A mutation in DOK7 in congenital myasthenic syndrome forms aggresome in cultured cells, and reduces DOK7 expression and MuSK phosphorylation in patient-derived iPS cells.

At the neuromuscular junction, the downstream of tyrosine kinase 7 (DOK7) enhances the phosphorylation of muscle-specific kinase (MuSK) and induces clustering of acetylcholine receptors (AChRs). We identified a patient with congenital myasthenic syndrome (CMS) with two heteroallelic mutations in DOK7, c.653-1G>C in intron 5 and c.190G>A predicting p.G64R in the pleckstrin homology domain. iPS cells established from the patient (CMS-iPSCs) showed that c.653-1G>C caused in-frame skipping of exon 6 (120 bp) and frame-shifting activation of a cryptic splice site deleting seven nucleotides in exon 6. p.G64R reduced the expression of DOK7 to 10% of wild-type DOK7, and markedly compromised AChR clustering in transfected C2C12 myotubes. p.G64R-DOK7 made insoluble aggresomes at the juxtanuclear region in transfected C2C12 myoblasts and COS7 cells, which were co-localized with molecules in the autophagosome system. A protease inhibitor MG132 reduced the soluble fraction of p.G64R-DOK7 and enhanced the aggresome formation of p.G64R-DOK7. To match the differentiation levels between patient-derived and control induced pluripotent stem cells (iPSCs), we corrected c.190G>A (p.G64R) by CRISPR/Cas9 to make isogenic iPSCs while retaining c.653-1G>C (CMS-iPSCsCas9). Myogenically differentiated CMS-iPSCs showed juxtanuclear aggregates of DOK7, reduced expression of endogenous DOK7 and reduced phosphorylation of endogenous MuSK. Another mutation, p.T77M, also made aggresome to a less extent compared with p.G64R in transfected COS7 cells. These results suggest that p.G64R-DOK7 makes aggresomes in cultured cells and is likely to compromise MuSK phosphorylation for AChR clustering.

A novel heterozygous TMEM63A variant in a familial case with early onset nystagmus, severe hypomyelination, and a favorable adult prognosis.

Heterozygous transmembrane protein 63A (TMEM63A) variants cause transient infantile hypomyelinating leukodystrophy-19, which features remarkable natural resolution of clinical and imaging findings during childhood. Previous reports have mainly described de novo variants lacking detailed familial cases. Herein, we describe the clinical course of familial cases with a TMEM63A variant. A 5-month-old girl presented with nystagmus, global hypotonia, and difficulty swallowing since birth. Brain magnetic resonance imaging at 1.5 and 5 months revealed diffuse hypomyelination. Her mother, maternal aunt, and grandfather had nystagmus and motor developmental delays in infancy, which resolved spontaneously during childhood. Compared with these cases, the proband's motor developmental delay was profound, and she was the only one with feeding difficulties, necessitating nasogastric tube feeding. Genetic testing revealed a heterozygous TMEM63A variant (NM_014698.3:c.1658G>A, p.(Gly553Asp)) in the proband and her family. This is the first three-generation familial report of a TMEM63A variant that provides insight into its history and heterogeneity.

Myelin abnormalities in merosin-deficient congenital muscular dystrophy.

INTRODUCTION/AIMS: Merosin is a protein complex located in the basement membrane of skeletal muscles and laminin α2-containing regions of the central and peripheral nervous systems. However, because of the prominence of muscle-related symptoms, peripheral neuropathy associated with merosin-deficient congenital muscular dystrophy type 1A (MDC1A) has received little clinical attention. This study aimed to present pathological changes in intramuscular nerves of three patients with MDC1A and discuss their relationship with electrophysiological findings to provide new evidence of peripheral nerve involvement in MDC1A. METHODS: MDC1A was confirmed by clinical features, muscle biopsy, and genetic testing for variants in LAMA2. To clarify peripheral nerve involvement, we statistically evaluated electrophysiological and muscle pathology findings of intramuscular nerves. These findings were compared with those of age-matched boys with Duchenne muscular dystrophy (DMD) as controls with normal nerves. Nerve conduction studies (NCS) were performed before biopsy. Biopsied intramuscular nerves were examined with electron microscopy using g-ratio, which is the ratio of axon diameter to myelinated fiber diameter. RESULTS: The myelin sheaths were significantly thinner in MDC1A patients than in age-matched DMD patients, with a mean g-ratio of 0.76 ± 0.07 in MDC1A patients and 0.65 ± 0.14 in DMD patients (p < .0001). No neuropathic changes were identified in muscle pathology. Low compound muscle action potential amplitudes, positive sharp waves and fibrillation potentials, and low-amplitude motor unit potentials with increased polyphasia indicated myopathic changes; no neurogenic changes were seen. DISCUSSION: We postulate that the thin myelin associated with MDC1A reflects the role of merosin in myelin maturation.

RNA-seq analysis, targeted long-read sequencing and in silico prediction to unravel pathogenic intronic events and complicated splicing abnormalities in dystrophinopathy.

Dystrophinopathy is caused by alterations in DMD. Approximately 1% of patients remain genetically undiagnosed, because intronic variations are not detected by standard methods. Here, we combined laboratory and in silico analyses to identify disease-causing genomic variants in genetically undiagnosed patients and determine the regulatory mechanisms underlying abnormal DMD transcript generation. DMD transcripts from 20 genetically undiagnosed dystrophinopathy patients in whom no exon variants were identified, despite dystrophin deficiency on muscle biopsy, were analyzed by transcriptome sequencing. Genome sequencing captured intronic variants and their effects were interpreted using in silico tools. Targeted long-read sequencing was applied in cases with suspected structural genomic abnormalities. Abnormal DMD transcripts were detected in 19 of 20 cases; Exonization of intronic sequences in 15 cases, exon skipping in one case, aberrantly spliced and polyadenylated transcripts in two cases and transcription termination in one case. Intronic single nucleotide variants, chromosomal rearrangements and nucleotide repeat expansion were identified in DMD gene as pathogenic causes of transcript alteration. Our combined analysis approach successfully identified pathogenic events. Detection of diseasing-causing mechanisms in DMD transcripts could inform the therapeutic options for patients with dystrophinopathy.

Factors influencing the decision to introduce alternative nutrition in patients with Duchenne muscular dystrophy.

INTRODUCTION/AIMS: Nutritional management of adults with Duchenne muscular dystrophy (DMD) is an important clinical issue. However, it is not clear which dysphagia-related factors should prompt introduction of alternative nutrition (AN). We aimed to determine which patients with DMD were introduced to AN. METHODS: This retrospective study included 56 patients with DMD (median age, 23.5 years). They were divided into patients able to continue oral feeding (OF) and those introduced to AN. Body weight, frequency of ventilator use, daily meals, history of steroid treatment, results of videofluoroscopic examination of swallowing (VF), and awareness of dysphagia were evaluated. RESULTS: Of 56 patients, 19 were in the AN group. After AN introduction, 93% of the patients continued oral intake. The proportion of patients who consumed chopped and liquid diets was higher, and body weight was lower, in the AN than in the OF group. There were no significant differences in age, upper limb function of feeding, frequency of ventilator use, or history of steroid therapy between the two groups. The frequencies of aspiration and residue in the pyriform sinus in VF were higher in the AN group than in the OF group. Decision-tree analysis showed that food form and subjective difficulty swallowing solid foods were the most important factors affecting the decision-making for AN. DISCUSSION: Patients with DMD who had difficulty eating solid foods were started on AN because they were unable to maintain their weight. These findings provide information for future longitudinal studies to assess the value of AN.

Establishment of a Triple Quadrupole HPLC-MS Quantitation Method for Dystrophin Protein in Mouse and Human Skeletal Muscle.

Duchenne muscular dystrophy (DMD) is the most common type of neuromuscular disease caused by mutations in the DMD gene encoding dystrophin protein. To quantitively assess human dystrophin protein in muscle biopsy samples, it is imperative to consistently detect as low as 0.003% of the dystrophin protein relative to the total muscle protein content. The quantitation of dystrophin protein has traditionally been conducted using semiquantitative immunoblotting or immunohistochemistry; however, there is a growing need to establish a more precise quantitative method by employing liquid chromatography-mass spectrometry (LC-MS) to measure dystrophin protein. In this study, a novel quantification method was established using a mouse experiment platform applied to the clinical quantification of human dystrophin protein. The method using a spike-in approach with a triple quadrupole LC-MS quantitated the amount of dystrophin in wild-type and human DMD transgenic mice but not in DMD-null mice. In conclusion, we established a quantitating method of dystrophin using HPLC-LC-MS with a novel spike-in approach. These results indicate that our methodology could be applied to several LC-MS devices to enable the accurate measurement of dystrophin protein in patients with DMD.

Genetic and clinical landscape of childhood cerebellar hypoplasia and atrophy.

PURPOSE: Cerebellar hypoplasia and atrophy (CBHA) in children is an extremely heterogeneous group of disorders, but few comprehensive genetic studies have been reported. Comprehensive genetic analysis of CBHA patients may help differentiating atrophy and hypoplasia and potentially improve their prognostic aspects. METHODS: Patients with CBHA in 176 families were genetically examined using exome sequencing. Patients with disease-causing variants were clinically evaluated. RESULTS: Disease-causing variants were identified in 96 of the 176 families (54.5%). After excluding 6 families, 48 patients from 42 families were categorized as having syndromic associations with CBHA, whereas the remaining 51 patients from 48 families had isolated CBHA. In 51 patients, 26 aberrant genes were identified, of which, 20 (76.9%) caused disease in 1 family each. The most prevalent genes were CACNA1A, ITPR1, and KIF1A. Of the 26 aberrant genes, 21 and 1 were functionally annotated to atrophy and hypoplasia, respectively. CBHA+S was more clinically severe than CBHA-S. Notably, ARG1 and FOLR1 variants were identified in 2 families, leading to medical treatments. CONCLUSION: A wide genetic and clinical diversity of CBHA was revealed through exome sequencing in this cohort, which highlights the importance of comprehensive genetic analyses. Furthermore, molecular-based treatment was available for 2 families.

MLIP causes recessive myopathy with rhabdomyolysis, myalgia and baseline elevated serum creatine kinase.

Striated muscle needs to maintain cellular homeostasis in adaptation to increases in physiological and metabolic demands. Failure to do so can result in rhabdomyolysis. The identification of novel genetic conditions associated with rhabdomyolysis helps to shed light on hitherto unrecognized homeostatic mechanisms. Here we report seven individuals in six families from different ethnic backgrounds with biallelic variants in MLIP, which encodes the muscular lamin A/C-interacting protein, MLIP. Patients presented with a consistent phenotype characterized by mild muscle weakness, exercise-induced muscle pain, variable susceptibility to episodes of rhabdomyolysis, and persistent basal elevated serum creatine kinase levels. The biallelic truncating variants were predicted to result in disruption of the nuclear localizing signal of MLIP. Additionally, reduced overall RNA expression levels of the predominant MLIP isoform were observed in patients' skeletal muscle. Collectively, our data increase the understanding of the genetic landscape of rhabdomyolysis to now include MLIP as a novel disease gene in humans and solidifies MLIP's role in normal and diseased skeletal muscle homeostasis.

Exon skipping induced by nonsense/frameshift mutations in DMD gene results in Becker muscular dystrophy.

Duchenne muscular dystrophy (DMD) is caused by a nonsense or frameshift mutation in the DMD gene, while its milder form, Becker muscular dystrophy (BMD) is caused by an in-frame deletion/duplication or a missense mutation. Interestingly, however, some patients with a nonsense mutation exhibit BMD phenotype, which is mostly attributed to the skipping of the exon containing the nonsense mutation, resulting in in-frame deletion. This study aims to find BMD cases with nonsense/frameshift mutations in DMD and to investigate the exon skipping rate of those nonsense/frameshift mutations. We searched for BMD cases with nonsense/frameshift mutations in DMD in the Japanese Registry of Muscular Dystrophy. For each DMD mutation identified, we constructed minigene plasmids containing one exon with/without a mutation and its flanking intronic sequence. We then introduced them into HeLa cells and measured the skipping rate of transcripts of the minigene by RT-qPCR. We found 363 cases with a nonsense/frameshift mutation in DMD gene from a total of 1497 dystrophinopathy cases in the registry. Among them, 14 had BMD phenotype. Exon skipping rates were well correlated with presence or absence of dystrophin, suggesting that 5% exon skipping rate is critical for the presence of dystrophin in the sarcolemma, leading to milder phenotypes. Accurate quantification of the skipping rate is important in understanding the exact functions of the nonsense/frameshift mutations in DMD and for interpreting the phenotypes of the BMD patients.

The nSMase2/Smpd3 gene modulates the severity of muscular dystrophy and the emotional stress response in mdx mice.

BACKGROUND: Duchenne muscular dystrophy (DMD) is a progressive, degenerative muscular disorder and cognitive dysfunction caused by mutations in the dystrophin gene. It is characterized by excess inflammatory responses in the muscle and repeated degeneration and regeneration cycles. Neutral sphingomyelinase 2/sphingomyelin phosphodiesterase 3 (nSMase2/Smpd3) hydrolyzes sphingomyelin in lipid rafts. This protein thus modulates inflammatory responses, cell survival or apoptosis pathways, and the secretion of extracellular vesicles in a Ca2+-dependent manner. However, its roles in dystrophic pathology have not yet been clarified. METHODS: To investigate the effects of the loss of nSMase2/Smpd3 on dystrophic muscles and its role in the abnormal behavior observed in DMD patients, we generated mdx mice lacking the nSMase2/Smpd3 gene (mdx:Smpd3 double knockout [DKO] mice). RESULTS: Young mdx:Smpd3 DKO mice exhibited reduced muscular degeneration and decreased inflammation responses, but later on they showed exacerbated muscular necrosis. In addition, the abnormal stress response displayed by mdx mice was improved in the mdx:Smpd3 DKO mice, with the recovery of brain-derived neurotrophic factor (Bdnf) expression in the hippocampus. CONCLUSIONS: nSMase2/Smpd3-modulated lipid raft integrity is a potential therapeutic target for DMD.

COX6A2 variants cause a muscle-specific cytochrome c oxidase deficiency.

OBJECTIVE: Cytochrome c oxidase (COX) deficiency is a major mitochondrial respiratory chain defect that has vast genetic and phenotypic heterogeneity. This study aims to identify novel causative genes of COX deficiency with only striated muscle-specific symptoms. METHODS: Whole exome sequencing was performed in 2 unrelated individuals who were diagnosed with congenital myopathy and presented COX deficiency in muscle pathology. We assessed the COX6A2 variants using measurements of enzymatic activities and assembly of mitochondrial respiratory chain complexes in the samples from the patients and knockout mice. RESULTS: Both patients presented muscle weakness and hypotonia in 4 limbs along with facial muscle weakness. One patient had cardiomyopathy. Neither patient exhibited involvement from other organs. Whole exome sequencing identified biallelic missense variants in COX6A2, which is expressed only in the skeletal muscle and heart. The variants detected were homozygous c.117C > A (p.Ser39Arg) and compound heterozygous c.117C > A (p.Ser39Arg) and c.127T > C (p.Cys43Arg). We found specific reductions in complex IV activities in the skeletal muscle of both individuals. Assembly of complex IV and its supercomplex formation were impaired in the muscle. INTERPRETATION: This study indicates that biallelic variants in COX6A2 cause a striated muscle-specific form of COX deficiency. ANN NEUROL 2019;86:193-202.

Renal dysfunction can occur in advanced-stage Duchenne muscular dystrophy.

INTRODUCTION: With improved treatments, patients with Duchenne muscular dystrophy (DMD) can survive far beyond adolescence. However, advanced-stage DMD patients are at risk of developing renal dysfunction. In this study, long-term renal function outcomes and associated risk factors in advanced stage DMD were analyzed. METHODS: Fifty-one patients were classified into three different age groups (<20, 20-29, and ≥30 years of age), and cystatin C (CysC) levels were compared among groups. RESULTS: Median serum CysC levels were 0.74 mg/L, 0.63 mg/L, and 0.76 mg/L in the age groups of <20, 20-29, and ≥30 years, respectively (P = .003). Five of the nine patients in the ≥30 years age group showed elevated serum CysC and decreased cardiac function compared with the other four in the group (P = .014). DISCUSSION: Our results indicate an association between cardiac and renal dysfunction in patients with advanced-stage DMD.

Static Leukoencephalopathy Associated with 17p13.3 Microdeletion Syndrome: A Case Report.

BACKGROUND: Leukoencephalopathy associated with dysmorphic features may be attributed to chromosomal abnormalities such as 17p13.3 microdeletion syndrome. CASE: A 19-year-old female patient was referred to our hospital for diagnostic evaluation of her leukoencephalopathy. She demonstrated moderate intellectual disability, minor dysmorphic features, and short stature. Serial brain magnetic resonance images obtained within a 16-year interval revealed prolonged T2 signals in the deep cerebral white matter with enlarged Virchow-Robin spaces. A nonsymptomatic atlas anomaly was also noted. Using microarray-based comparative genomic hybridization, we identified a 2.2-Mb terminal deletion at 17p13.3, encompassing YWHAE, CRK, and RTN4RL1 but not PAFAH1B1. CONCLUSION: Except for atlas anomaly, the patient's clinical and imaging findings were compatible with the diagnosis of 17p13.3 microdeletion syndrome. The white matter abnormality was static and nonprogressive. The association between the atlas abnormality and this deletion remains elusive. We note the importance of exploring submicroscopic chromosomal imbalance when patients show prominent but static white matter abnormalities with discrepantly mild and stable neurological signs.

Quantitative Analysis of Surface Electromyography for Pediatric Neuromuscular Disorders.

INTRODUCTION: Needle electromyography (EMG) has been an important diagnostic tool, although discomfort may limit its use in some children. We investigated the diagnostic utility of the clustering index (CI) method, a quantitative analysis for surface EMG (SEMG), in children. METHODS: SEMG was recorded from the tibialis anterior muscle. Discriminant analysis between patients with neurogenic disorders and patients with myopathy was performed for whole epochs by using the CI and area values. RESULTS: Forty-five children (29 with myopathy, 16 with neurogenic disorders; age 9 ± 3.9 years) were enrolled. The mean discriminant function value of the neurogenic group was 0.58 ± 0.88 (-0.48-2.30), whereas that of the myopathic group was -0.55 ± 0.70 (-2.38-0.68). When the cutoff value was set at the limit of the other group, 17 of 29 children with myopathy and 7 of 16 children with neurogenic disorders were correctly classified. DISCUSSION: The CI method can be a useful noninvasive diagnostic tool in children with neuromuscular disorders. Muscle Nerve 58:824-827, 2018.

Seizure imitators monitored using video-EEG in children with intellectual disabilities.

Diagnosis of seizure imitators in children is often challenging, and individuals with intellectual disability (ID) could be at additional risk of seizure imitator misdiagnosis. We aimed to elucidate distinct features of clinical semiology among children of different intellectual levels, which may help in distinguishing seizure imitators from epilepsy in such individuals. We retrospectively compared semiological features of seizure imitators in children with and without ID captured using video-electroencephalography (video-EEG). Seizure imitators were classified based on the definition of the International League Against Epilepsy (ILAE). A total of 67 individuals (mean age: 8.4 years, SD: 4.2 years) with seizure imitators documented using long-term video-EEG were identified, in which 27 patients had normal IQ/DQ, 20 had moderate ID, and 20 had severe ID. There was no statistically significant difference in the semiological features of seizure imitators between individuals with ID and those without ID; similarly, no difference was found between those with moderate ID and severe ID compared with individuals with normal IQ/DQ. Among all the patients, altered responsiveness mimicking cognitive or absence seizures was most frequently observed (36%), followed by jerks mimicking myoclonic seizures (22%). The most common seizure imitators among all the patients were unclassifiable nonepileptic seizures per the ILAE definition (28 cases, 42%), followed by day dreaming (24 cases, 36%) and physiological myoclonus (14 cases, 21%). In summary, the present study found no marked difference in semiological features of seizure imitators between patients with ID and those without ID regardless of ID severity, suggesting the necessity of early video-EEG for correct diagnosis.

A Nationwide Survey on Danon Disease in Japan.

Danon disease, an X-linked dominant cardioskeletal myopathy, is caused by primary deficiency of lysosome-associated membrane protein-2 (LAMP-2). To clarify the clinicopathological features and management, we performed the first nationwide, questionnaire-based survey on Danon disease in Japan. A total of 39 patients (17 males, 22 females) from 20 families were identified in the analysis. All patients had cardiomyopathy. Of the 21 patients who died, 20 (95%) died of cardiac failure or sudden cardiac arrest. Most patients had hypertrophic cardiomyopathy. Wolf⁻Parkinson⁻White syndrome was present at a comparatively high incidence (54% in males, 22% in females). Only one female patient received a heart transplant, which is the most effective therapy. Histopathologically, all male patients showed autophagic vacuoles with sarcolemmal features in muscle. Half of the probands showed de novo mutations. Male patients showed completely absent LAMP-2 expression in muscle. In contrast, female patients showed decreased LAMP-2 expression, which is suggested to reflect LAMP-2 haploinsufficiency due to a heterozygous null mutation. In conclusion, Danon disease is an extremely rare muscular disorder in Japan. Cardiomyopathy is the most significant prognostic factor and the main cause of death. Our findings suggest that the present survey can extend our understanding of the clinical features of this rare disease.

Pediatric necrotizing myopathy associated with anti-3-hydroxy-3-methylglutaryl-coenzyme A reductase antibodies.

OBJECTIVE: Antibodies against 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR) have recently been associated with immune-mediated necrotizing myopathy, especially in patients with statin exposure. As the data are very limited concerning phenotypes and treatment in paediatric patients, we aimed to identify the paediatric patients positive for anti-HMGCR antibodies and clarify their features and therapeutic strategies. METHODS: We screened 62 paediatric patients who were clinically and/or pathologically suspected to have inflammatory myopathy for anti-HMGCR antibodies. We further re-assessed the clinical and histological findings and the treatment of the patients positive for anti-HMGCR antibodies. RESULTS: We identified nine paediatric patients with anti-HMGCR antibodies (15%). This was more frequent than anti-signal recognition particle antibodies (four patients, 6%) in our cohort. The onset age ranged from infancy to 13 years. Five patients were initially diagnosed with muscular dystrophy, including congenital muscular dystrophy. Most patients responded to high-dose corticosteroid therapy first but often needed adjuvant immunosuppressants to become stably controlled. CONCLUSION: Paediatric necrotizing myopathy associated with anti-HMGCR antibodies may not be very rare. Phenotypes are similar to those of adult patients, but a chronic slowly progressive course may be more frequent. Some patients share the clinicopathological features of muscular dystrophy indicating that recognizing inflammatory aetiology would be challenging without autoantibody information. On the other hand, most patients responded to treatment, especially those who were diagnosed early. Our results suggest the importance of early autoantibody testing in paediatric patients who have manifestations apparently compatible with muscular dystrophy in addition to those who have typical features of inflammatory myopathy.

Mutations Causing Slow-Channel Myasthenia Reveal That a Valine Ring in the Channel Pore of Muscle AChR is Optimized for Stabilizing Channel Gating.

We identify two novel mutations in acetylcholine receptor (AChR) causing a slow-channel congenital myasthenia syndrome (CMS) in three unrelated patients (Pts). Pt 1 harbors a heterozygous ßV266A mutation (p.Val289Ala) in the second transmembrane domain (M2) of the AChR ß subunit (CHRNB1). Pts 2 and 3 carry the same mutation at an equivalent site in the ε subunit (CHRNE), εV265A (p.Val285Ala). The mutant residues are conserved across all AChR subunits of all species and are components of a valine ring in the channel pore, which is positioned four residues above the leucine ring. Both ßV266A and εV265A reduce the amino acid size and lengthen the channel opening bursts by fourfold by enhancing gating efficiency by approximately 30-fold. Substitution of alanine for valine at the corresponding position in the δ and α subunit prolongs the burst duration four- and eightfold, respectively. Replacing valine at ε codon 265 either by a still smaller glycine or by a larger leucine also lengthens the burst duration. Our analysis reveals that each valine in the valine ring contributes to channel kinetics equally, and the valine ring has been optimized in the course of evolution to govern channel gating.

Trends in steroid therapy for Duchenne muscular dystrophy in Japan.

INTRODUCTION: We conducted a study to reveal trends in steroid prescription for Duchenne muscular dystrophy (DMD) patients in Japan. METHODS: We asked patients (ages 5-20 years) identified in the patient registry and their clinicians about steroid therapy experiences. Regimen, dose, and starting age were compared among 3 subgroups according to prednisolone initiation year (2000-2004, 2005-2009, and 2010-2013). RESULTS: Among 157 prednisolone users, 4 different regimens were used. Dose frequencies were: every other day (98 patients), daily (44 patients), 10 days on 20 days off (14 patients), and weekly (1 patient). Median starting age was 6 years, and median dose was 0.42 mg/kg/day. There was an increase in daily regimen use from 2005-2009 (n = 9, 16%) to 2010-2013 (n = 33, 36%). CONCLUSIONS: This study revealed a transition over time in steroid use from expert opinion to evidence-based recommendation. Clinical research should be encouraged to optimize medication worldwide. Muscle Nerve 54: 673-680, 2016.