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Background:Spinal muscular atrophy (SMA) is a neuromuscular disorder characterized by the degeneration of the second motor neuron. The phenotype ranges from very severe to very mild forms. All patients have the homozygous loss of the SMN1 gene and a variable number of SMN2 (generally 2–4 copies), inversely related to the severity. The amazing results of the available treatments have made compelling the need of prognostic biomarkers to predict the progression trajectories of patients. Besides the SMN2 products, few other biomarkers have been evaluated so far, including some miRs.Methods:We performed whole miRNome analysis of muscle samples of patients and controls (14 biopsies and 9 cultures). The levels of muscle differentially expressed miRs were evaluated in serum samples (51 patients and 37 controls) and integrated with SMN2 copies, SMN2 full-length transcript levels in blood and age (SMA-score).Results:Over 100 miRs were differentially expressed in SMA muscle; 3 of them (hsa-miR-181a-5p, -324-5p, -451a; SMA-miRs) were significantly upregulated in the serum of patients. The severity predicted by the SMA-score was related to that of the clinical classification at a correlation coefficient of 0.87 (p-5).Conclusions:miRNome analyses suggest the primary involvement of skeletal muscle in SMA pathogenesis. The SMA-miRs are likely actively released in the blood flow; their function and target cells require to be elucidated. The accuracy of the SMA-score needs to be verified in replicative studies: if confirmed, its use could be crucial for the routine prognostic assessment, also in presymptomatic patients.Funding:Telethon Italia (grant #GGP12116).

Aims Catecholaminergic polymorphic ventricular tachycardia (CPVT) and short QT syndrome (SQTS) are inherited arrhythmogenic disorders that can cause sudden death. Numerous genes have been reported to cause these conditions, but evidence supporting these gene–disease relationships varies considerably. To ensure appropriate utilization of genetic information for CPVT and SQTS patients, we applied an evidence-based reappraisal of previously reported genes. Methods and results Three teams independently curated all published evidence for 11 CPVT and 9 SQTS implicated genes using the ClinGen gene curation framework. The results were reviewed by a Channelopathy Expert Panel who provided the final classifications. Seven genes had definitive to moderate evidence for disease causation in CPVT, with either autosomal dominant (RYR2, CALM1, CALM2, CALM3) or autosomal recessive (CASQ2, TRDN, TECRL) inheritance. Three of the four disputed genes for CPVT (KCNJ2, PKP2, SCN5A) were deemed by the Expert Panel to be reported for phenotypes that were not representative of CPVT, while reported variants in a fourth gene (ANK2) were too common in the population to be disease-causing. For SQTS, only one gene (KCNH2) was classified as definitive, with three others (KCNQ1, KCNJ2, SLC4A3) having strong to moderate evidence. The majority of genetic evidence for SQTS genes was derived from very few variants (five in KCNJ2, two in KCNH2, one in KCNQ1/SLC4A3). Conclusions Seven CPVT and four SQTS genes have valid evidence for disease causation and should be included in genetic testing panels. Additional genes associated with conditions that may mimic clinical features of CPVT/SQTS have potential utility for differential diagnosis.

The advent of clinical trials has highlighted the need for natural history studies reporting disease progression in type 1 spinal muscular atrophy. The aim of this study was to assess functional changes using the Children's Hospital of Philadelphia Infant Test of Neuromuscular Disorders (CHOP INTEND) scale in a cohort of type 1 infants. Nutritional and respiratory longitudinal data were also recorded. Patients were classified according to the severity of the phenotype and age of onset. SMN2 copies were also assessed. Twenty patients were included, eight with early onset most severe phenotype, eight with the more typical type 1 phenotype and 4, who achieved some head control, with a milder phenotype. Both baseline values and trajectories of progression were different in the three subgroups (p = 0.0001). Infants with the most severe phenotype had the lowest scores (below 20) on their first assessment and had the most rapid decline. Those with the typical phenotype had scores generally between 20 and 40 and also had a fast decline. The infants with the milder phenotype had the highest scores, generally above 35, and a much slower deterioration. Infants with three SMN2 copies had an overall milder phenotype and milder progression while two SMN2 copies were found in all three subgroups.

Supplemental Digital Content is available in the text., Background: Long QT syndrome (LQTS) is the first described and most common inherited arrhythmia. Over the last 25 years, multiple genes have been reported to cause this condition and are routinely tested in patients. Because of dramatic changes in our understanding of human genetic variation, reappraisal of reported genetic causes for LQTS is required. Methods: Utilizing an evidence-based framework, 3 gene curation teams blinded to each other’s work scored the level of evidence for 17 genes reported to cause LQTS. A Clinical Domain Channelopathy Working Group provided a final classification of these genes for causation of LQTS after assessment of the evidence scored by the independent curation teams. Results: Of 17 genes reported as being causative for LQTS, 9 (AKAP9, ANK2, CAV3, KCNE1, KCNE2, KCNJ2, KCNJ5, SCN4B, SNTA1) were classified as having limited or disputed evidence as LQTS-causative genes. Only 3 genes (KCNQ1, KCNH2, SCN5A) were curated as definitive genes for typical LQTS. Another 4 genes (CALM1, CALM2, CALM3, TRDN) were found to have strong or definitive evidence for causality in LQTS with atypical features, including neonatal atrioventricular block. The remaining gene (CACNA1C) had moderate level evidence for causing LQTS. Conclusions: More than half of the genes reported as causing LQTS have limited or disputed evidence to support their disease causation. Genetic variants in these genes should not be used for clinical decision-making, unless accompanied by new and sufficient genetic evidence. The findings of insufficient evidence to support gene-disease associations may extend to other disciplines of medicine and warrants a contemporary evidence-based evaluation for previously reported disease-causing genes to ensure their appropriate use in precision medicine.

BackgroundSpinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder, due to the loss of function of the survival motor neuron (SMN1) gene. The first treatment for the condition, recently approved, is based on the reduction of exon 7 skipping in mRNAs produced by a highly homologous gene (SMN2). The primary objective of the present study was to evaluate the applicability of the dosage of SMN gene produts in blood, as biomarker for SMA, and the safety of oral salbutamol, a beta2-adrenergic agonist modulating SMN2 levels.MethodsWe have performed a 1-year multicentre, double-blind, placebo-controlled study with salbutamol in 45 adult patients with SMA. Patients assumed 4 mg of salbutamol or placebo/three times a day. Molecular tests were SMN2 copy number, SMN transcript and protein levels. We have also explored the clinical effect, by the outcome measures available at the time of study design.ResultsThirty-six patients completed the study. Salbutamol was safe and well tolerated. We observed a significant and progressive increase in SMN2 full-length levels in peripheral blood of the salbutamol-treated patients (pConclusionsOur data demonstrate safety and molecular efficacy of salbutamol. We provide the first longitudinal evaluation of SMN levels (both transcripts and protein) in placebo and in response to a compound modulating the gene expression: SMN transcript dosage in peripheral blood is reliable and may be used as pharmacodynamic marker in clinical trials with systemic compounds modifying SMN2levels.Trial registration numberEudraCT no. 2007-001088-32.

Arthrogryposis multiplex congenita (AMC) is a developmental condition characterized by multiple joint contractures resulting from reduced or absent fetal movements. Homozygosity mapping of disease loci combined with whole exome sequencing in a consanguineous family presenting with lethal AMC allowed the identification of a homozygous frameshift deletion in UNC50 gene (c.750_751del:p.Cys251Phefs*4) in the index case. To assess the effect of the mutation, an equivalent mutation in the Caenorhabditis elegans orthologous gene was created using CRISPR/Cas9. We demonstrated that unc-50(kr331) modification caused the loss of acetylcholine receptor (AChR) expression in C. elegans muscle. unc-50(kr331) animals were as resistant to the cholinergic agonist levamisole as unc-50 null mutants suggesting that AChRs were no longer expressed in this animal model. This was confirmed by using a knock-in strain in which a red fluorescent protein was inserted into the AChR locus: no signal was detected in unc-50(kr331) background, suggesting that UNC-50, a protein known to be involved in AChR trafficking, was no longer functional. These data indicate that biallelic mutation in the UNC50 gene underlies AMC through a probable loss of AChR expression at the neuromuscular junction which is essential for the cholinergic transmission during human muscle development.

We report longitudinal clinical and neurophysiological assessments in twins affected by spinal muscular atrophy (SMA) with discordant phenotypes. The boy had the homozygous deletion of SMN1, a typical type 1 SMA course, and died at the age of eight months. His twin sister, asymptomatic at the time of the diagnosis in her brother, had the same genetic defect but she developed clinical and electrophysiological signs of type 2 SMA. The reduction of tendon reflexes was the first clinical sign at the age of 4 months, followed within few weeks, by a mild decrement in the amplitude of the compound motor action potentials. After the age of 9 months, she showed a sudden clinical and electrophysiological deterioration. Among molecular tests, we determined SMN2 copy number, SMN2 and Plastin 3 transcript levels in peripheral blood, and observed no relevant differences between twins.

Proximal spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder caused by mutations of the SMN1 gene. Based on severity, three forms of SMA are recognised (types I-III). All patients usually have 2-4 copies of a highly homologous gene (SMN2) which produces insufficient levels of functional survival motor neuron (SMN) protein, due to the alternative splicing of exon 7. The availability of potential candidates to the treatment of SMA has raised a number of issues, including the availability of biomarkers. This study was aimed at evaluating whether the quantification of SMN2 products in peripheral blood is a suitable biomarker for SMA. Forty-five adult type III patients were evaluated by Manual Muscle Testing, North Star Ambulatory Assessment scale, 6-Minute Walk Test, myometry, forced vital capacity, and dual energy X-ray-absorptiometry. Molecular assessments included SMN2 copy number, levels of full length SMN2 (SMN2-fl) transcripts and those lacking exon 7, and SMN protein. Clinical outcome measures strongly correlated to each other. Lean body mass correlated inversely with years from diagnosis and with several aspects of motor performance. SMN2 copy number and SMN protein levels were not associated with motor performance or transcript levels. SMN2-fl levels correlated with motor performance in ambulant patients. Our results indicate that SMN2-fl levels correlate with motor performance only in patients preserving higher levels of motor function, while motor performance was strongly influenced by disease duration and lean body mass. If not taken into account, the confounding effect of disease duration may impair the identification of potential SMA biomarkers.

SummaryObjective To present the clinical features and the results of laboratory investigations in three patients with spinal muscular atrophy associated with progressive myoclonic epilepsy (SMA-PME), a rare condition caused by mutations in the N-acylsphingosine amidohydrosilase 1 (ASAH1) gene. Methods The patients were submitted to clinical evaluation, neurophysiologic investigations (that included wakefulness and sleep electroencephalography [EEG], video-polygraphic recording with jerk-locked back-averaging, multimodal evoked potentials, and electromyography), brain magnetic resonance imaging (MRI), biochemical screening, muscle and skin biopsies, and molecular genetic analysis. Results The main clinical features were onset in childhood with proximal muscular weakness, generalized epilepsy with absences and myoclonic seizures, cognitive impairment of variable degree; the course was progressive with muscle wasting and uncontrolled epileptic seizures. In one patient, earlier onset before the age of 2 years was associated with a more complex clinical picture, with abnormal eye movements, progressive cognitive impairment, and a more rapid and severe course. EEG/polygraphic data were consistent with PME, demonstrating generalized spike-and-wave discharges, evidence of positive and negative myoclonia, and prominent photosensitivity. In one patient, transcranial magnetic stimulation showed a hyperexcitable motor cortex, whereas somatosensory evoked potentials were unaffected. Possible involvement of the central acoustic and visual pathways was suggested by abnormal auditory and visual evoked potentials. Muscle biopsies showed typical signs of neurogenic damage. Molecular genetic analysis showed mutations of the ASAH1 gene. Significance Our data indicate that SMA-PME associated with ASAH1 mutations is a genetically distinct condition with specific clinical and neurophysiologic features. Further studies are warranted to explore the role of the ASAH1 gene in muscle and brain function.

We have conducted a study to evaluate tolerability and efficacy of salbutamol in 45 type III adult SMA patients. Patients enrolled in the study were randomly divided into two groups treated, under double-blind conditions, with salbutamol or identical placebo tablets at the same dosing schedule. Patients were evaluated at baseline, and after 3, 6, and 12 months of treatment by Manual Muscle Testing, North Star Ambulatory Assessment scale, 6-Minute Walk Test and forced vital capacity. Molecular analyses included SMN2 gene copy number, SMN2 transcript, and SMN protein levels. Thirty-six patients completed the study (19 in the treated-group and 17 in the placebo-group): 8 patients were lost at the follow-up, 1 salbutamol-treated patient was withdrawn from the trial after 6 months because of diffuse erythematic eruption, improbably related to the drug. SMN2-fl transcript levels progressively increased in all salbutamol-treated patients (p 0.61). SMN protein levels in longitudinal samples were highly variable in both groups. In the treated-group, 8/11 walking patients showed a statistically significant increase in NSAA score (p = 0.03) and in the 6MWT (p = 0.02). In both ambulant and non-ambulant subjects, MRC total score significantly increased in 11/19 patients from the treated-group (p = 0.003) but not in the placebo group (p = 0.80). We did not observe any correlation between SMN2-fl level increase and variation of clinical outcome measures. Of the 36 patients who had completed the double-blind study, 21 continued the follow-up and were re-evaluated after 1 year, with the same protocol. Our study shows that salbutamol is well tolerated, significantly increases SMN2-fl transcripts, and induces a clinically meaningful improvement of motor performance in the majority of patients.