cohort of Sanfilippo patients we investigated the utility of primary and secondary biomarkers to inform on disease activity. These included enzyme activity, specific oligosaccharides with non-reducing end residues reflective of the enzyme deficiency, and gangliosides. The diagnostic oligosaccharides - a HS disaccharide and tetrasaccharide - were elevated in the urine, plasma and CSF of all MPS IIIA and IIIB patients, respectively. There was no correlation between the concentrations in any of the matrices suggesting they reflect specific tissues and not overall disease burden. Enzyme activity did not inform on disease severity, with no measurable activity in CSF and activity approaching normal in MPS IIIA plasma. The concentration of gangliosides, GM2 and GM3, were significantly higher in the CSF of all MPS III subjects when compared to controls and correlated with the age of onset of first symptoms. Given that these gangliosides reflect delayed brain development they may be useful measures of disease burden, within the limitations of the clinical surrogates. Observation of these biochemical measurements in MPS III patients enrolled in clinical trials may determine whether they represent true pharmacodynamics biomarkers.

Long-range genomic regulators of THBS1 and LTBP4 modify disease severity in duchenne muscular dystrophy.

OBJECTIVE: Duchenne muscular dystrophy (DMD) is a severe X-linked recessive disease caused by loss-of-function dystrophin (DMD) mutations in boys, who typically suffer loss of ambulation by age 12. Previously, we reported that coding variants in latent transforming growth factor beta (TGFß)-binding protein 4 (LTBP4) were associated with reduced TGFß signaling and prolonged ambulation (p = 1.0 × 10-3 ) in DMD patients; this result was subsequently replicated by other groups. In this study, we evaluated whether additional DMD modifier genes are observed using whole-genome association in the original cohort. METHODS: We performed a genome-wide association study (GWAS) for single-nucleotide polymorphisms (SNPs) influencing loss of ambulation (LOA) in the same cohort of 253 DMD patients used to detect the candidate association with LTBP4 coding variants. Gene expression and chromatin interaction databases were used to fine-map association signals above the threshold for genome-wide significance. RESULTS: Despite the small sample size, two loci associated with prolonged ambulation met genome-wide significance and were tagged by rs2725797 (chr15, p = 6.6 × 10-9 ) and rs710160 (chr19, p = 4.7 × 10-8 ). Gene expression and chromatin interaction data indicated that the latter SNP tags regulatory variants of LTBP4, whereas the former SNP tags regulatory variants of thrombospondin-1 (THBS1): an activator of TGFß signaling by direct binding to LTBP4 and an inhibitor of proangiogenic nitric oxide signaling. INTERPRETATION: Together with previous evidence implicating LTBP4, the THBS1 modifier locus emphasizes the role that common regulatory variants in gene interaction networks can play in mitigating disease progression in muscular dystrophy. Ann Neurol 2018;84:234-245.

Twice-weekly glucocorticosteroids in infants and young boys with Duchenne muscular dystrophy.

INTRODUCTION: Glucocorticosteroids (GC) are effective in slowing weakness in boys with Duchenne muscular dystrophy (DMD). METHODS: This is a multisite, 1-year, open-label trial of twice-weekly prednisolone (5 mg/kg/dose) in infants/young boys (0.4-2.4 years) with DMD. We compared changes in Bayley III Scales of Infant Development (Bayley-III) with untreated boys followed for 1 year (historical control cohort [HCC]). Twenty-three of 25 participants completed the study. RESULTS: Treated boys gained an average of 0.5 points on the Bayley-III gross motor scaled score (GMSS) compared with the HCC who, on average, declined 1.3 points (P = 0.03). All boys maintained linear growth, and none developed Cushingoid features. Excessive weight gain occurred in 13 of 23 (56%) boys. DISCUSSION: This study provides evidence that twice-weekly GC is well tolerated in infants and young boys with DMD and improves GMSS. Excessive weight gain is a potential risk. Longer follow-up is required to determine whether early GC initiation is feasible in most infants/boys with DMD. Muscle Nerve 59:650-657, 2019.

Diagnostic Utility of Whole Exome Sequencing in the Neuromuscular Clinic.

Next-generation sequencing is a powerful diagnostic tool, yet it has proven inadequate to establish a diagnosis in all cases of congenital hypotonia or childhood onset weakness. We sought to describe the impact of whole exome sequencing (WES), which has only recently become widely available clinically, on molecular diagnosis in the Nationwide Children's Hospital Neuromuscular clinics. We reviewed records of all patients in our clinic with pediatric onset of symptoms who had WES done since 2013. Patients were included if clinical suspicion was high for a neuromuscular disease. Clinical WES was performed in 30 families, representing 31 patients, all of whom were seen for hypotonia, weakness, or gait disturbance. Probands had between 2 and 12 genetic diagnostic tests prior to obtaining WES. A genetic diagnosis was established in 11 families (37%), and in 12 patients (39%), with mutations in 10 different genes. Five of these genes have only been associated with disease since 2013, and were not previously represented on clinically available disease gene panels. Our results confirm the utility of WES in the clinical setting, particularly for genetically heterogeneous syndromes. The availability of WES can provide an end to the diagnostic odyssey for parents and allow for expansion of phenotypes.

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.